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Sample records for encoding sterol regulatory

  1. A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in Saccharomyces cerevisiae

    PubMed Central

    Crowley, James H.; Leak, Frank W.; Shianna, Kevin V.; Tove, Shirley; Parks, Leo W.

    1998-01-01

    Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca2+. Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1. PMID:9696767

  2. Sterols regulate 3β-hydroxysterol Δ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by Sterol Regulatory Element Binding Proteins.

    PubMed

    Zerenturk, Eser J; Sharpe, Laura J; Brown, Andrew J

    2012-10-01

    3β-Hydroxysterol Δ24-reductase (DHCR24) catalyzes a final step in cholesterol synthesis, and has been ascribed diverse functions, such as being anti-apoptotic and anti-inflammatory. How this enzyme is regulated transcriptionally by sterols is currently unclear. Some studies have suggested that its expression is regulated by Sterol Regulatory Element Binding Proteins (SREBPs) while another suggests it is through the Liver X Receptor (LXR). However, these transcription factors have opposing effects on cellular sterol levels, so it is likely that one predominates. Here we establish that sterol regulation of DHCR24 occurs predominantly through SREBP-2, and identify the particular region of the DHCR24 promoter to which SREBP-2 binds. We demonstrate that sterol regulation is mediated by two sterol regulatory elements (SREs) in the promoter of the gene, assisted by two nearby NF-Y binding sites. Moreover, we present evidence that the dual SREs work cooperatively to regulate DHCR24 expression by comparison to two known SREBP target genes, the LDL receptor with one SRE, and farnesyl-diphosphate farnesyltransferase 1, with two SREs. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum.

    PubMed

    Warnecke, D; Erdmann, R; Fahl, A; Hube, B; Müller, F; Zank, T; Zähringer, U; Heinz, E

    1999-05-07

    Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.

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

    PubMed

    Petersen, Josiah; Drake, Mary Jane; Bruce, Emily A; Riblett, Amber M; Didigu, Chukwuka A; Wilen, Craig B; Malani, Nirav; Male, Frances; Lee, Fang-Hua; Bushman, Frederic D; Cherry, Sara; Doms, Robert W; Bates, Paul; Briley, Kenneth

    2014-02-01

    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.

  5. Sterol Regulatory Element Binding Protein Is a Principal Regulator of Anaerobic Gene Expression in Fission Yeast†

    PubMed Central

    Todd, Bridget L.; Stewart, Emerson V.; Burg, John S.; Hughes, Adam L.; Espenshade, Peter J.

    2006-01-01

    Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced ≥2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption. PMID:16537923

  6. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    PubMed Central

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  7. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

    PubMed

    Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin; Choi, Ah-Reum; Lee, Sook-Jeong; Hoe, Kwang-Lae; Kim, Dong-Uk

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Regulation of Calcium-Independent Phospholipase A2 Expression by Adrenoceptors and Sterol Regulatory Element Binding Protein-Potential Crosstalk Between Sterol and Glycerophospholipid Mediators.

    PubMed

    Chew, Wee-Siong; Ong, Wei-Yi

    2016-01-01

    Calcium-independent phospholipase A2 (iPLA2) is an 85-kDa enzyme that releases docosahexaenoic acid (DHA) from glycerophospholipids. DHA can be metabolized to resolvins and neuroprotectins that have anti-inflammatory properties and effects on neural plasticity. Recent studies show an important role of prefrontal cortical iPLA2 in hippocampo-prefrontal cortical LTP and antidepressant-like effect of the norepinephrine reuptake inhibitor (NRI) antidepressant, maprotiline. In this study, we elucidated the cellular mechanisms through which stimulation of adrenergic receptors could lead to increased iPLA2 expression. Treatment of SH-SY5Y neuroblastoma cells with maprotiline, another tricyclic antidepressant with noradrenaline reuptake inhibiting properties, nortriptyline, and the adrenergic receptor agonist, phenylephrine, resulted in increased iPLA2β mRNA expression. This increase was blocked by inhibitors to alpha-1 adrenergic receptor, mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) 1/2, and sterol regulatory element-binding protein (SREBP). Maprotiline and phenylephrine induced binding of SREBP-2 to sterol regulatory element (SRE) region on the iPLA2 promoter, as determined by electrophoretic mobility shift assay (EMSA). Together, results indicate that stimulation of adrenoreceptors causes increased iPLA2 expression via MAP kinase/ERK 1/2 and SREBP, and suggest a possible mechanism for effect of CNS noradrenaline on neural plasticity and crosstalk between sterol and glycerophospholipid mediators, that may play a role in physiological or pathophysiological processes in the brain and other organs.

  9. Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay

    PubMed Central

    He, Miao; Kratz, Lisa E.; Michel, Joshua J.; Vallejo, Abbe N.; Ferris, Laura; Kelley, Richard I.; Hoover, Jacqueline J.; Jukic, Drazen; Gibson, K. Michael; Wolfe, Lynne A.; Ramachandran, Dhanya; Zwick, Michael E.; Vockley, Jerry

    2011-01-01

    Defects in cholesterol synthesis result in a wide variety of symptoms, from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in individuals with Smith-Lemli-Opitz syndrome. We report here the identification of mutations in sterol-C4-methyl oxidase–like gene (SC4MOL) as the cause of an autosomal recessive syndrome in a human patient with psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl oxidase (SMO), which catalyzes demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-Methylsterols are meiosis-activating sterols (MASs). They exist at high concentrations in the testis and ovary and play roles in meiosis activation. In this study, we found that an accumulation of MASs in the patient led to cell overproliferation in both skin and blood. SMO deficiency also substantially altered immunocyte phenotype and in vitro function. MASs serve as ligands for liver X receptors α and β (LXRα and LXRβ), which are important in regulating not only lipid transport in the epidermis, but also innate and adaptive immunity. Deficiency of SMO represents a biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined. PMID:21285510

  10. Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish

    PubMed Central

    Ashikawa, Yoshifumi; Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish

  11. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway.

    PubMed

    Bhattacharya, Bonhi S; Sweby, Peter K; Minihane, Anne-Marie; Jackson, Kim G; Tindall, Marcus J

    2014-05-21

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

  12. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

    PubMed Central

    Bhattacharya, Bonhi S.; Sweby, Peter K.; Minihane, Anne-Marie; Jackson, Kim G.; Tindall, Marcus J.

    2014-01-01

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature. PMID:24444765

  13. MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

    PubMed Central

    Horie, Takahiro; Nishino, Tomohiro; Baba, Osamu; Kuwabara, Yasuhide; Nakao, Tetsushi; Nishiga, Masataka; Usami, Shunsuke; Izuhara, Masayasu; Sowa, Naoya; Yahagi, Naoya; Shimano, Hitoshi; Matsumura, Shigenobu; Inoue, Kazuo; Marusawa, Hiroyuki; Nakamura, Tomoyuki; Hasegawa, Koji; Kume, Noriaki; Yokode, Masayuki; Kita, Toru; Kimura, Takeshi; Ono, Koh

    2013-01-01

    MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo. PMID:24300912

  14. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    SciTech Connect

    Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin; Choi, Ah-Reum; Lee, Sook-Jeong; Hoe, Kwang-Lae; Kim, Dong-Uk

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

  15. Sterols Regulate Development and Gene Expression in Arabidopsis1

    PubMed Central

    He, Jun-Xian; Fujioka, Shozo; Li, Tsai-Chi; Kang, Shin Gene; Seto, Hideharu; Takatsuto, Suguru; Yoshida, Shigeo; Jang, Jyan-Chyun

    2003-01-01

    Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::β-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways. PMID:12644676

  16. Sterols regulate development and gene expression in Arabidopsis.

    PubMed

    He, Jun-Xian; Fujioka, Shozo; Li, Tsai-Chi; Kang, Shin Gene; Seto, Hideharu; Takatsuto, Suguru; Yoshida, Shigeo; Jang, Jyan-Chyun

    2003-03-01

    Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.

  17. CYP710A genes encoding sterol C22-desaturase in Physcomitrella patens as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants.

    PubMed

    Morikawa, Tomomi; Saga, Hirohisa; Hashizume, Hiroko; Ohta, Daisaku

    2009-05-01

    We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC-MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56-60% of total sterol) and sitosterol to a lesser extent (8-12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with beta-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K(m) values for beta-sitosterol of CYP710A13 (1.0 +/- 0.043 microM) and CYP710A14 (2.1 +/- 0.17 microM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants.

  18. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    PubMed Central

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  19. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation.

    PubMed

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-08-14

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

  20. The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

    PubMed Central

    Kidani, Yoko; Elsaesser, Heidi; Hock, M Benjamin; Vergnes, Laurent; Williams, Kevin J; Argus, Joseph P; Marbois, Beth N; Komisopoulou, Evangelia; Wilson, Elizabeth B; Osborne, Timothy F; Graeber, Thomas G; Reue, Karen; Brooks, David G; Bensinger, Steven J

    2013-01-01

    Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation. PMID:23563690

  1. Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism.

    PubMed

    Lebeau, Paul; Al-Hashimi, Ali; Sood, Sudesh; Lhoták, Šárka; Yu, Pei; Gyulay, Gabriel; Paré, Guillaume; Chen, S R Wayne; Trigatti, Bernardo; Prat, Annik; Seidah, Nabil G; Austin, Richard C

    2017-01-27

    Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9 Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca(2+) depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca(2+) depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca(2+) inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.

  2. Sterol Synthesis in Diverse Bacteria

    PubMed Central

    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 identified genes encoding homologs of sterol biosynthesis proteins in the genomes of several additional species, indicating that sterol production may be more widespread in the bacterial domain than previously thought. Although the occurrence of sterol synthesis genes in a genome indicates the potential for sterol production, it provides neither conclusive evidence of sterol synthesis nor information about the composition and abundance of basic and modified sterols that are actually being produced. Here, we coupled bioinformatics with lipid analyses to investigate the scope of bacterial sterol production. We identified oxidosqualene cyclase (Osc), which catalyzes the initial cyclization of oxidosqualene to the basic sterol structure, in 34 bacterial genomes from five phyla (Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria, and Verrucomicrobia) and in 176 metagenomes. Our data indicate that bacterial sterol synthesis likely occurs in diverse organisms and environments and also provides evidence that there are as yet uncultured groups of bacterial sterol producers. Phylogenetic analysis of bacterial and eukaryotic Osc sequences confirmed a complex evolutionary history of sterol synthesis in this domain. Finally, we characterized the lipids produced by Osc-containing bacteria and found that we could generally predict the ability to synthesize sterols. However, predicting the final modified sterol based on our current knowledge of sterol synthesis was difficult. Some bacteria

  3. Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Deng, Xiong; Beranova-Giorgianni, Sarka; Bridges, Dave; Park, Edwards A; Raghow, Rajendra; Elam, Marshall B

    2014-07-11

    The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.

  4. An expansive human regulatory lexicon encoded in transcription factor footprints

    PubMed Central

    Neph, Shane; Vierstra, Jeff; Stergachis, Andrew B.; Reynolds, Alex P.; Haugen, Eric; Vernot, Benjamin; Thurman, Robert E.; Sandstrom, Richard; Johnson, Audra K.; Maurano, Matthew T.; Humbert, Richard; Rynes, Eric; Wang, Hao; Vong, Shinny; Lee, Kristen; Bates, Daniel; Diegel, Morgan; Roach, Vaughn; Dunn, Douglas; Neri, Jun; Schafer, Anthony; Hansen, R. Scott; Kutyavin, Tanya; Giste, Erika; Weaver, Molly; Canfield, Theresa; Sabo, Peter; Zhang, Miaohua; Balasundaram, Gayathri; Byron, Rachel; MacCoss, Michael J.; Akey, Joshua M.; Bender, Michael; Groudine, Mark; Kaul, Rajinder; Stamatoyannopoulos, John A.

    2013-01-01

    Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNaseI, leaving nucleotide-resolution footprints. Using genomic DNaseI footprinting across 41 diverse cell and tissue types, we detected 45 million factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNaseI cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50 base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation, and pluripotency. PMID:22955618

  5. An expansive human regulatory lexicon encoded in transcription factor footprints.

    PubMed

    Neph, Shane; Vierstra, Jeff; Stergachis, Andrew B; Reynolds, Alex P; Haugen, Eric; Vernot, Benjamin; Thurman, Robert E; John, Sam; Sandstrom, Richard; Johnson, Audra K; Maurano, Matthew T; Humbert, Richard; Rynes, Eric; Wang, Hao; Vong, Shinny; Lee, Kristen; Bates, Daniel; Diegel, Morgan; Roach, Vaughn; Dunn, Douglas; Neri, Jun; Schafer, Anthony; Hansen, R Scott; Kutyavin, Tanya; Giste, Erika; Weaver, Molly; Canfield, Theresa; Sabo, Peter; Zhang, Miaohua; Balasundaram, Gayathri; Byron, Rachel; MacCoss, Michael J; Akey, Joshua M; Bender, M A; Groudine, Mark; Kaul, Rajinder; Stamatoyannopoulos, John A

    2012-09-06

    Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

  6. Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase.

    PubMed

    Inoue, J; Sato, R; Maeda, M

    1998-06-01

    The expression of the human SREBP-2 gene is transcriptionally regulated in a cooperative manner by sterol regulatory element-binding proteins (SREBPs) and the general transcription factor NF-Y [Sato, R., Inoue, J., Kawabe, Y., Kodama, T., Takano, T., and Maeda, M. (1996) J. Biol. Chem. 271, 26461-26464]. To understand the sterol-dependent transcriptional regulation by these factors in detail, we have examined the regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and squalene synthase genes, whose promoters have multiple potential sterol regulatory elements (SRE, SREBP binding site) and NF-Y binding sites. The promoter of the human HMG CoA synthase gene was cloned, sequenced, and functionally characterized by means of reporter gene assays. The results indicate that an inverted CCAAT box, two SRE motifs and two Sp1 sites localized in a 90-bp region coordinately regulate the transcription. In the case of the human squalene synthase promoter, two SRE motifs and an inverted CCAAT box between the motifs localized in a 51-bp region are responsible for the sterol-regulated transcription of the gene. Gel mobility shift assay reveals that these two inverted CCAAT boxes are recognized by NF-Y. The involvement of multiple responsive elements in the transcription of HMG CoA synthase and squalene synthase seems to induce a higher level of sterol-dependent regulation (3.5 to 5. 8-fold) compared with that of the SREBP-2 promoter, which contains a single pair of SRE motif and CCAAT box (1.8 to 2.6-fold). Reporter gene assays using constructs containing various nucleotide spacing lengths between the SRE motif and the CCAAT box demonstrate that the 16 to 20-bp spacing range is required for maximal transcriptional regulation. These results agree with the findings that the distances between the two motifs in the known sterol responsive elements in several genes, including the human HMG CoA synthase and squalene synthase genes, are in this range.

  7. In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression

    SciTech Connect

    Takeuchi, Yoshinori; Yahagi, Naoya; Nakagawa, Yoshimi; Matsuzaka, Takashi; Shimizu, Ritsuko; Sekiya, Motohiro; Iizuka, Yoko; Ohashi, Ken; Gotoda, Takanari; Yamamoto, Masayuki; Nagai, Ryozo; Kadowaki, Takashi; Yamada, Nobuhiro; Osuga, Jun-ichi; Shimano, Hitoshi

    2007-11-16

    Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown. To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model that carries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter in liver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstrate that the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5'-flanking region is sufficient for this regulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated by serum insulin level. These mice are the first models to provide the mechanistic insight into the transcriptional regulation of SREBP-1c gene in vivo.

  8. Plant Sterols: Diversity, Biosynthesis, and Physiological Functions.

    PubMed

    Valitova, J N; Sulkarnayeva, A G; Minibayeva, F V

    2016-08-01

    Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3-methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.

  9. Activation of sterol regulatory element-binding proteins in mice exposed to perfluorooctanoic acid for 28 days.

    PubMed

    Yan, Shengmin; Wang, Jianshe; Dai, Jiayin

    2015-09-01

    Perfluoroalkyl acids are widely used in numerous industrial and commercial applications due to their unique physical and chemical characteristics. Although perfluorooctanoic acid (PFOA) is associated with hepatomegaly through peroxisome proliferator-activated receptor α (PPARα) activation, liver fat accumulation and changes in gene expression related to fatty acid metabolism could still be found in PPARα-null mice exposed to PFOA. To explore the potential effects of PFOA on sterol regulatory element-binding proteins (SREBPs) activity, male mice were dosed with either Milli-Q water or PFOA at doses of 0.08, 0.31, 1.25, 5, and 20 mg/kg/day by gavage for 28 days. Liver total cholesterol concentrations and PFOA contents showed a dose-dependent decrease and increase, respectively. Transcriptional activity of PPARα and SREBPs was significantly enhanced in livers. Protein expression analyzed by Western blotting showed that PFOA exposure stimulated SREBP maturation. Furthermore, proteins blocked SREBP precursor transport, insulin-induced gene 1 (INSIG1) and INSIG2 proteins, as well as a protein-mediated nuclear SREBP proteolysis, F-box and WD-40 domain protein 7, decreased in mouse liver exposed to PFOA. The expression levels of the miR-183-96-182 cluster, which is possibly involved in a regulatory loop intermediated by SREBPs maturation, were also increased in the mouse liver after PFOA exposure. We also observed that PFOA induced lipid content and PPARα in Hepa 1-6 cells after exposure to PFOA for 72 h but SREBPs were not activated in vitro. These results demonstrated that SREBPs were maturated by activating the miR-183-96-182 cluster-SREBP regulatory loop in PFOA-exposed mouse liver.

  10. Forkhead transcription factor 1 inhibits endometrial cancer cell proliferation via sterol regulatory element-binding protein 1

    PubMed Central

    Zhang, Yifang; Zhang, Lili; Sun, Hengzi; Lv, Qingtao; Qiu, Chunping; Che, Xiaoxia; Liu, Zhiming; Jiang, Jie

    2017-01-01

    The morbidity and mortality associated with endometrial cancer (EC) has increased in recent years. Regarded as a tumor suppressor, forkhead transcription factor 1 (FOXO1) has various biological activities and participates in cell cycle progression, apoptosis and differentiation. Notably, FOXO1 also functions in the regulation of lipogenesis and energy metabolism. Lipogenesis is a feature of cancer and is upregulated in EC. Sterol regulatory element-binding protein 1 (SREBP1) is a transcription factor that is also able to regulate lipogenesis. Increased expression of SREBP1 is directly correlated with malignant transformation of tumors. A previous study demonstrated that SREBP1 was highly expressed in EC and directly resulted in tumorigenesis. However, the association between FOXO1 and SREBP1 in EC is not clear. In the present study, lentiviruses overexpressing FOXO1 were used in cell transfection and transduction. Cell viability assays demonstrated that the overexpression of FOXO1 was able to suppress cell proliferation significantly in Ishikawa and AN3 CA cell lines. In addition, FOXO1 overexpression significantly inhibited cell migration and invasion ability in vitro. In xenograft models, overexpression of FOXO1 suppressed cell tumorigenesis, and western blot analysis demonstrated that SREBP1 expression was markedly reduced in the FOXO1-overexpressing cells. It may therefore be concluded that FOXO1 is able to inhibit the proliferative capacity of cells in vitro and in vivo, in addition to the migratory and invasive capacities in vitro by directly targeting SREBP1. PMID:28356952

  11. Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation.

    PubMed

    Lian, Li-Hua; Wu, Yan-Ling; Song, Shun-Zong; Wan, Ying; Xie, Wen-Xue; Li, Xin; Bai, Ting; Ouyang, Bing-Qing; Nan, Ji-Xing

    2010-12-22

    This study was undertaken to investigate the protective effects of Gentiana manshurica Kitagawa (GM) on acute alcohol-induced fatty liver. Mice were treated with ethanol (5 g/kg of body weight) by gavage every 12 h for a total of three doses to induce acute fatty liver. Methanol extract of GM (50, 100, or 200 mg/kg) or silymarin (100 mg/kg) was gavaged simultaneously with ethanol for three doses. GM administration significantly reduced the increases in serum ALT and AST levels, the serum and hepatic triglyceride levels, at 4 h after the last ethanol administration. GM was also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Additionally, GM suppressed the elevation of malondialdehyde (MDA) levels, restored the glutathione (GSH) levels, and enhanced the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities. The concurrent administration of GM efficaciously abrogated cytochrome P450 2E1 (CYP2E1) induction. Moreover, GM significantly reduced the nuclear translocation of sterol regulatory element-binding protein-1 (nSREBP-1) in ethanol-treated mice. These data indicated that GM possessed the ability to prevent ethanol-induced acute liver steatosis, possibly through blocking CYP2E1-mediated free radical scavenging effects and SREBP-1-regulated fatty acid synthesis. Especially, GM may be developed as a potential therapeutic candidate for ethanol-induced oxidative damage in liver.

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

    SciTech Connect

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

    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.

  13. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway*

    PubMed Central

    Zhou, Jie; Zhai, Yonggong; Mu, Ying; Gong, Haibiao; Uppal, Hirdesh; Toma, David; Ren, Songrong; Evans, Ronald M.; Xie, Wen

    2014-01-01

    The pregnane X receptor (PXR) was isolated as a xenosensor regulating xenobiotic responses. In this study, we show that PXR plays an endobiotic role by impacting lipid homeostasis. Expression of an activated PXR in the livers of transgenic mice resulted in an increased hepatic deposit of triglycerides. This PXR-mediated lipid accumulation was independent of the activation of the lipogenic transcriptional factor SREBP-1c (sterol regulatory element-binding protein 1c) and its primary lipogenic target enzymes, including fatty-acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC-1). Instead, the lipid accumulation in transgenic mice was associated with an increased expression of the free fatty acid transporter CD36 and several accessory lipogenic enzymes, such as stearoyl-CoA desaturase-1 (SCD-1) and long chain free fatty acid elongase. Studies using transgenic and knock-out mice showed that PXR is both necessary and sufficient for Cd36 activation. Promoter analyses revealed a DR-3-type of PXR-response element in the mouse Cd36 promoter, establishing Cd36 as a direct transcriptional target of PXR. The hepatic lipid accumulation and Cd36 induction were also seen in the hPXR “humanized” mice treated with the hPXR agonist rifampicin. The activation of PXR was also associated with an inhibition of pro-β-oxidative genes, such as peroxisome proliferator-activated receptor α (PPARα) and thiolase, and an up-regulation of PPARγ, a positive regulator of CD36. The cross-regulation of CD36 by PXR and PPARγ suggests that this fatty acid transporter may function as a common target of orphan nuclear receptors in their regulation of lipid homeostasis. PMID:16556603

  14. Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    PubMed Central

    2013-01-01

    Background Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. Results We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction. Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1

  15. Activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis C virus non-structural protein 2.

    PubMed

    Oem, Jae-Ku; Jackel-Cram, Candice; Li, Yi-Ping; Zhou, Yan; Zhong, Jin; Shimano, Hitoshi; Babiuk, Lorne A; Liu, Qiang

    2008-05-01

    Transcriptional factor sterol regulatory element-binding protein 1c (SREBP-1c) activates the transcription of lipogenic genes, including fatty acid synthase (FAS). Hepatitis C virus (HCV) infection is often associated with lipid accumulation within the liver, known as steatosis in the clinic. The molecular mechanisms of HCV-associated steatosis are not well characterized. Here, we showed that HCV non-structural protein 2 (NS2) activated SREBP-1c transcription in human hepatic Huh-7 cells as measured by using a human SREBP-1c promoter-luciferase reporter plasmid. We further showed that sterol regulatory element (SRE) and liver X receptor element (LXRE) in the SREBP-1c promoter were involved in SREBP-1c activation by HCV NS2. Furthermore, expression of HCV NS2 resulted in the upregulation of FAS transcription. We also showed that FAS upregulation by HCV NS2 was SREBP-1-dependent since deleting the SRE sequence in a FAS promoter and expressing a dominant-negative SREBP-1 abrogated FAS promoter upregulation by HCV NS2. Taken together, our results suggest that HCV NS2 can upregulate the transcription of SREBP-1c and FAS, and thus is probably a contributing factor for HCV-associated steatosis.

  16. MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro.

    PubMed

    Roth, G; Kotzka, J; Kremer, L; Lehr, S; Lohaus, C; Meyer, H E; Krone, W; Müller-Wieland, D

    2000-10-27

    Sterol regulatory element-binding protein (SREBP)-1a is a transcription factor sensing cellular cholesterol levels and integrating gene regulatory signals mediated by MAP kinase cascades. Here we report the identification of serine 117 in SREBP-1a as the major phosphorylation site of the MAP kinases Erk1/2. This site was identified by nanoelectrospray mass spectrometry and peptide sequencing of recombinant fusion proteins phosphorylated by Erk1/2 in vitro. Serine 117 was verified as the major phosphorylation site by in vitro mutagenesis. Mutation of serine 117 to alanine abolished Erk2-mediated phosphorylation in vitro and the MAP kinase-related transcriptional activation of SREBP-1a by insulin and platelet-derived growth factor in vivo. Our data indicate that the MAP kinase-mediated effects on SREBP-1a-regulated target genes are linked to this phosphorylation site.

  17. Architecture of the human regulatory network derived from ENCODE data.

    PubMed

    Gerstein, Mark B; Kundaje, Anshul; Hariharan, Manoj; Landt, Stephen G; Yan, Koon-Kiu; Cheng, Chao; Mu, Xinmeng Jasmine; Khurana, Ekta; Rozowsky, Joel; Alexander, Roger; Min, Renqiang; Alves, Pedro; Abyzov, Alexej; Addleman, Nick; Bhardwaj, Nitin; Boyle, Alan P; Cayting, Philip; Charos, Alexandra; Chen, David Z; Cheng, Yong; Clarke, Declan; Eastman, Catharine; Euskirchen, Ghia; Frietze, Seth; Fu, Yao; Gertz, Jason; Grubert, Fabian; Harmanci, Arif; Jain, Preti; Kasowski, Maya; Lacroute, Phil; Leng, Jing Jane; Lian, Jin; Monahan, Hannah; O'Geen, Henriette; Ouyang, Zhengqing; Partridge, E Christopher; Patacsil, Dorrelyn; Pauli, Florencia; Raha, Debasish; Ramirez, Lucia; Reddy, Timothy E; Reed, Brian; Shi, Minyi; Slifer, Teri; Wang, Jing; Wu, Linfeng; Yang, Xinqiong; Yip, Kevin Y; Zilberman-Schapira, Gili; Batzoglou, Serafim; Sidow, Arend; Farnham, Peggy J; Myers, Richard M; Weissman, Sherman M; Snyder, Michael

    2012-09-06

    Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

  18. Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

    PubMed

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2016-05-01

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

  19. Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)*

    PubMed Central

    Shao, Wei; Espenshade, Peter J.

    2014-01-01

    Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism. PMID:24478315

  20. Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo.

    PubMed

    Li, Enhu; Cui, Miao; Peter, Isabelle S; Davidson, Eric H

    2014-03-11

    By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo.

  1. Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo

    PubMed Central

    Li, Enhu; Cui, Miao; Peter, Isabelle S.; Davidson, Eric H.

    2014-01-01

    By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo. PMID:24556994

  2. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    PubMed

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  4. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells.

    PubMed

    Abd Eldaim, Mabrouk A; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-11-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP-la, expression of FAS genes and lipid accumulation in 3T3-L1 cells in the presence of RA and/or H2O2. RA (1 microM) treatment suppressed expression of SREBP-1a and FAS genes and lipid accumulation. H2O2 (2 microM) treatment induced increased cleavage of SREBP-1a, without affecting amounts of SREBP-1a mRNA and precursor protein, and enhanced expression of FAS gene and lipid accumulation. Increased cleavage of SREBP-1a by H2O2 was also observed even in the presence of RA. These results suggest that H2O2, enhances a cleavage of SREBP-1a precursor protein, which independently occurs with the RA suppression of SREBP-1a gene expression, and that RA itself has no role in the SREBP-1a activation in adipocytes.

  5. Neuronal Activity-Induced Sterol Regulatory Element Binding Protein-1 (SREBP1) is Disrupted in Dysbindin-Null Mice-Potential Link to Cognitive Impairment in Schizophrenia.

    PubMed

    Chen, Yong; Bang, Sookhee; McMullen, Mary F; Kazi, Hala; Talbot, Konrad; Ho, Mei-Xuan; Carlson, Greg; Arnold, Steven E; Ong, Wei-Yi; Kim, Sangwon F

    2017-04-01

    Schizophrenia is a chronic debilitating neuropsychiatric disorder that affects about 1 % of the population. Dystrobrevin-binding protein 1 (DTNBP1 or dysbindin) is one of the Research Domain Constructs (RDoC) associated with cognition and is significantly reduced in the brain of schizophrenia patients. To further understand the molecular underpinnings of pathogenesis of schizophrenia, we have performed microarray analyses of the hippocampi from dysbindin knockout mice, and found that genes involved in the lipogenic pathway are suppressed. Moreover, we discovered that maturation of a master transcriptional regulator for lipid synthesis, sterol regulatory element binding protein-1 (SREBP1) is induced by neuronal activity, and is required for induction of the immediate early gene ARC (activity-regulated cytoskeleton-associated protein), necessary for synaptic plasticity and memory. We found that nuclear SREBP1 is dramatically reduced in dysbindin-1 knockout mice and postmortem brain tissues from human patients with schizophrenia. Furthermore, activity-dependent maturation of SREBP1 as well as ARC expression were attenuated in dysbindin-1 knockout mice, and these deficits were restored by an atypical antipsychotic drug, clozapine. Together, results indicate an important role of dysbindin-1 in neuronal activity induced SREBP1 and ARC, which could be related to cognitive deficits in schizophrenia.

  6. Pu-erh tea down-regulates sterol regulatory element-binding protein and stearyol-CoA desaturase to reduce fat storage in Caenorhaditis elegans.

    PubMed

    Ding, YiHong; Zou, XiaoJu; Jiang, Xue; Wu, JieYu; Zhang, YuRu; Chen, Dan; Liang, Bin

    2015-01-01

    Consumption of Pu-erh has been reported to result in numerous health benefits, but the mechanisms underlying purported weight-loss and lowering of lipid are poorly understood. Here, we used the nematode Caenorhaditis elegans to explore the water extract of Pu-erh tea (PTE) functions to reduce fat storage. We found that PTE down-regulates the expression of the master fat regulator SBP-1, a homologue of sterol regulatory element binding protein (SREBP) and its target stearoyl-CoA desaturase (SCD), a key enzyme in fat biosynthesis, leading to an increased ratio of stearic acid (C18:0) to oleic acid (C18:1n-9), and subsequently decreased fat storage. We also found that both the pharyngeal pumping rate and food uptake of C. elegans decreased with exposure to PTE. Collectively, these results provide an experimental basis for explaining the ability of Pu-erh tea in promoting inhibition of food uptake and the biosynthesis of fat via SBP-1 and SCD, thereby reducing fat storage.

  7. Pu-Erh Tea Down-Regulates Sterol Regulatory Element-Binding Protein and Stearyol-CoA Desaturase to Reduce Fat Storage in Caenorhaditis elegans

    PubMed Central

    Ding, YiHong; Zou, XiaoJu; Jiang, Xue; Wu, JieYu; Zhang, YuRu; Chen, Dan; Liang, Bin

    2015-01-01

    Consumption of Pu-erh has been reported to result in numerous health benefits, but the mechanisms underlying purported weight-loss and lowering of lipid are poorly understood. Here, we used the nematode Caenorhaditis elegans to explore the water extract of Pu-erh tea (PTE) functions to reduce fat storage. We found that PTE down-regulates the expression of the master fat regulator SBP-1, a homologue of sterol regulatory element binding protein (SREBP) and its target stearoyl-CoA desaturase (SCD), a key enzyme in fat biosynthesis, leading to an increased ratio of stearic acid (C18:0) to oleic acid (C18:1n-9), and subsequently decreased fat storage. We also found that both the pharyngeal pumping rate and food uptake of C. elegans decreased with exposure to PTE. Collectively, these results provide an experimental basis for explaining the ability of Pu-erh tea in promoting inhibition of food uptake and the biosynthesis of fat via SBP-1 and SCD, thereby reducing fat storage. PMID:25659129

  8. Maturation and activity of sterol regulatory element binding protein 1 is inhibited by acyl-CoA binding domain containing 3.

    PubMed

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

  9. Sterol regulatory element-binding protein-1 determines plasma remnant lipoproteins and accelerates atherosclerosis in low-density lipoprotein receptor-deficient mice.

    PubMed

    Karasawa, Tadayoshi; Takahashi, Akimitsu; Saito, Ryo; Sekiya, Motohiro; Igarashi, Masaki; Iwasaki, Hitoshi; Miyahara, Shoko; Koyasu, Saori; Nakagawa, Yoshimi; Ishii, Kiyoaki; Matsuzaka, Takashi; Kobayashi, Kazuto; Yahagi, Naoya; Takekoshi, Kazuhiro; Sone, Hirohito; Yatoh, Shigeru; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2011-08-01

    Sterol regulatory element-binding protein-1 (SREBP-1) is nutritionally regulated and is known to be a key transcription factor regulating lipogenic enzymes. The goal of this study was to evaluate the roles of SREBP-1 in dyslipidemia and atherosclerosis. Transgenic mice that overexpress SREBP-1c in the liver and SREBP-1-deficient mice were crossed with low-density lipoprotein receptor (LDLR)-deficient mice, and the plasma lipids and atherosclerosis were analyzed. Hepatic SREBP-1c overexpression in LDLR-deficient mice caused postprandial hypertriglyceridemia, increased very-low-density lipoprotein (VLDL) cholesterol, and decreased high-density lipoprotein cholesterol in plasma, which resulted in accelerated aortic atheroma formation. Conversely, absence of SREBP-1 suppressed Western diet-induced hyperlipidemia in LDLR-deficient mice and ameliorated atherosclerosis. In contrast, bone marrow-specific SREBP-1 deficiency did not alter the development of atherosclerosis. The size of nascent VLDL particles secreted from the liver was increased in SREBP-1c transgenic mice and reduced in SREBP-1-deficient mice, accompanied by upregulation and downregulation of phospholipid transfer protein expression, respectively. Hepatic SREBP-1c determines plasma triglycerides and remnant cholesterol and contributes to atherosclerosis in hyperlipidemic states. Hepatic SREBP-1c also regulates the size of nascent VLDL particles.

  10. Preventing phosphorylation of sterol regulatory element-binding protein 1a by MAP-kinases protects mice from fatty liver and visceral obesity.

    PubMed

    Kotzka, Jorg; Knebel, Birgit; Haas, Jutta; Kremer, Lorena; Jacob, Sylvia; Hartwig, Sonja; Nitzgen, Ulrike; Muller-Wieland, Dirk

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP-1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP-1a mice the phosphorylation-deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

  11. A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

    PubMed Central

    Liu, Jing; Yuan, Yongze; Wu, Zhi; Li, Na; Chen, Yuanlei; Qin, Tingting; Geng, Hui; Xiong, Li; Liu, Deli

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression. PMID:25699519

  12. Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

    PubMed Central

    Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

    2017-01-01

    Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

  13. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    PubMed

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-02

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  14. Regulatory roles of LINE-1-encoded reverse transcriptase in cancer onset and progression

    PubMed Central

    Sciamanna, Ilaria; Gualtieri, Alberto; Piazza, Pier Vincenzo; Spadafora, Corrado

    2014-01-01

    LINE-1 retrotransposons encode the reverse transcriptase (RT) enzyme, required for their own mobility, the expression of which is inhibited in differentiated tissues while being active in tumors. Experimental evidence indicate that the inhibition of LINE-1-derived RT restores differentiation in cancer cells, inhibits tumor progression and yields globally reprogrammed transcription profiles. Newly emerging data suggest that LINE-1-encoded RT modulates the biogenesis of miRNAs, by governing the balance between the production of regulatory double-stranded RNAs and RNA:DNA hybrid molecules, with a direct impact on global gene expression. Abnormally high RT activity unbalances the transcriptome in cancer cells, while RT inhibition restores ‘normal’ miRNA profiles and their regulatory networks. This RT-dependent mechanism can target the myriad of transcripts - both coding and non-coding, sense and antisense - in eukaryotic transcriptomes, with a profound impact on cell fates. LINE-1-encoded RT emerges therefore as a key regulator of a previously unrecognized mechanism in tumorigenesis PMID:25478632

  15. Regulatory roles of LINE-1-encoded reverse transcriptase in cancer onset and progression.

    PubMed

    Sciamanna, Ilaria; Gualtieri, Alberto; Piazza, Pier Francesco; Spadafora, Corrado

    2014-09-30

    LINE-1 retrotransposons encode the reverse transcriptase (RT) enzyme, required for their own mobility, the expression of which is inhibited in differentiated tissues while being active in tumors. Experimental evidence indicate that the inhibition of LINE-1-derived RT restores differentiation in cancer cells, inhibits tumor progression and yields globally reprogrammed transcription profiles. Newly emerging data suggest that LINE-1-encoded RT modulates the biogenesis of miRNAs, by governing the balance between the production of regulatory double-stranded RNAs and RNA:DNA hybrid molecules, with a direct impact on global gene expression. Abnormally high RT activity unbalances the transcriptome in cancer cells, while RT inhibition restores "normal" miRNA profiles and their regulatory networks. This RT-dependent mechanism can target the myriad of transcripts - both coding and non-coding, sense and antisense - in eukaryotic transcriptomes, with a profound impact on cell fates. LINE-1-encoded RT emerges therefore as a key regulator of a previously unrecognized mechanism in tumorigenesis.

  16. U18666A, an Activator of Sterol Regulatory Element Binding Protein (SREBP) Pathway Modulates Presynaptic Dopaminergic Phenotype of SH-SY5Y Neuroblastoma Cells.

    PubMed

    Schmitt, Mathieu; Dehay, Benjamin; Bezard, Erwan; Garcia-Ladona, F Javier

    2017-04-13

    The therapeutic use of statins has been associated to a reduced risk of Parkinson's disease (PD) and may hold neuroprotective potential by counteracting the degeneration of dopaminergic neurons. Transcriptional activation of the sterol regulatory element-binding protein (SREBP) is one of the major downstream signalling pathways triggered by the cholesterol-lowering effect of statins. In a previous study in neuroblastoma cells, we have shown that statins consistently induce the up-regulation of presynaptic dopaminergic proteins as well as changes of their function and these effects were accompanied by downstream activation of SREBP. In current study, we aimed to determine the direct role of SREBP pathway in the modulation of dopaminergic phenotype. We demonstrate that treatment of SH-SY5Y cells with U18666A, a SREBP activator, increases the translocation of SREBPs into the nucleus, increases expression of SREBP-1, SREBP-2 and of the presynaptic dopaminergic markers such as vesicular monoamine transporter 2, synaptic vesicle glycoprotein 2A and 2C, synaptogyrin-3 and tyrosine hydroxylase. The addition of SREBP inhibitor, PF-429242, blocks the increase of U18666A-induced expression of SREBPs and of presynaptic markers. Our results, in line with previously reported effects of statins, demonstrate that direct stimulation of SREBP translocation is associated to differentiation towards a dopaminergic-like phenotype and suggest that SREBP-mediated transcriptional activity may lead to the restoration of the presynaptic dopamine markers and may contribute to neuroprotection of dopaminergic neurons. These findings further support the potential protective role of statin in PD and shed light upon SREBP as a potential new target for developing disease-modifying treatment in PD. This article is protected by copyright. All rights reserved.

  17. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs.

    PubMed

    Chen, J; Yang, X J; Xia, D; Chen, J; Wegner, J; Jiang, Z; Zhao, R Q

    2008-01-01

    Two experiments were performed to elucidate the role of sterol regulatory element binding transcription factor 1 (SREBF1) in i.m. fat (IMF) deposition in pigs. In Exp. 1, LM samples were removed from 4 male and 4 female Erhualian piglets at 3, 20, and 45 d of age, and SREBF1 mRNA expression level and IMF content were measured. Intramuscular fat content and expression of SREBF1 mRNA was greater (P < 0.05) in females than males at all 3 stages of age, providing initial evidence that the level of SREBF1 mRNA expression is related to IMF deposition in muscle of suckling pigs. Additionally, in Exp. 2 there was a positive correlation between the SREBF1 mRNA level and IMF content (r = 0.67, P < 0.01) in 100 Sutai finishing pigs, a synthetic line produced by crossing Erhualian and Duroc pigs. Single-strand conformation polymorphism (SSCP) analysis of the reverse transcription PCR products of the SREBF1 gene revealed 3 genotypes in Sutai pigs with frequencies of 50% for AA, 36% for AB, and 14% for BB, respectively. Both SREBF1 mRNA level and IMF content in muscle were greater (P < 0.05) in AB and BB animals than in AA animals, whereas no difference in backfat thickness was observed among the 3 genotypes. Sequencing analysis identified 2 SNP at T1006C and C1033T within the open reading frame of the SREBF1 gene (NM_214157). Although both are silent mutations, they affected the secondary structure of SREBF1 mRNA. These results suggest that SREBF1 might play an important role in regulation of muscle fat deposition during postnatal growth of pigs. The SNP identified in the SREBF1 gene suggest that it could be used as a genetic marker to improve IMF content in pigs.

  18. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  19. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation.

    PubMed

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-09-15

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation.

  20. Expression of Sterol Regulatory Element-Binding Proteins in epicardial adipose tissue in patients with coronary artery disease and diabetes mellitus: preliminary study

    PubMed Central

    Pérez-Belmonte, Luis M.; Moreno-Santos, Inmaculada; Cabrera-Bueno, Fernando; Sánchez-Espín, Gemma; Castellano, Daniel; Such, Miguel; Crespo-Leiro, María G; Carrasco-Chinchilla, Fernando; Alonso-Pulpón, Luis; López-Garrido, Miguel; Ruiz-Salas, Amalio; Becerra-Muñoz, Víctor M.; Gómez-Doblas, Juan J.; de Teresa-Galván, Eduardo; Jiménez-Navarro, Manuel

    2017-01-01

    Objectives: Sterol regulatory element-binding proteins (SREBP) genes are crucial in lipid biosynthesis and cardiovascular homeostasis. Their expression in epicardial adipose tissue (EAT) and their influence in the development of coronary artery disease (CAD) and type-2 diabetes mellitus remain to be determined. The aim of our study was to evaluate the expression of SREBP genes in EAT in patients with CAD according to diabetes status and its association with clinical and biochemical data. Methods: SREBP-1 and SREBP-2 mRNA expression levels were measured in EAT from 49 patients with CAD (26 with diabetes) and 23 controls without CAD or diabetes. Results: Both SREBPs mRNA expression were significantly higher in patients with CAD and diabetes (p<0.001) and were identified as independent cardiovascular risk factor for coronary artery disease in patients with type-2 diabetes (SREBP-1: OR 1.7, 95%CI 1.1-2.5, p=0.02; SREBP-2: OR 1.6, 95%CI 1.2-3, p=0.02) and were independently associated with the presence of multivessel CAD, left main and anterior descending artery stenosis, and higher total and LDL cholesterol levels, and lower HDL cholesterol levels, in patients with CAD and diabetes. Conclusions: SREBP genes are expressed in EAT and were higher in CAD patients with diabetes than those patients without CAD or diabetes. SREBP expression was associated as cardiovascular risk factor for the severity of CAD and the poor lipid control. In this preliminary study we suggest the importance of EAT in the lipid metabolism and cardiovascular homeostasis for coronary atherosclerosis of patients with diabetes and highlight a future novel therapeutic target. PMID:28367087

  1. Association Study Between Metabolic Syndrome and rs8066560 Polymorphism in the Promoter Region of Sterol Regulatory Element-binding Transcription Factor 1 Gene in Iranian Children and Adolescents

    PubMed Central

    Miranzadeh-Mahabadi, Hajar; Emadi-Baygi, Modjtaba; Nikpour, Parvaneh; Kelishadi, Roya

    2016-01-01

    Background: Metabolic syndrome (MetS) is a prevalent disorder in pediatric age groups, described by a combination of genetic and environmental factors. Sterol regulatory element-binding transcription factor 1 (SREBF-1) induces the expression of a family of genes involved in fatty acid synthesis. Moreover, dysregulation of miR-33b, which is located within the intron 17 of the SREBF-1 gene, disrupts fatty acid oxidation and insulin signaling, thus leading to MetS. The aim of the present study was to investigate the association between SREBF-1 rs8066560 polymorphism and MetS in Iranian children and adolescents. Methods: This study includes 100 MetS and 100 normal individuals aged 9–19 years. Anthropological and biochemical indexes were measured. The -1099G > A polymorphism was genotyped by TaqMan real-time polymerase chain reaction. Results: Significant differences were observed in anthropometric measurements and lipid profiles between MetS and normal children. There were no differences in the genotype frequencies or allele distribution for -1099G > A polymorphism between MetS and control groups. High-density lipoprotein cholesterol levels were significantly higher in the MetS GG group than in the A allele carrier group. The genotype AA controls had significantly increased cholesterol and low-density lipoprotein cholesterol levels than AG genotypes. By logistic regression using different genetic models, no significant association was observed between SREBF-1 rs8066560 polymorphism and the risk of MetS. Conclusions: We conclude that the -1099G > A variant on SREBF-1 gene associated with serum lipid profiles, however, it may not be a major risk factor for the MetS in Iranian children and adolescents. PMID:27076879

  2. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    PubMed

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  3. Elevated sterol regulatory elementary binding protein 1 and GluA2 levels in the hippocampal nuclear fraction of Genetic Absence Epilepsy Rats from Strasbourg.

    PubMed

    Sekar, Sathiya; Omran, Entesar; Gopalakrishnan, Venkat; Howland, John G; Snutch, Terrance P; Taghibiglou, Changiz

    2017-10-01

    Studies in animal models and human tissues show that nuclear translocation of sterol regulatory element binding protein 1 (SREBP1) and glutamate A2 subunit (GluA2) of cell-surface AMPA receptor (AMPAR) trigger neuronal excitotoxicity-induced apoptosis in stroke. However, it is not known whether a similar type of underlying pathophysiology occurs in absence epilepsy. To explore this issue, we examined the levels of mature SREBP1, GluA2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p53, and activated to total caspase 3 ratio in nuclear fractions (NF) of hippocampal homogenate from 8 to 10 week old male Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) strains. Mature SREBP1 and GluA2 levels were elevated approximately two-fold in NFs of GAERS hippocampal homogenates compared to NEC animals. Significant increases in GAPDH (∼15-fold) and total caspase 3 (∼10-fold) levels were also found in NFs of GAERS hippocampal homogenates in comparison to the non-epileptic strain. Data from the current study suggest that absence epilepsy in GAERS is associated with nuclear translocation of mature SREBP1, GluA2 subunit of AMPARs, and recruitment of pro-cell death signaling proteins such as GAPDH and caspase 3. These changes may contribute to hippocampal neuronal/glial cell death in GAERS. Therefore, inhibiting the nuclear accumulation of mature SREBP1 and GluA2 translocation may reduce the pathophysiology of absence epilepsy. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Lipid extract of Nostoc commune var. sphaeroides Kutzing, a blue-green alga, inhibits the activation of sterol regulatory element binding proteins in HepG2 cells.

    PubMed

    Rasmussen, Heather E; Blobaum, Kara R; Park, Young-Ki; Ehlers, Sarah J; Lu, Fan; Lee, Ji-Young

    2008-03-01

    Nostoc commune var. sphaeroides Kützing (N. commune), a blue-green alga, has been used as both a food ingredient and in medicine for centuries. To determine the effect of N. commune on cholesterol metabolism, N. commune lipid extract was incubated at increasing concentrations (25-100 mg/L) with HepG2 cells, a human hepatoma cell line. The addition of N. commune lipid extract markedly reduced mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDLR) (P < 0.05), with a concomitant decrease in their protein expression (P < 0.001). Reduced HMGR activity by 90% with N. commune lipid extract confirmed the inhibitory role of N. commune in cholesterol synthesis (P < 0.006). To elucidate a molecular mechanism underlying the repression of HMGR and LDLR by N. commune lipid extract, expression of sterol regulatory element binding protein 2 (SREBP-2) was assessed. Whereas mRNA for SREBP-2 remained unchanged, SREBP-2 mature protein was reduced by N. commune (P < 0.009). In addition, N. commune lipid extract also decreased SREBP-1 mature protein by approximately 30% (P < 0.002) and reduced the expression of SREBP-1-responsive genes such as fatty acid synthase and stearoyl CoA desaturase 1 (SCD-1) (P < 0.05). Therefore, our results demonstrate that N. commune lipid extract inhibits the maturation process of both SREBP-1 and -2, resulting in a decrease in expression of genes involved in cholesterol and fatty acid metabolism.

  5. Time of Day and Nutrients in Feeding Govern Daily Expression Rhythms of the Gene for Sterol Regulatory Element-binding Protein (SREBP)-1 in the Mouse Liver*

    PubMed Central

    Matsumoto, Eriko; Ishihara, Akinori; Tamai, Saki; Nemoto, Ayako; Iwase, Katsuro; Hiwasa, Takaki; Shibata, Shigenobu; Takiguchi, Masaki

    2010-01-01

    Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, “when to eat in a day (the light/dark cycle),” rather than “whenever to eat in a day,” is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of “what to eat.” Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients. PMID:20720008

  6. p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3*

    PubMed Central

    Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P.; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T.; Arumäe, Urmas; Lindholm, Dan

    2016-01-01

    Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. PMID:26984409

  7. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.

    PubMed

    Xu, J; Nakamura, M T; Cho, H P; Clarke, S D

    1999-08-13

    Polyunsaturated fatty acids (PUFA) coordinately suppress the transcription of a wide array of hepatic lipogenic genes including fatty acid synthase (FAS) and acetyl-CoA carboxylase. Interestingly, the over-expression of sterol regulatory element binding protein-1 (SREBP-1) induces the expression of all of the enzymes suppressed by PUFA. This observation led us to hypothesize that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1. Our initial studies revealed that the SREBP-1 and FAS mRNA contents of HepG2 cells were reduced by 20:4(n-6) in a dose-dependent manner (i.e. EC(50) approximately 10 microM), whereas 18:1(n-9) had no effect. Similarly, supplementing a fat-free, high glucose diet with oils rich in (n-6) or (n-3) PUFA reduced the hepatic content of precursor and nuclear SREBP-1 60 and 85%, respectively; however, PUFA had no effect on the nuclear content of upstream stimulatory factor (USF)-1. The PUFA-dependent decrease in nuclear content of mature SREBP-1 was paralleled by a 70-90% suppression in FAS gene transcription. In contrast, dietary 18:1(n-9), i.e. triolein, had no inhibitory influence on the expression of SREBP-1 or FAS. The decrease in hepatic expression of SREBP-1 and FAS associated with PUFA ingestion was mimicked by supplementing the fat-free diet with the PPARalpha-activator, WY 14, 643. Interestingly, nuclear run-on assays revealed that changes in SREBP-1 mRNA abundance were not accompanied by changes in SREBP-1 gene transcription. These results support the concept that PUFA coordinately inhibit lipogenic gene transcription by suppressing the expression of SREBP-1 and that the PUFA regulation of SREBP-1 appears to occur at the post-transcriptional level.

  8. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism*

    PubMed Central

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-01-01

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  9. Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation

    PubMed Central

    Lee, Jae Ho; Lee, Gha Young; Jang, Hagoon; Choe, Sung Sik; Koo, Seung-Hoi; Kim, Jae Bum

    2014-01-01

    Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. Conclusion: RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation

  10. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

    PubMed

    Deng, Xiong; Dong, Qingming; Bridges, Dave; Raghow, Rajendra; Park, Edwards A; Elam, Marshall B

    2015-12-01

    In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

  11. Sterol Regulatory Element-Binding Protein-1c Regulates Inflammasome Activation in Gingival Fibroblasts Infected with High-Glucose-Treated Porphyromonas gingivalis

    PubMed Central

    Kuo, Hsing-Chun; Chang, Li-Ching; Chen, Te-Chuan; Lee, Ko-Chao; Lee, Kam-Fai; Chen, Cheng-Nan; Yu, Hong-Ren

    2016-01-01

    Background: Porphyromonas gingivalis is a major bacterial species implicated in the progression of periodontal disease, which is recognized as a common complication of diabetes. The interleukin (IL)-1β, processed by the NLR family pyrin domain containing 3 (NLRP3) inflammasome, has been identified as a target for pathogenic infection of the inflammatory response. However, the effect of P. gingivalis in a high-glucose situation in the modulation of inflammasome activation in human gingival fibroblasts (HGFs) is not well-understood. Methods: P. gingivalis strain CCUG25226 was used to study the mechanisms underlying the regulation of HGF NLRP3 expression by the infection of high-glucose-treated P. gingivalis (HGPg). Results: HGF infection with HGPg increases the expression of IL-1β and NLRP3. We further demonstrated that the upregulation of sterol regulatory element-binding protein (SREBP)-1c by activation of the Akt and p70S6K pathways is critical for HGPg-induced NLRP3 expression. We showed that the inhibition of Janus kinase 2 (JAK2) blocks the Akt- and p70S6K-mediated SREBP-1c, NLRP3, and IL-1β expression. The effect of HGPg on HGF signaling and NLRP3 expression is mediated by β1 integrin. In addition, gingival tissues from diabetic patients with periodontal disease exhibited higher NLRP3 and SREBP-1c expression. Conclusions: Our findings identify the molecular pathways underlying HGPg-dependent NLRP3 inflammasome expression in HGFs, providing insight into the effect of P. gingivalis invasion in HGFs. PMID:28083517

  12. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    PubMed Central

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  13. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    SciTech Connect

    Yin Huquan; Kim, Youn-Chul; Chung, Young-Suk; Kim, Young-Chul; Shin, Young-Kee; Lee, Byung-Hoon

    2009-04-01

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-{alpha} levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-{alpha}, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c.

  14. Oxysterol sulfation by cytosolic sulfotransferase suppresses liver X receptor/sterol regulatory element binding protein-1c signaling pathway and reduces serum and hepatic lipids in mouse models of nonalcoholic fatty liver disease.

    PubMed

    Bai, Qianming; Zhang, Xin; Xu, Leyuan; Kakiyama, Genta; Heuman, Douglas; Sanyal, Arun; Pandak, William M; Yin, Lianhua; Xie, Wen; Ren, Shunlin

    2012-06-01

    Cytosolic sulfotransferase (SULT2B1b) catalyzes oxysterol sulfation. 5-Cholesten-3β-25-diol-3-sulfate (25HC3S), one product of this reaction, decreases intracellular lipids in vitro by suppressing liver X receptor/sterol regulatory element binding protein (SREBP)-1c signaling, with regulatory properties opposite to those of its precursor 25-hydroxycholesterol. Upregulation of SULT2B1b may be an effective strategy to treat hyperlipidemia and hepatic steatosis. The objective of the study was to explore the effect and mechanism of oxysterol sulfation by SULT2B1b on lipid metabolism in vivo. C57BL/6 and LDLR(-/-) mice were fed with high-cholesterol diet or high-fat diet for 10 weeks and infected with adenovirus encoding SULT2B1b. SULT2B1b expressions in different tissues were determined by immunohistochemistry and Western blot. Sulfated oxysterols in liver were analyzed by high-pressure liquid chromatography. Serum and hepatic lipid levels were determined by kit reagents and hematoxylin and eosin staining. Gene expressions were determined by real-time reverse transcriptase polymerase chain reaction and Western Blot. Following infection, SULT2B1b was successfully overexpressed in the liver, aorta, and lung tissues, but not in the heart or kidney. SULT2B1b overexpression, combined with administration of 25-hydroxycholesterol, significantly increased the formation of 25HC3S in liver tissue and significantly decreased serum and hepatic lipid levels, including triglycerides, total cholesterol, free cholesterol, and free fatty acids, as compared with controls in both C57BL/6 and LDLR(-/-) mice. Gene expression analysis showed that increases in SULT2B1b expression were accompanied by reduction in key regulators and enzymes involved in lipid metabolism, including liver X receptor α, SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, and fatty acid synthase. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid biosynthesis.

  15. Insect growth regulatory effects of some extracts and sterols from Myrtillocactus geometrizans (Cactaceae) against Spodoptera frugiperda and Tenebrio molitor.

    PubMed

    Céspedes, Carlos L; Salazar, J Rodrigo; Martínez, Mariano; Aranda, Eduardo

    2005-10-01

    A methanol extract from the roots and aerial parts of Myrtillocactus geometrizans (Cactaceae) yielded peniocerol 1, macdougallin 2, and chichipegenin 3. The natural products 1, 2 their mixtures, MeOH and CH(2)Cl(2) extracts showed insecticidal and insect growth regulatory activity against fall armyworm [Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)], an important insect pest of corn, and [Tenebrio molitor (Coleoptera)], a pest of stored grains in Mexico. The most active compounds were 1, 2, and a mixture (M(2)) of 1 and 2 (6:4). All these extracts, compounds and the mixture had insect growth regulating (IGR) activity between 5.0 and 50.0 ppm and insecticidal effects between 50 and 300 ppm in diets. The extracts were insecticidal to larvae, with lethal doses between 100 and 200 ppm. These compounds appear to have selective effects on the pre-emergence metabolism of Coleoptera, because in all treatments of the larvae of T. molitor, pupation were shortened and this process show precociousness in relation to controls. In contrast to S. frugiperda larvae, onset of pupation was noticeably delayed. Emergence in both cases was drastically diminished. In both pupae and in the few adults that were able to emerge, many deformations were observed. The results of these assays indicated that the compounds were more active than other known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. Peniocerol, macdougallin and chichipegenin, as well as mixtures of these substances, may be useful as natural insecticidal agents.

  16. Identification of functional elements and regulatory circuits by Drosophila modENCODE.

    PubMed

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L; Landolin, Jane M; Bristow, Christopher A; Ma, Lijia; Lin, Michael F; Washietl, Stefan; Arshinoff, Bradley I; Ay, Ferhat; Meyer, Patrick E; Robine, Nicolas; Washington, Nicole L; Di Stefano, Luisa; Berezikov, Eugene; Brown, Christopher D; Candeias, Rogerio; Carlson, Joseph W; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y; Will, Sebastian; Alekseyenko, Artyom A; Artieri, Carlo; Booth, Benjamin W; Brooks, Angela N; Dai, Qi; Davis, Carrie A; Duff, Michael O; Feng, Xin; Gorchakov, Andrey A; Gu, Tingting; Henikoff, Jorja G; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K; Riddle, Nicole C; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E; Schwartz, Yuri B; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E; Brent, Michael R; Cherbas, Lucy; Elgin, Sarah C R; Gingeras, Thomas R; Grossman, Robert; Hoskins, Roger A; Kaufman, Thomas C; Kent, William; Kuroda, Mitzi I; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J; Celniker, Susan E; Henikoff, Steven; Karpen, Gary H; Lai, Eric C; MacAlpine, David M; Stein, Lincoln D; White, Kevin P; Kellis, Manolis

    2010-12-24

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

  17. Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE

    PubMed Central

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L.; Landolin, Jane M.; Bristow, Christopher A.; Ma, Lijia; Lin, Michael F.; Washietl, Stefan; Arshinoff, Bradley I.; Ay, Ferhat; Meyer, Patrick E.; Robine, Nicolas; Washington, Nicole L.; Di Stefano, Luisa; Berezikov, Eugene; Brown, Christopher D.; Candeias, Rogerio; Carlson, Joseph W.; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y.; Will, Sebastian; Alekseyenko, Artyom A.; Artieri, Carlo; Booth, Benjamin W.; Brooks, Angela N.; Dai, Qi; Davis, Carrie A.; Duff, Michael O.; Feng, Xin; Gorchakov, Andrey A.; Gu, Tingting; Henikoff, Jorja G.; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K.; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K.; Riddle, Nicole C.; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E.; Schwartz, Yuri B.; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H.; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E.; Brent, Michael R.; Cherbas, Lucy; Elgin, Sarah C. R.; Gingeras, Thomas R.; Grossman, Robert; Hoskins, Roger A.; Kaufman, Thomas C.; Kent, William; Kuroda, Mitzi I.; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W.; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R.; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J.; Celniker, Susan E.; Henikoff, Steven; Karpen, Gary H.; Lai, Eric C.; MacAlpine, David M.; Stein, Lincoln D.; White, Kevin P.; Kellis, Manolis

    2011-01-01

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. PMID:21177974

  18. The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

    PubMed

    Giraudo, A T; Calzolari, A; Cataldi, A A; Bogni, C; Nagel, R

    1999-08-01

    Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

  19. Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.

    PubMed

    Piłsyk, Sebastian; Perlińska-Lenart, Urszula; Górka-Nieć, Wioletta; Graczyk, Sebastian; Antosiewicz, Beata; Zembek, Patrycja; Palamarczyk, Grażyna; Kruszewska, Joanna S

    2014-07-10

    The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.

  20. Detection of regulatory SNPs in human genome using ChIP-seq ENCODE data.

    PubMed

    Bryzgalov, Leonid O; Antontseva, Elena V; Matveeva, Marina Yu; Shilov, Alexander G; Kashina, Elena V; Mordvinov, Viatcheslav A; Merkulova, Tatyana I

    2013-01-01

    A vast amount of SNPs derived from genome-wide association studies are represented by non-coding ones, therefore exacerbating the need for effective identification of regulatory SNPs (rSNPs) among them. However, this task remains challenging since the regulatory part of the human genome is annotated much poorly as opposed to coding regions. Here we describe an approach aggregating the whole set of ENCODE ChIP-seq data in order to search for rSNPs, and provide the experimental evidence of its efficiency. Its algorithm is based on the assumption that the enrichment of a genomic region with transcription factor binding loci (ChIP-seq peaks) indicates its regulatory function, and thereby SNPs located in this region are more likely to influence transcription regulation. To ensure that the approach preferably selects functionally meaningful SNPs, we performed enrichment analysis of several human SNP datasets associated with phenotypic manifestations. It was shown that all samples are significantly enriched with SNPs falling into the regions of multiple ChIP-seq peaks as compared with the randomly selected SNPs. For experimental verification, 40 SNPs falling into overlapping regions of at least 7 TF binding loci were selected from OMIM. The effect of SNPs on the binding of the DNA fragments containing them to the nuclear proteins from four human cell lines (HepG2, HeLaS3, HCT-116, and K562) has been tested by EMSA. A radical change in the binding pattern has been observed for 29 SNPs, besides, 6 more SNPs also demonstrated less pronounced changes. Taken together, the results demonstrate the effective way to search for potential rSNPs with the aid of ChIP-seq data provided by ENCODE project.

  1. Detection of Regulatory SNPs in Human Genome Using ChIP-seq ENCODE Data

    PubMed Central

    Matveeva, Marina Yu.; Shilov, Alexander G.; Kashina, Elena V.; Mordvinov, Viatcheslav A.; Merkulova, Tatyana I.

    2013-01-01

    A vast amount of SNPs derived from genome-wide association studies are represented by non-coding ones, therefore exacerbating the need for effective identification of regulatory SNPs (rSNPs) among them. However, this task remains challenging since the regulatory part of the human genome is annotated much poorly as opposed to coding regions. Here we describe an approach aggregating the whole set of ENCODE ChIP-seq data in order to search for rSNPs, and provide the experimental evidence of its efficiency. Its algorithm is based on the assumption that the enrichment of a genomic region with transcription factor binding loci (ChIP-seq peaks) indicates its regulatory function, and thereby SNPs located in this region are more likely to influence transcription regulation. To ensure that the approach preferably selects functionally meaningful SNPs, we performed enrichment analysis of several human SNP datasets associated with phenotypic manifestations. It was shown that all samples are significantly enriched with SNPs falling into the regions of multiple ChIP-seq peaks as compared with the randomly selected SNPs. For experimental verification, 40 SNPs falling into overlapping regions of at least 7 TF binding loci were selected from OMIM. The effect of SNPs on the binding of the DNA fragments containing them to the nuclear proteins from four human cell lines (HepG2, HeLaS3, HCT-116, and K562) has been tested by EMSA. A radical change in the binding pattern has been observed for 29 SNPs, besides, 6 more SNPs also demonstrated less pronounced changes. Taken together, the results demonstrate the effective way to search for potential rSNPs with the aid of ChIP-seq data provided by ENCODE project. PMID:24205329

  2. Expression of the rat sterol regulatory element-binding protein-1c gene in response to insulin is mediated by increased transactivating capacity of specificity protein 1 (Sp1).

    PubMed

    Deng, Xiong; Yellaturu, Chandrahasa; Cagen, Lauren; Wilcox, Henry G; Park, Edwards A; Raghow, Rajendra; Elam, Marshall B

    2007-06-15

    The induction of genes involved in lipid biosynthesis by insulin is mediated in part by the sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c is directly regulated by insulin by transcriptional and post-transcriptional mechanisms. Previously, we have demonstrated that the insulin-responsive cis-acting unit of the rat SREBP-1c promoter is composed of several elements that include a sterol regulatory element, two liver X receptor elements, and a number of conserved GC boxes. Here we systematically dissected the role of these GC boxes and report that five bona fide Sp1-binding elements of the SREBP-1c promoter determine its basal and insulin-induced activation. Luciferase expression driven by the rat SREBP-1c promoter was accelerated by ectopic expression of Sp1, and insulin further enhanced the transactivation potential of Sp1. Introduction of a small interfering RNA against Sp1 reduced both basal and insulin-induced activation of the SREBP-1c promoter. We also found that Sp1 interacted with both SREBP-1c and LXRalpha proteins and that insulin promoted these interactions. Chromatin immunoprecipitation studies revealed that insulin facilitated the recruitment of the steroid receptor coactivator-1 to the SREBP-1c promoter. These studies identify a novel mechanism by which maximal activation of the rat SREBP-1c gene expression by insulin is mediated by Sp1 and its enhanced ability to interact with other transcriptional regulatory proteins.

  3. Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells.

    PubMed

    Xu, H F; Luo, J; Zhao, W S; Yang, Y C; Tian, H B; Shi, H B; Bionaz, M

    2016-01-01

    Sterol regulatory element binding protein 1 (SREBP1; gene name SREBF1) is known to be the master regulator of lipid homeostasis in mammals, including milk fat synthesis. The major role of SREBP1 in controlling milk fat synthesis has been demonstrated in bovine mammary epithelial cells. Except for a demonstrated role in controlling the expression of FASN, a regulatory role of SREBP1 on milk fat synthesis is very likely, but has not yet been demonstrated in goat mammary epithelial cells (GMEC). To explore the regulatory function of SREBP1 on de novo fatty acids and triacylglycerol synthesis in GMEC, we overexpressed the mature form of SREBP1 (active NH2-terminal fragment) in GMEC using a recombinant adenovirus vector (Ad-nSREBP1), with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and infected the GMEC for 48 h. In infected cells, we assessed the expression of 20 genes related to milk fat synthesis using real time-quantitative PCR, the protein abundance of SREBP1 and FASN by Western blot, the production of triacylglycerol, and the fatty acid profile. Expression of SREBF1 was modest in mammary compared with the other tissues in dairy goats but its expression increased approximately 30-fold from pregnancy to lactation. The overexpression of the mature form of SREBP1 was confirmed by >200-fold higher expression of SREBF1 in Ad-nSREBP1 compared with Ad-GFP. We observed no changes in amount of the precursor form of SREBP1 protein but a >10-fold increase of the mature form of SREBP1 protein with Ad-nSREBP1. Compared with Ad-GFP cells (control), Ad-nSREBP1 cells had a significant increase in expression of genes related to long-chain fatty acid activation (ACSL1), transport (FABP3), desaturation (SCD1), de novo synthesis of fatty acids (ACSS2, ACLY, IDH1, ACACA, FASN, and ELOVL6), and transcriptional factors (NR1H3 and PPARG). We observed a >10-fold increase in expression of INSIG1 but SCAP was downregulated by Ad-nSREBP1. Among genes related to

  4. Sterol regulatory element binding protein (SREBP)-1 expression in brain is affected by age but not by hormones or metabolic changes.

    PubMed

    Okamoto, Kenjirou; Kakuma, Tetsuya; Fukuchi, Satoshi; Masaki, Takayuki; Sakata, Toshiie; Yoshimatsu, Hironobu

    2006-04-07

    Sterol regulatory element binding protein (SREBP)-1 is a membrane-bound transcription factor that regulates the expression of several genes involved in cellular fatty acid synthesis in the peripheral tissues, including liver. Although SREBP-1 is expressed in brain, little is known about its function. The aim of the present study was to clarify the characteristics of SREBP-1 mRNA expression in rat brain under various nutritional and hormonal conditions. In genetically obese (fa/fa) Zucker rats, expression of SREBP-1 mRNA was greater in liver than in hypothalamus or cerebrum compared to the lean littermates of these rats. Fasting for 45 h and refeeding for 3 h did not affect expression in brains of Wistar rats of SREBP-1 mRNA or the mRNAs of lipogenic enzymes that are targets of SREBP-1, i.e., fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). Infusion of 2.0 mIU insulin or 3.0 microg leptin into the third cerebroventricle did not affect SREBP-1 mRNA expression in either hypothalamus or cerebrum. SREBP-1 mRNA expression in brains of transgenic mice that overexpressed leptin did not differ from that of wild-type mice. However, we observed a unique age-related alteration in SREBP-1 mRNA expression in brains of Sprague-Dawley rats. Specifically, SREBP-1 mRNA expression increased between 1 and 20 months of age, while there was no such change in the expression of FAS or ACC. This raises the possibility that increased SREBP-1 expression secondary to aging-related decline of polyunsaturated fatty acid (PUFA) might compensate for the reduction of FAS expression in brain. These findings suggest that the expression of SREBP-1 and downstream lipogenic enzymes in brain is probably not regulated by peripheral nutritional conditions or humoral factors. Aging-related changes in SREBP-1 mRNA expression may be involved in developmental changes in brain lipid metabolism.

  5. Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation.

    PubMed

    Lee, Jae Ho; Lee, Gha Young; Jang, Hagoon; Choe, Sung Sik; Koo, Seung-Hoi; Kim, Jae Bum

    2014-09-01

    Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation. Copyright

  6. Genetic variations in sterol regulatory element binding protein cleavage-activating protein (SCAP) are associated with blood pressure in overweight/obese Chinese children.

    PubMed

    Yang, Yi-De; Song, Jie-Yun; Wang, Shuo; Liu, Fang-Hong; Zhang, Yi-Ning; Shang, Xiao-Rui; Wang, Hai-Jun; Ma, Jun

    2017-01-01

    Previous studies demonstrated a role of variations in sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) in obesity and blood lipids. But the associations between SCAP polymorphisms and blood pressure (BP) are not clear. This study aimed to investigate the relationship between genetic variations in SCAP and BP phenotypes in a Chinese pediatric population. A case-control study on 702 high blood pressure (HBP) children and 1319 controls was conducted to explore the correlation between single nucleotide polymorphism markers (rs12487736 and rs12490383) of SCAP and BP phenotypes. The associations with continuous and categorical variables were examined by linear regression and logistic regression models under a dominant genetic model for the minor rs12487736 A allele and rs12490383 T allele. The rs12487736 polymorphism was significantly associated with systolic BP (SBP) (β = 1.66, P = 0.003) and diastolic BP (DBP) (β = 1.35, P = 0.024) with age, age-squared, sex, study population and body mass index (BMI) adjusted under the dominant genetic model. The rs12490383 polymorphism was significantly associated with SBP (β = 1.71, P = 0.004) and SHBP (OR = 1.39, 95%CI: 1.04-1.86, P = 0.027). When analyzed by BMI categories, in the normal-weight children, no significant association between the SCAP polymorphisms and BP phenotypes was observed (all P > 0.05). However, in the overweight/obese children, rs12487736 was significantly associated with SBP (β = 1.6, P = 0.019) and SHBP (OR = 1.36, 95%CI: 1.02-1.82; P = 0.037), rs12490383 was associated with SBP (β = 2.04, P = 0.004) and SHBP (OR = 1.50, 95%CI: 1.10-2.05; P = 0.01). This study demonstrated that SCAP rs12487736 and rs12490383 were significantly associated with SBP and SHBP in overweight/obese Chinese children. It provided the evidence for association of SCAP with SBP.

  7. FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c.

    PubMed

    Deng, Xiong; Zhang, Wenwei; O-Sullivan, InSug; Williams, J Bradley; Dong, Qingming; Park, Edwards A; Raghow, Rajendra; Unterman, Terry G; Elam, Marshall B

    2012-06-08

    Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

  8. Identification of functional elements and regulatory circuits by Drosophila modENCODE

    SciTech Connect

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L.; Landolin, Jane M.; Bristow, Christopher A.; Ma, Lijia; Lin, Michael F.; Washietl, Stefan; Arshinoff, Bradley I.; Ay, Ferhat; Meyer, Patrick E.; Robine, Nicolas; Washington, Nicole L.; Stefano, Luisa Di; Berezikov, Eugene; Brown, Christopher D.; Candeias, Rogerio; Carlson, Joseph W.; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y.; Will, Sebastian; Alekseyenko, Artyom A.; Artieri, Carlo; Booth, Benjamin W.; Brooks, Angela N.; Dai, Qi; Davis, Carrie A.; Duff, Michael O.; Feng, Xin; Gorchakov, Andrey A.; Gu, Tingting; Henikoff, Jorja G.; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K.; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K.; Riddle, Nicole C.; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E.; Schwartz, Yuri B.; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H.; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E.; Brent, Michael R.; Cherbas, Lucy; Elgin, Sarah C. R.; Gingeras, Thomas R.; Grossman, Robert; Hoskins, Roger A.; Kaufman, Thomas C.; Kent, William; Kuroda, Mitzi I.; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W.; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R.; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J.; Celniker, Susan E.; Henikoff, Steven; Karpen, Gary H.; Lai, Eric C.; MacAlpine, David M.; Stein, Lincoln D.; White, Kevin P.; Kellis, Manolis

    2010-12-22

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions

  9. Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

    USDA-ARS?s Scientific Manuscript database

    Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

  10. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    PubMed

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  11. Involvement of the Phospholipid Sterol Acyltransferase1 in Plant Sterol Homeostasis and Leaf Senescence1[W

    PubMed Central

    Bouvier-Navé, Pierrette; Berna, Anne; Noiriel, Alexandre; Compagnon, Vincent; Carlsson, Anders S.; Banas, Antoni; Stymne, Sten; Schaller, Hubert

    2010-01-01

    Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging. PMID:19923239

  12. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

    PubMed

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-09-29

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA(+)) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

    PubMed

    Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

    2014-01-01

    This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

  14. The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

    PubMed Central

    Reinhart, Alexandria A.; Powell, Daniel A.; Nguyen, Angela T.; O'Neill, Maura; Djapgne, Louise; Wilks, Angela; Ernst, Robert K.

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. PMID:25510881

  15. STARD4 Membrane Interactions and Sterol Binding

    PubMed Central

    2016-01-01

    The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix. PMID:26168008

  16. STARD4 Membrane Interactions and Sterol Binding.

    PubMed

    Iaea, David B; Dikiy, Igor; Kiburu, Irene; Eliezer, David; Maxfield, Frederick R

    2015-08-04

    The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.

  17. Sterol methyltransferase 1 controls the level of cholesterol in plants.

    PubMed

    Diener, A C; Li, H; Zhou, W; Whoriskey, W J; Nes, W D; Fink, G R

    2000-06-01

    The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.

  18. STEROL METHYLTRANSFERASE 1 Controls the Level of Cholesterol in Plants

    PubMed Central

    Diener, Andrew C.; Li, Haoxia; Zhou, Wen-xu; Whoriskey, Wendy J.; Nes, W. David; Fink, Gerald R.

    2000-01-01

    The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes—sterol methyltransferase 1 (SMT1), SMT2, and SMT3—homologous to yeast ERG6, which is known to encode an S-adenosylmethionine–dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition. PMID:10852933

  19. Dietary fiber prevents obesity-related liver lipotoxicity by modulating sterol-regulatory element binding protein pathway in C57BL/6J mice fed a high-fat/cholesterol diet

    PubMed Central

    Han, Shufen; Jiao, Jun; Zhang, Wei; Xu, Jiaying; Wan, Zhongxiao; Zhang, Weiguo; Gao, Xiaoran; Qin, Liqiang

    2015-01-01

    Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/choleserol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibtied lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles. PMID:26510459

  20. Dietary fiber prevents obesity-related liver lipotoxicity by modulating sterol-regulatory element binding protein pathway in C57BL/6J mice fed a high-fat/cholesterol diet.

    PubMed

    Han, Shufen; Jiao, Jun; Zhang, Wei; Xu, Jiaying; Wan, Zhongxiao; Zhang, Weiguo; Gao, Xiaoran; Qin, Liqiang

    2015-10-29

    Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/cholesterol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibited lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles.

  1. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

    PubMed Central

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression. PMID:25429295

  2. A Positive Regulatory Loop Controls Expression of the Locus of Enterocyte Effacement-Encoded Regulators Ler and GrlA

    PubMed Central

    Barba, Jeannette; Bustamante, Víctor H.; Flores-Valdez, Mario A.; Deng, Wanyin; Finlay, B. Brett; Puente, José L.

    2005-01-01

    The formation of attaching and effacing (A/E) lesions on intestinal epithelial cells is an essential step in the pathogenesis of human enteropathogenic and enterohemorrhagic Escherichia coli and of the mouse pathogen Citrobacter rodentium. The genes required for the development of the A/E phenotype are located within a pathogenicity island known as the locus of enterocyte effacement (LEE). The LEE-encoded transcriptional regulators Ler, an H-NS-like protein, and GrlA, a member of a novel family of transcriptional activators, positively control the expression of the genes located in the LEE and their corresponding virulence. In this study, we used C. rodentium as a model to study the mechanisms controlling the expression of Ler and GrlA. By deletion analysis of the ler and grlRA regulatory regions and complementation experiments, negative and positive cis-acting regulatory motifs were identified that are essential for the regulation of both genes. This analysis confirmed that GrlA is required for the activation of ler, but it also showed that Ler is required for the expression of grlRA, revealing a novel regulatory loop controlling the optimal expression of virulence genes in A/E pathogens. Furthermore, our results indicate that Ler and GrlA induce the expression of each other by, at least in part, counteracting the repression mediated by H-NS. However, whereas GrlA is still required for the optimal expression of ler even in the absence of H-NS, Ler is not needed for the expression of grlRA in the absence of H-NS. This type of transcriptional positive regulatory loop represents a novel mechanism in pathogenic bacteria that is likely required to maintain an appropriate spatiotemporal transcriptional response during infection. PMID:16291665

  3. Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers

    PubMed Central

    Crocker, Justin; Potter, Nathan; Erives, Albert

    2010-01-01

    Concentration gradients of morphogenic proteins pattern the embryonic axes of Drosophila by activating different genes at different concentrations. The neurogenic ectoderm enhancers (NEEs) activate different genes at different threshold levels of the Dorsal (Dl) morphogen, which patterns the dorsal/ventral axis. NEEs share a unique arrangement of highly constrained DNA-binding sites for Dl, Twist (Twi), Snail (Sna) and Suppressor of Hairless (Su(H)), and encode the threshold variable in the precise length of DNA that separates one well-defined Dl element from a Twi element. However, NEEs also possess dense clusters of variant Dl sites. Here, we show that these increasingly variant sites are eclipsed relic elements, which were superseded by more recently evolved threshold encodings. Given the divergence in egg size during Drosophila lineage evolution, the observed characteristic clusters of divergent sites indicate a history of frequent selection for changes in threshold responses to the Dl morphogen gradient and confirm the NEE structure/function model. PMID:20981027

  4. The effects of sterol structure upon sterol esterification.

    PubMed

    Lin, Don S; Steiner, Robert D; Merkens, Louise S; Pappu, Anuradha S; Connor, William E

    2010-01-01

    Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT(1) and ACAT(2) (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67% and in tissues by ACAT was 64%. Esterification of nine sterols (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined. The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82, respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74, respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29% and decreased the esterification by LCAT by 5.9%. Esterification by ACAT and LCAT was reduced, respectively, by 25 and 11% by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.

  5. Berberine-improved visceral white adipose tissue insulin resistance associated with altered sterol regulatory element-binding proteins, liver x receptors, and peroxisome proliferator-activated receptors transcriptional programs in diabetic hamsters.

    PubMed

    Li, Guo-Sheng; Liu, Xu-Han; Zhu, Hua; Huang, Lan; Liu, Ya-Li; Ma, Chun-Mei; Qin, Chuan

    2011-01-01

    The diabetic "lipotoxicity" hypothesis presents that fat-induced visceral white adipose tissue insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Berberine, a hypolipidemic agent, has been reported to have antidiabetic activities. The molecular mechanisms for this property are, however, not well clarified. Therefore in this study type 2 diabetic hamsters were induced by high-fat diet with low-dose streptozotocin. Then, we investigated the gene expression alterations and explored the molecular mechanisms underlying the therapeutic effect of berberine on fat-induced visceral white adipose tissue insulin resistance in diabetic hamsters by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmation. Type 2 diabetic hamsters exhibited hyperglycemia and relative hyperinsulinemia, glucose intolerance, insulin resistance, intra-adipocyte lipid accumulation, significant increase in body weight and visceral white adipose tissue weight, abnormal serum adipokines levels, and deleterious dyslipidemia. Furthermore, they had increased sterol regulatory element-binding proteins (SREBPs) expression and decreased liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) expression in visceral white adipose tissue. After 9-week berberine treatment, fat-induced insulin resistance and diabetic phenotype in type 2 diabetic hamsters were significantly improved. Compared with diabetic hamsters, expression of LXRs and PPARs significantly increased and SREBPs significantly decreased in visceral white adipose tissue from berberine-treated diabetic hamsters. These results suggest that altered visceral white adipose tissue LXRs, PPARs, and SREBPs transcriptional programs are involved in the therapeutic mechanisms of berberine on fat-induced visceral white adipose tissue insulin resistance in type 2 diabetic hamsters.

  6. Dysregulation of sterol regulatory element binding protein-1c in livers of morbidly obese women is associated with altered suppressor of cytokine signaling-3 and signal transducer and activator of transcription-1 signaling.

    PubMed

    Elam, Marshall B; Yellaturu, Chandrahasa; Howell, George E; Deng, Xiong; Cowan, George S; Kumar, Poonam; Park, Edwards A; Hiler, M Lloyd; Wilcox, Henry G; Hughes, Thomas A; Cook, George A; Raghow, Rajendra

    2010-04-01

    We compared hepatic expression of genes that regulate lipid biosynthesis and metabolic signaling in liver biopsy specimens from women who were undergoing gastric bypass surgery (GBP) for morbid obesity with that in women undergoing ventral hernia repair who had experienced massive weight loss (MWL) after prior GBP. Comprehensive metabolic profiles of morbidly obese (MO) (22 subjects) and MWL (9 subjects) were also compared. Analyses of gene expression in liver biopsies from MO and MWL were accomplished by Affymetrix microarray, real-time polymerase chain reaction, and Western blotting techniques. After GBP, MWL subjects had lost on average 102 lb as compared with MO subjects. This was accompanied by effective reversal of the dyslipidemia and insulin resistance that were present in MO. As compared with MWL, livers of MO subjects exhibited increased expression of sterol regulatory element binding protein (SREBP)-1c and its downstream lipogenic targets, fatty acid synthase and acetyl-coenzyme A-carboxylase-1. Livers of MO subjects also exhibited enhanced expression of suppressor of cytokine signaling-3 protein and attenuated Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling. Consistent with these findings, we found that the human SREBP-1c promoter was positively regulated by insulin and negatively regulated by STAT3. These data support the hypothesis that suppressor of cytokine signaling-3-mediated attenuation of the STAT signaling pathway and resulting enhanced expression of SREBP-1c, a key regulator of de novo lipid biosynthesis, are mechanistically related to the development of hepatic insulin resistance and dyslipidemia in MO women.

  7. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

    PubMed

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-03-04

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

  8. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter*

    PubMed Central

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5′-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript. PMID:26728456

  9. Caloric restriction-associated remodeling of rat white adipose tissue: effects on the growth hormone/insulin-like growth factor-1 axis, sterol regulatory element binding protein-1, and macrophage infiltration.

    PubMed

    Chujo, Yoshikazu; Fujii, Namiki; Okita, Naoyuki; Konishi, Tomokazu; Narita, Takumi; Yamada, Atsushi; Haruyama, Yushi; Tashiro, Kosuke; Chiba, Takuya; Shimokawa, Isao; Higami, Yoshikazu

    2013-08-01

    The role of the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis in the lifelong caloric restriction (CR)-associated remodeling of white adipose tissue (WAT), adipocyte size, and gene expression profiles was explored in this study. We analyzed the WAT morphology of 6-7-month-old wild-type Wistar rats fed ad libitum (WdAL) or subjected to CR (WdCR), and of heterozygous transgenic dwarf rats bearing an anti-sense GH transgene fed ad libitum (TgAL) or subjected to CR (TgCR). Although less effective in TgAL, the adipocyte size was significantly reduced in WdCR compared with WdAL. This CR effect was blunted in Tg rats. We also used high-density oligonucleotide microarrays to examine the gene expression profile of WAT of WdAL, WdCR, and TgAL rats. The gene expression profile of WdCR, but not TgAL, differed greatly from that of WdAL. The gene clusters with the largest changes induced by CR but not by Tg were genes involved in lipid biosynthesis and inflammation, particularly sterol regulatory element binding proteins (SREBPs)-regulated and macrophage-related genes, respectively. Real-time reverse-transcription polymerase chain reaction analysis confirmed that the expression of SREBP-1 and its downstream targets was upregulated, whereas the macrophage-related genes were downregulated in WdCR, but not in TgAL. In addition, CR affected the gene expression profile of Tg rats similarly to wild-type rats. Our findings suggest that CR-associated remodeling of WAT, which involves SREBP-1-mediated transcriptional activation and suppression of macrophage infiltration, is regulated in a GH-IGF-1-independent manner.

  10. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes.

    PubMed

    Rees, Matthew G; Ng, David; Ruppert, Sarah; Turner, Clesson; Beer, Nicola L; Swift, Amy J; Morken, Mario A; Below, Jennifer E; Blech, Ilana; Mullikin, James C; McCarthy, Mark I; Biesecker, Leslie G; Gloyn, Anna L; Collins, Francis S

    2012-01-01

    Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

  11. Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription.

    PubMed

    Mascaró, C; Ortiz, J A; Ramos, M M; Haro, D; Hegardt, F G

    2000-02-15

    The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol.

  12. Identification of a novel regulatory sequence of actin nucleation promoting factor encoded by Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Wang, Yun; Zhang, Yongli; Han, Shili; Hu, Xue; Zhou, Yuan; Mu, Jingfang; Pei, Rongjuan; Wu, Chunchen; Chen, Xinwen

    2015-04-10

    Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.

  13. Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments

    PubMed Central

    Kheradpour, Pouya; Kellis, Manolis

    2014-01-01

    Recent advances in technology have led to a dramatic increase in the number of available transcription factor ChIP-seq and ChIP-chip data sets. Understanding the motif content of these data sets is an important step in understanding the underlying mechanisms of regulation. Here we provide a systematic motif analysis for 427 human ChIP-seq data sets using motifs curated from the literature and also discovered de novo using five established motif discovery tools. We use a systematic pipeline for calculating motif enrichment in each data set, providing a principled way for choosing between motif variants found in the literature and for flagging potentially problematic data sets. Our analysis confirms the known specificity of 41 of the 56 analyzed factor groups and reveals motifs of potential cofactors. We also use cell type-specific binding to find factors active in specific conditions. The resource we provide is accessible both for browsing a small number of factors and for performing large-scale systematic analyses. We provide motif matrices, instances and enrichments in each of the ENCODE data sets. The motifs discovered here have been used in parallel studies to validate the specificity of antibodies, understand cooperativity between data sets and measure the variation of motif binding across individuals and species. PMID:24335146

  14. Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

    PubMed Central

    Vos, Seychelle M.; Lyubimov, Artem Y.; Hershey, David M.; Schoeffler, Allyn J.; Sengupta, Sugopa; Nagaraja, Valakunja; Berger, James M.

    2014-01-01

    Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state. PMID:24990966

  15. Intracellular Sterol Dynamics

    PubMed Central

    Mesmin, Bruno; Maxfield, Frederick R.

    2009-01-01

    We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated. PMID:19286471

  16. "Dinoflagellate Sterols" in marine diatoms.

    PubMed

    Giner, José-Luis; Wikfors, Gary H

    2011-10-01

    Sterol compositions for three diatom species, recently shown to contain sterols with side chains typically found in dinoflagellates, were determined by HPLC and ¹H NMR spectroscopic analyses. The centric diatom Triceratium dubium (=Biddulphia sp., CCMP 147) contained the highest percentage of 23-methylated sterols (37.2% (24R)-23-methylergosta-5,22-dienol), whereas the pennate diatom Delphineis sp. (CCMP 1095) contained the cyclopropyl sterol gorgosterol, as well as the 27-norsterol occelasterol. The sterol composition of Ditylum brightwellii (CCMP 358) was the most complex, containing Δ⁰- and Δ⁷-sterols, in addition to the predominant Δ⁵-sterols. A pair of previously unknown sterols, stigmasta-5,24,28-trienol and stigmasta-24,28-dienol, were detected in D. brightwellii and their structures were determined by NMR spectroscopic analysis and by synthesis of the former sterol from saringosterol. Also detected in D. brightwellii was the previously unknown 23-methylcholesta-7,22-dienol. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Regulation of Sterol Biosynthesis in the Human Fungal Pathogen Aspergillus fumigatus: Opportunities for Therapeutic Development

    PubMed Central

    Dhingra, Sourabh; Cramer, Robert A.

    2017-01-01

    Sterols are a major component of eukaryotic cell membranes. For human fungal infections caused by the filamentous fungus Aspergillus fumigatus, antifungal drugs that target sterol biosynthesis and/or function remain the standard of care. Yet, an understanding of A. fumigatus sterol biosynthesis regulatory mechanisms remains an under developed therapeutic target. The critical role of sterol biosynthesis regulation and its interactions with clinically relevant azole drugs is highlighted by the basic helix loop helix (bHLH) class of transcription factors known as Sterol Regulatory Element Binding Proteins (SREBPs). SREBPs regulate transcription of key ergosterol biosynthesis genes in fungi including A. fumigatus. In addition, other emerging regulatory pathways and target genes involved in sterol biosynthesis and drug interactions provide additional opportunities including the unfolded protein response, iron responsive transcriptional networks, and chaperone proteins such as Hsp90. Thus, targeting molecular pathways critical for sterol biosynthesis regulation presents an opportunity to improve therapeutic options for the collection of diseases termed aspergillosis. This mini-review summarizes our current understanding of sterol biosynthesis regulation with a focus on mechanisms of transcriptional regulation by the SREBP family of transcription factors. PMID:28203225

  18. hydra Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signaling

    PubMed Central

    Souter, Martin; Topping, Jennifer; Pullen, Margaret; Friml, Jiri; Palme, Klaus; Hackett, Rachel; Grierson, Don; Lindsey, Keith

    2002-01-01

    The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Δ8-Δ7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file–specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function. PMID:12034894

  19. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  20. Effects of bovine fatty acid synthase, stearoyl-coenzyme A desaturase, sterol regulatory element-binding protein 1, and growth hormone gene polymorphisms on fatty acid composition and carcass traits in Japanese Black cattle.

    PubMed

    Matsuhashi, T; Maruyama, S; Uemoto, Y; Kobayashi, N; Mannen, H; Abe, T; Sakaguchi, S; Kobayashi, E

    2011-01-01

    The quality of fat is an important factor in defining the quality of meat. Fat quality is determined by the composition of fatty acids. Among lipid metabolism-related genes, including fatty acid synthesis genes, several genetic variations have been reported in the bovine fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), sterol regulatory element-binding protein 1 (SREBP1), and GH genes. In the present study, we evaluated the single and epistatic effects of 5 genetic variations (4 SNP and 1 insertion/deletion) in 4 genes (FASN, SCD, SREBP1, and GH) on the fatty acid composition of the longissimus thoracis muscle and carcass and meat quality traits in 480 commercial Japanese Black cattle. Significant single effects of FASN, SCD, and GH(L127V) polymorphisms on the fatty acid composition of the longissimus thoracis muscle were detected. The A293V polymorphism of SCD had the largest effect on myristic acid (C14:0, P < 0.001), myristoleic acid (C14:1, P < 0.001), stearic acid (C18:0, P < 0.001), oleic acid (C18:1, P < 0.001), and MUFA (P < 0.001). Polymorphisms in the FASN, SCD, and SREBP1 genes showed no effect on any meat yield trait. There were no significant epistatic effects on fatty acid composition among pairs of the 3 genes (FASN, SCD, and SREBP1) involved in fatty acid synthesis. No epistatic interactions (P > 0.1) were detected between FASN and SCD for any carcass trait. When the genotypes of 3 markers (FASN, SCD, and GH(L127V)) were substituted from the lesser effect allele to the greater effect allele, the proportion of C18:1 increased by 4.46%. More than 20% of the genetic variance in the C18:1 level could be accounted for by these 3 genetic markers. The present results revealed that polymorphisms in 2 fatty acid synthesis genes (FASN and SCD) independently influenced fatty acid composition in the longissimus thoracis muscle. These results suggest that SNP in the FASN and SCD genes are useful markers for the improvement of fatty acid composition in

  1. Survival strategies of a sterol auxotroph

    PubMed Central

    Carvalho, Maria; Schwudke, Dominik; Sampaio, Julio L.; Palm, Wilhelm; Riezman, Isabelle; Dey, Gautam; Gupta, Gagan D.; Mayor, Satyajit; Riezman, Howard; Shevchenko, Andrej; Kurzchalia, Teymuras V.; Eaton, Suzanne

    2010-01-01

    The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis. PMID:20940226

  2. Induction and Relaxation Dynamics of the Regulatory Network Controlling the Type III Secretion System encoded within Salmonella Pathogenicity Island 1

    PubMed Central

    Temme, Karsten; Salis, Howard; Tullman-Ercek, Danielle; Levskaya, Anselm; Hong, Soon-Ho; Voigt, Christopher A.

    2008-01-01

    Summary Bacterial pathogenesis requires the precise spatial and temporal control of gene expression, the dynamics of which are controlled by regulatory networks. A network encoded within Salmonella Pathogenicity Island 1 controls the expression of a type III protein secretion system involved in the invasion of host cells. The dynamics of this network are measured in single cells using promoter-green fluorescent protein (gfp) reporters and flow cytometry. During induction, there is a temporal order of gene expression, with transcriptional inputs turning on first, followed by structure, and effector genes. The promoters show varying stochastic properties, where graded inputs are converted into all-or-none and hybrid responses. The relaxation dynamics are measured by shifting cells from inducing into non-inducing conditions and measuring the fluorescence decay. The gfp expressed from promoters controlling the transcriptional inputs (hilC and hilD) and structural genes (prgH) decay exponentially with a characteristic time of 50–55 minutes. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110 ± 9 minutes. This promoter is controlled by a genetic circuit formed by a transcription factor (InvF), chaperone (SicA) and secreted protein (SipC) that regulates effector expression in response to the secretion capacity of the cell. A mathematical model of this circuit demonstrates that the delay is due to a split positive feedback loop. This model is tested in a ΔsicA knockout where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Further, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers to an operator in the sicA promoter. This prediction is used to construct a targeted library, which contains mutants with both longer and shorter delays. This combination of theory and

  3. Phylogenetic Distribution of Fungal Sterols

    PubMed Central

    Weete, John D.; Abril, Maritza; Blackwell, Meredith

    2010-01-01

    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 Δ5 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 -Δ5 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 the synthesis of

  4. Sterols of the cultured dinoflagellate Pyrocystis lunula.

    PubMed

    Kokke, W C; Fenical, W; Djerassi, C

    1982-09-01

    Eighteen components of the sterol fraction of Pyrocystis lunula have been identified. In addition to 4 alpha-methyl sterols (typical dinoflagellate sterols), regular sterols, both with a saturated and delta 5-unsaturated skeleton, were isolated, together with delta 4-3-keto steroids including the hitherto unknown 23,24R-dimethyl-4,22E-cholestadien-3-one.

  5. Study of Behavior of Sterols at Interfaces

    NASA Technical Reports Server (NTRS)

    Klein, P. D.; Knight, J. C.; Szczepanik, P. A.

    1968-01-01

    Behavior of sterols and sterol acetates on various types of interfaces indicates that the function of a sterol depends upon a surface orientation and surface energy of the interface. Column-chromatographic techniques determine the retention volume of various sterols under standard conditions.

  6. Interferon Control of the Sterol Metabolic Network: Bidirectional Molecular Circuitry-Mediating Host Protection

    PubMed Central

    Robertson, Kevin A.; Ghazal, Peter

    2016-01-01

    The sterol metabolic network is emerging center stage in inflammation and immunity. Historically, observational clinical studies show that hypocholesterolemia is a common side effect of interferon (IFN) treatment. More recently, comprehensive systems-wide investigations of the macrophage IFN response reveal a direct molecular link between cholesterol metabolism and infection. Upon infection, flux through the sterol metabolic network is acutely moderated by the IFN response at multiple regulatory levels. The precise mechanisms by which IFN regulates the mevalonate-sterol pathway—the spine of the network—are beginning to be unraveled. In this review, we discuss our current understanding of the multifactorial mechanisms by which IFN regulates the sterol pathway. We also consider bidirectional communications resulting in sterol metabolism regulation of immunity. Finally, we deliberate on how this fundamental interaction functions as an integral element of host protective responses to infection and harmful inflammation. PMID:28066443

  7. Interferon Control of the Sterol Metabolic Network: Bidirectional Molecular Circuitry-Mediating Host Protection.

    PubMed

    Robertson, Kevin A; Ghazal, Peter

    2016-01-01

    The sterol metabolic network is emerging center stage in inflammation and immunity. Historically, observational clinical studies show that hypocholesterolemia is a common side effect of interferon (IFN) treatment. More recently, comprehensive systems-wide investigations of the macrophage IFN response reveal a direct molecular link between cholesterol metabolism and infection. Upon infection, flux through the sterol metabolic network is acutely moderated by the IFN response at multiple regulatory levels. The precise mechanisms by which IFN regulates the mevalonate-sterol pathway-the spine of the network-are beginning to be unraveled. In this review, we discuss our current understanding of the multifactorial mechanisms by which IFN regulates the sterol pathway. We also consider bidirectional communications resulting in sterol metabolism regulation of immunity. Finally, we deliberate on how this fundamental interaction functions as an integral element of host protective responses to infection and harmful inflammation.

  8. Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis.

    PubMed

    Bouvier-Navé, P; Husselstein, T; Benveniste, P

    1998-08-15

    Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smtl and smt2; in addition, smt2 cDNAs were shown to encode a 24-methylene lophenol C24 methyltransferase [Bouvier-Navé, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518-529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1-1 which belongs to the first SMT cDNA family and Ntsmt2-1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24-alkyl sterols, erg6 Ntsmt1-1 contained a majority of 24-methylene sterols and erg6 Ntsmt2-1, a majority of 24-ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsm1-1 efficiently converted cycloartenol into 24-methylene cycloartanol, but did not produce any 24-ethylidene lophenol upon incubation with 24-methylene lophenol. This demonstrates that cDNA Ntsmt1-1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2-1 were shown to methylate preferentially 24-methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1-1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2-1) belongs to the second family and was shown to encode

  9. Specific Sterols Required for the Internalization Step of Endocytosis in Yeast

    PubMed Central

    Munn, Alan L.; Heese-Peck, Antje; Stevenson, Brian J.; Pichler, Harald; Riezman, Howard

    1999-01-01

    Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Δ, erg6Δ, and erg2Δerg6Δ) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergΔ mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergΔ mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37°C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization. PMID:10564282

  10. Sterol metabolism in the oomycete Aphanomyces euteiches, a legume root pathogen.

    PubMed

    Madoui, Mohammed-Amine; Bertrand-Michel, Justine; Gaulin, Elodie; Dumas, Bernard

    2009-01-01

    Sterols are isoprenoid-derived molecules that have essential functions in eukaryotes but whose metabolism remains largely unknown in a large number of organisms. Oomycetes are fungus-like microorganisms that are evolutionarily related to stramenopile algae, a large group of organisms for which no sterol metabolic pathway has been reported. Here, we present data that support a model of sterol biosynthesis in Aphanomyces euteiches, an oomycete species causing devastating diseases in legume crops. In silico analyses were performed to identify genes encoding enzymes involved in the conversion of the isoprenoid precursor 3-hydroxy-3-methylglutaryl coenzyme A to isoprenoids. Several metabolic intermediates and two major sterol end-products were identified by gas chromatography-mass spectroscopy. We show that A. euteiches is able to produce fucosterol (a sterol initially identified in brown algae) and cholesterol (the major animal sterol). Mycelium development is inhibited by two sterol demethylase inhibitors used as fungicides, namely tebuconazole and epoxiconazole. We propose the first sterol biosynthetic pathway identified in a stramenopile species. Phylogenetic analyses revealed close relationships between A. euteiches enzyme sequences and those found in stramenopile algae, suggesting that part of this pathway could be conserved in the Stramenopila kingdom.

  11. Sterols as Complex-forming Species

    NASA Astrophysics Data System (ADS)

    Ioffe, D. V.

    1986-02-01

    The formation of complexes of sterols with different compounds determines the biological properties of both sterols and various natural substances such as saponins and polyene antibiotics. Complex formation by sterols with phospholipids, steroid saponins, and polyene antibiotics is determined by the same characteristic features of the structure of the sterol molecule. The principal role in complex formation is played by the hydrophobic reaction of the cyclopentanoperhydrophenanthrene ring. The formation of a hydrogen bond between the hydroxyl group of the sterol and a proton acceptor, which is assumed in most complexes, has been proved only in the complexes of sterols with water and acids. The bibliography contains 122 references.

  12. FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR regulatory mechanism.

    PubMed

    Lee, E-H; Rouquette-Loughlin, C; Folster, J P; Shafer, W M

    2003-12-01

    The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

  13. Sterol biosynthesis in oomycete pathogens.

    PubMed

    Gaulin, Elodie; Bottin, Arnaud; Dumas, Bernard

    2010-03-01

    Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores, but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such as diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Saprolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs, suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales.

  14. Sterol biosynthesis in oomycete pathogens

    PubMed Central

    Gaulin, Elodie; Bottin, Arnaud

    2010-01-01

    Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores, but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such as diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Saprolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs, suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales. PMID:20023385

  15. Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Morris, Lindsey L; Hartman, Isamu Z; Jun, Dong-Jae; Seemann, Joachim; DeBose-Boyd, Russell A

    2014-07-04

    Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Cholesterol-lowering effect of plant sterols.

    PubMed

    AbuMweis, Suhad S; Jones, Peter J H

    2008-12-01

    Plant sterols are plant components that have a chemical structure similar to cholesterol except for the addition of an extra methyl or ethyl group; however, plant sterol absorption in humans is considerably less than that of cholesterol. In fact, plant sterols reduce cholesterol absorption and thus reduce circulating levels of cholesterol. Earlier studies that have tested the efficacy of plant sterols as cholesterol-lowering agents incorporated plant sterols into fat spreads. Later on, plant sterols were added to other food matrices, including juices, nonfat beverages, milk and yogurt, cheese, meat, croissants and muffins, and cereal and chocolate bars. The beneficial physiologic effects of plant sterols could be further enhanced by combining them with other beneficial substances, such as olive and fish oils, fibers, and soy proteins, or with exercise. The addition of plant sterols to the diet is suggested by health experts as a safe and effective way to reduce the risk of coronary heart disease.

  17. Distribution and functions of sterols and sphingolipids.

    PubMed

    Hannich, J Thomas; Umebayashi, Kyohei; Riezman, Howard

    2011-05-01

    Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.

  18. Sterols of the fungi - Distribution and biosynthesis

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

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

  19. Sterols of the fungi - Distribution and biosynthesis.

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

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

  20. Sterols of the fungi - Distribution and biosynthesis.

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

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

  1. Sterols of the fungi - Distribution and biosynthesis

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

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

  2. Distribution and Functions of Sterols and Sphingolipids

    PubMed Central

    Hannich, J. Thomas; Umebayashi, Kyohei; Riezman, Howard

    2011-01-01

    Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking. PMID:21454248

  3. The pathogenicity island encoded PvrSR/RcsCB regulatory network controls biofilm formation and dispersal in Pseudomonas aeruginosa PA14.

    PubMed

    Mikkelsen, Helga; Hui, Kailyn; Barraud, Nicolas; Filloux, Alain

    2013-08-01

    Pseudomonas aeruginosa biofilm formation is linked to persistent infections in humans. Biofilm formation is facilitated by extracellular appendages, some of which are assembled by the Chaperone Usher Pathway (Cup). The cupD gene cluster is located on the PAPI-1 pathogenicity island of strain PA14 and has probably been acquired together with four genes encoding two-component signal transduction proteins. We have previously showed that the RcsB response regulator activates expression of the cupD genes, which leads to the production of CupD fimbriae and increased attachment. Here we show that RcsB activity is tightly modulated by two sensors, RcsC and PvrS. While PvrS acts as a kinase that enhances RcsB activity, RcsC has a dual function, first as a phosphorelay, and second as a phosphatase. We found that, under certain growth conditions, overexpression of RcsB readily induces biofilm dispersal. Microarray analysis shows that RcsB positively controls expression of pvrR that encodes the phosphodiesterase required for this dispersal process. Finally, in addition to the PAPI-1 encoded cupD genes, RcsB controls several genes on the core genome, some of which encode orphan response regulators. We thus discovered that RcsB is central to a large regulatory network that fine-tunes the switch between biofilm formation and dispersal. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  4. Sterol methylation in Saccharomyces cerevisiae.

    PubMed Central

    McCammon, M T; Hartmann, M A; Bottema, C D; Parks, L W

    1984-01-01

    Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5. PMID:6363386

  5. Plant Sterol Diversity in Pollen from Angiosperms.

    PubMed

    Villette, Claire; Berna, Anne; Compagnon, Vincent; Schaller, Hubert

    2015-08-01

    Here we have examined the composition of free sterols and steryl esters of pollen from selected angiosperm species, as a first step towards a comprehensive analysis of sterol biogenesis in the male gametophyte. We detected four major sterol structural groups: cycloartenol derivatives bearing a 9β,19-cyclopropyl group, sterols with a double bond at C-7(8), sterols with a double bond at C-5(6), and stanols. All these groups were unequally distributed among species. However, the distribution of sterols as free sterols or as steryl esters in pollen grains indicated that free sterols were mostly Δ(5)-sterols and that steryl esters were predominantly 9β,19-cyclopropyl sterols. In order to link the sterol composition of a pollen grain at anthesis with the requirement for membrane lipid constituents of the pollen tube, we germinated pollen grains from Nicotiana tabacum, a model plant in reproductive biology. In the presence of radiolabelled mevalonic acid and in a time course series of measurements, we showed that cycloeucalenol was identified as the major neosynthesized sterol. Furthermore, the inhibition of cycloeucalenol neosynthesis by squalestatin was in full agreement with a de novo biogenesis and an apparent truncated pathway in the pollen tube.

  6. The Arabidopsis pyruvate,orthophosphate dikinase regulatory proteins encode a novel, unprecedented Ser/Thr protein kinase primary structure

    USDA-ARS?s Scientific Manuscript database

    Pyruvate,orthophosphate dikinase (PPDK) is a ubiquitous, low abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual, bifuncti...

  7. Non-random distribution and co-localization of purine/pyrimidine-encoded information and transcriptional regulatory domains.

    PubMed

    Povinelli, C M

    1992-01-01

    In order to detect sequence-based information predictive for the location of eukaryotic transcriptional regulatory domains, the frequencies and distributions of the 36 possible purine/pyrimidine reverse complement hexamer pairs was determined for test sets of real and random sequences. The distribution of one of the hexamer pairs (RRYYRR/YYRRYY, referred to as M1) was further examined in a larger set of sequences (> 32 genes, 230 kb). Predominant clusters of M1 and the locations of eukaryotic transcriptional regulatory domains were found to be associated and non-randomly distributed along the DNA consistent with a periodicity of approximately 1.2 kb. In the context of higher ordered chromatin this would align promoters, enhancers and the predominant clusters of M1 longitudinally along one face of a 30 nm fiber. Using only information about the distribution of the M1 motif, 50-70% of a sequence could be eliminated as being unlikely to contain transcriptional regulatory domains with an 87% recovery of the regulatory domains present.

  8. On the sterols of some ascidians.

    PubMed

    Voogt, P A; van Rheenen, J W

    1975-08-01

    The lipid content of sea squirts is low, namely less than a half percent of the fresh weight. Lipids consist of about seventy percent of saponifiable lipids and of about twenty percent of non-saponifiable lipids. Both types of these lipids, including sterols, can be synthesized from acetate by these animals. Small amounts of C30 sterols were observed only in Microcosmus sulcatus and Halocynthia papillosa, the species with a low content of C27 sterols and a high content of C28 sterols. In addition these species contained considerable higher amounts of sterols with a double bond at the C22 position than Ciona intestinalis and Styela plicata did.

  9. Dietary plant sterols and cholesterol metabolism.

    PubMed

    Ellegård, Lars H; Andersson, Susan W; Normén, A Lena; Andersson, Henrik A

    2007-01-01

    Plant sterols, naturally occurring in foods of plant origin, reduce cholesterol absorption. Experimental studies show plant sterols to be an important part of the serum-cholesterol lowering effect of certain diets and dietary components. Epidemiological data show that individuals with higher intakes of plant sterols from their habitual diets have lower serum-cholesterol levels. To date, the role of naturally occurring plant sterols for lowering serum cholesterol has probably been underestimated. The consumption of dietary plant sterols should be a part of dietary advice to patients with hypercholesterolemia and the general public for the prevention and management of coronary heart disease.

  10. Multiple Functions of Sterols in Yeast Endocytosis

    PubMed Central

    Heese-Peck, Antje; Pichler, Harald; Zanolari, Bettina; Watanabe, Reika; Daum, Günther; Riezman, Howard

    2002-01-01

    Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergΔ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Δerg6Δ and erg3Δerg6Δ cells exhibit a strong internalization defect of the α-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand–receptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. ergΔ cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment. PMID:12181337

  11. The nadA gene of Aspergillus nidulans, encoding adenine deaminase, is subject to a unique regulatory pattern.

    PubMed

    Oestreicher, Nathalie; Ribard, Carin; Scazzocchio, Claudio

    2008-05-01

    The adenine deaminase of A. nidulans, encoded by nadA, can be considered both as a catabolic and a purine salvage enzyme. We show that its transcriptional regulation reflects this double metabolic role. As all other genes involved in purine utilisation it is induced by uric acid, and this induction is mediated by the UaY transcription factor. However, it is also independently and synergistically induced by adenosine by a UaY-independent mechanism. At variance with all other enzymes of purine catabolism it is not repressed but induced by ammonium. This is at least partly due to the ammonium responsive GATA factor, AreA, acting in the nadA promoter as a competitor rather than in synergy with UaY. The adB gene, encoding adenylo-succinate synthetase, which can be considered both a biosynthetic and a salvage pathway enzyme, shares with nadA both ammonium and adenosine induction.

  12. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    PubMed

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  13. Steroleosin, a sterol-binding dehydrogenase in seed oil bodies.

    PubMed

    Lin, Li-Jen; Tai, Sorgan S K; Peng, Chi-Chung; Tzen, Jason T C

    2002-04-01

    Besides abundant oleosin, three minor proteins, Sop 1, 2, and 3, are present in sesame (Sesamum indicum) oil bodies. The gene encoding Sop1, named caleosin for its calcium-binding capacity, has recently been cloned. In this study, Sop2 gene was obtained by immunoscreening, and it was subsequently confirmed by amino acid partial sequencing and immunological recognition of its overexpressed protein in Escherichia coli. Immunological cross recognition implies that Sop2 exists in seed oil bodies of diverse species. Along with oleosin and caleosin genes, Sop2 gene was transcribed in maturing seeds where oil bodies are actively assembled. Sequence analysis reveals that Sop2, tentatively named steroleosin, possesses a hydrophobic anchoring segment preceding a soluble domain homologous to sterol-binding dehydrogenases/reductases involved in signal transduction in diverse organisms. Three-dimensional structure of the soluble domain was predicted via homology modeling. The structure forms a seven-stranded parallel beta-sheet with the active site, S-(12X)-Y-(3X)-K, between an NADPH and a sterol-binding subdomain. Sterol-coupling dehydrogenase activity was demonstrated in the overexpressed soluble domain of steroleosin as well as in purified oil bodies. Southern hybridization suggests that one steroleosin gene and certain homologous genes may be present in the sesame genome. Comparably, eight hypothetical steroleosin-like proteins are present in the Arabidopsis genome with a conserved NADPH-binding subdomain, but a divergent sterol-binding subdomain. It is indicated that steroleosin-like proteins may represent a class of dehydrogenases/reductases that are involved in plant signal transduction regulated by various sterols.

  14. Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus.

    PubMed Central

    Raibaud, A; Zalacain, M; Holt, T G; Tizard, R; Thompson, C J

    1991-01-01

    Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide

  15. The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13.

    PubMed

    Ohtani, Kaori; Kawsar, Hameem I; Okumura, Kayo; Hayashi, Hideo; Shimizu, Tohru

    2003-05-16

    We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13.

  16. regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein.

    PubMed

    Kirk, M M; Stark, K; Miller, S M; Müller, W; Taillon, B E; Gruber, H; Schmitt, R; Kirk, D L

    1999-02-01

    Volvox has two cell types: mortal somatic cells and immortal germ cells. Here we describe the transposon-tagging, cloning and characterization of regA, which plays a central role as a master regulatory gene in Volvox germ-soma differentiation by suppressing reproductive activities in somatic cells. The 12.5 kb regA transcription unit generates a 6,725 nucleotide mRNA that appears at the beginning of somatic cell differentiation, and that encodes a 111 kDa RegA protein that localizes to the nucleus, and has an unusual abundance of alanine, glutamine and proline. This is a compositional feature shared by functional domains of many 'active' repressors. These findings are consistent with the hypothesis that RegA acts in somatic cells to repress transcription of genes required for growth and reproduction, including 13 genes whose products are required for chloroplast biogenesis.

  17. Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis*

    PubMed Central

    Jang, Kyung Ku; Gil, So Yeon; Lim, Jong Gyu; Choi, Sang Ho

    2016-01-01

    Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at −164.5 and −106, and CRP and SmcR bind specifically to the sequences centered at −68 and −45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis. PMID:26755724

  18. Regulatory domain or CpG site variation in SLC12A5, encoding the chloride transporter KCC2, in human autism and schizophrenia

    PubMed Central

    Merner, Nancy D.; Chandler, Madison R.; Bourassa, Cynthia; Liang, Bo; Khanna, Arjun R.; Dion, Patrick; Rouleau, Guy A.; Kahle, Kristopher T.

    2015-01-01

    Many encoded gene products responsible for neurodevelopmental disorders (NDs) like autism spectrum disorders (ASD), schizophrenia (SCZ), intellectual disability (ID), and idiopathic generalized epilepsy (IGE) converge on networks controlling synaptic function. An increase in KCC2 (SLC12A5) Cl− transporter activity drives the developmental GABA excitatory-inhibitory sequence, but the role of KCC2 in human NDs is essentially unknown. Here, we report two rare, non-synonymous (NS), functionally-impairing variants in the KCC2 C-terminal regulatory domain (CTRD) in human ASD (R952H and R1049C) and SCZ (R952H) previously linked with IGE and familial febrile seizures, and another novel NS KCC2 variant in ASD (R1048W) with highly-predicted pathogenicity. Exome data from 2517 simplex families in the ASD Simon Simplex Collection (SSC) revealed significantly more KCC2 CTRD variants in ASD cases than controls, and interestingly, these were more often synonymous and predicted to disrupt or introduce a CpG site. Furthermore, full gene analysis showed ASD cases are more likely to contain rare KCC2 variants affecting CpG sites than controls. These data suggest genetically-encoded dysregulation of KCC2-dependent GABA signaling may contribute to multiple human NDs. PMID:26528127

  19. The Goldfish SG2NA Gene Encodes Two α-Type Regulatory Subunits for PP-2A and Displays Distinct Developmental Expression Pattern

    PubMed Central

    Ma, Hai-Li; Peng, Yun-Lei; Gong, Lili; Liu, Wen-Bin; Sun, Shuming; Liu, Jiao; Zheng, Chun-Bing; Fu, Hu; Yuan, Dan; Zhao, Junqiong; Chen, Pei-Chao; Xie, Si-si; Zeng, Xiao-Ming; Xiao, Ya-Mei; Liu, Yun; Li, David Wan-Cheng

    2009-01-01

    SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NAα and SG2NAβ. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the α-type, and are named SG2NAα and SG2NAα+. RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues. PMID:19838339

  20. Exome Sequencing and cis-Regulatory Mapping Identify Mutations in MAK, a Gene Encoding a Regulator of Ciliary Length, as a Cause of Retinitis Pigmentosa

    PubMed Central

    Özgül, Rıza Köksal; Siemiatkowska, Anna M.; Yücel, Didem; Myers, Connie A.; Collin, Rob W.J.; Zonneveld, Marijke N.; Beryozkin, Avigail; Banin, Eyal; Hoyng, Carel B.; van den Born, L. Ingeborgh; Bose, Ron; Shen, Wei; Sharon, Dror; Cremers, Frans P.M.; Klevering, B. Jeroen; den Hollander, Anneke I.; Corbo, Joseph C.

    2011-01-01

    A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases. PMID:21835304

  1. Identification and molecular characterization of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus genomic region encoding the regulatory genes pkip, p47, lef-12, and gta.

    PubMed

    Lapointe, R; Back, D W; Ding, Q; Carstens, E B

    2000-05-25

    Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) is a baculovirus pathogenic to spruce budworm, the most damaging insect pest in Canadian forestry. CfMNPV is less virulent to its host insect and its replication cycle is slower than the baculovirus type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) but the basis of these characteristics is not known. We have now identified, localized, and determined the sequence of the region of CfMNPV carrying potentially important regulatory genes including p47, lef-12, gta, and pkip. DNA database searches revealed that this region of CfMNPV is most closely related to the homologous OpMNPV genes. Transcription analysis demonstrated that CfMNPV P47 is encoded by a 1.6-kb transcript, LEF-12 is encoded by a 2.6-kb transcript, and GTA is encoded by a 2.1-kb transcript. Transcripts for these genes were detectable at 6 h postinfection but all of them showed a burst in expression levels between 12 and 24 h postinfection corresponding to the time of initiation of CfMNPV DNA replication. A polyclonal antibody, raised against CfMNPV P47, detected a nuclear 43-kDa polypeptide from 12 to 72 h postinfection, demonstrating that the CfMNPV p47 gene product is first expressed at a time corresponding to the burst of transcriptional activity between the early and the late phases. Both AcMNPV and CfMNPV P47 translocate to the nucleus of infected cells.

  2. Analysis of molluscan sterols: Colorimetric methods.

    PubMed

    Swift, M L

    1984-08-01

    The wide variety of sterols normally found in extracts of bivalve molluscs leads to high variability in analytical data obtained with colorimetric (chole)sterol methods. Total sterol levels in oyster (Crassostrea virginica) extracts were determined using the Liebermann-Burchard reagent, an acid-FeCl3 reagent and a cholesterol oxidase procedure. The data from the latter two agreed to within 5.4% and yielded about 30% higher estimates of sterol content than the Liebermann-Burchard test. Gas-liquid chromatographic data also are compared.Several pure sterols, selected because of their presence in oyster sterol fractions or because of their structural similarities to such sterols, were examined using each of the three procedures. Sterols, differing from cholesterol only with regard to the side chain, reacted 80-102% as well as cholesterol with the acid-FeCl3 reagent and cholesterol oxidase. The Liebermann-Burchard reaction was more specific for cholesterol. The colorimetric cholesterol oxidase method is recommended for the estimation of total molluscan sterol content.

  3. The sterols of calcareous sponges (Calcarea, Porifera).

    PubMed

    Hagemann, Andrea; Voigt, Oliver; Wörheide, Gert; Thiel, Volker

    2008-11-01

    Sponges are sessile suspension-feeding organisms whose internal phylogenetic relationships are still the subject of intense debate. Sterols may have the potential to be used as independent markers to test phylogenetic hypotheses. Twenty representative specimens of calcareous sponges (class Calcarea, phylum Porifera) with a broad coverage within both subclasses Calcinea and Calcaronea were analysed for their sterol content. Two major pseudohomologous series were found, accompanied by some additional sterols. The first series encompassing conventional C(27) to C(29)Delta(5,7,22) sterols represented the major sterols, with ergosterol (ergosta-5,7,22-trien-3beta-ol, C(28)Delta(5,7,22)) being most prominent in many species. The second series consisted of unusual C(27) to C(29)Delta(5,7,9(11),22) sterols. Cholesterol occurred sporadically, mostly in trace amounts. The sterol patterns did not resolve intraclass phylogenetic relationships, namely the distinction between the subclasses, Calcinea and Calcaronea. This pointed towards major calcarean lipid traits being established prior to the separation of subclasses. Furthermore, calcarean sterol patterns clearly differ from those found in Hexactinellida, whereas partial overlap occurred with some Demospongiae. Hence, sterols only partly reflect the phylogenetic separation of Calcarea from both of the other poriferan classes that was proposed by recent molecular work and fatty acid analyses.

  4. C26 sterol in a human urine.

    PubMed

    Ikekawa, N; Fujimoto, Y; Isiguro, M; Suwa, S; Hirayama, Y; Mizunuma, H

    1979-06-15

    A new C26 sterol, 22-trans-27-norcholesta-5,22-dien-3 beta-ol, was found in the urine of a 6-year-old girl, with a clinical diagnosis of congenital adrenal hyperplasia of the salt losing type, accompanied by symptoms of mixed sex anatomy and skin pigmentation. The structure of the sterol was determined by comparison with the synthetic compound. The sterol was also detected in ther serum. This appears to be the first case in which a C26 sterol has occurred in mammalia.

  5. Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity.

    PubMed

    Deota, Shaunak; Chattopadhyay, Tandrika; Ramachandran, Deepti; Armstrong, Eric; Camacho, Beatriz; Maniyadath, Babukrishna; Fulzele, Amit; Gonzalez-de-Peredo, Anne; Denu, John M; Kolthur-Seetharam, Ullas

    2017-03-28

    The conserved NAD(+)-dependent deacylase SIRT1 plays pivotal, sometimes contrasting, roles in diverse physiological and pathophysiological conditions. In this study, we uncover a tissue-restricted isoform of SIRT1 (SIRT1-ΔE2) that lacks exon 2 (E2). Candidate-based screening of SIRT1 substrates demonstrated that the domain encoded by this exon plays a key role in specifying SIRT1 protein-protein interactions. The E2 domain of SIRT1 was both necessary and sufficient for PGC1α binding, enhanced interaction with p53, and thus downstream functions. Since SIRT1-FL and SIRT1-ΔE2 were found to have similar intrinsic catalytic activities, we propose that the E2 domain tethers specific substrate proteins. Given the absence of SIRT1-ΔE2 in liver, our findings provide insight into the role of the E2 domain in specifying "metabolic functions" of SIRT1-FL. Identification of SIRT1-ΔE2 and the conserved specificity domain will enhance our understanding of SIRT1 and guide the development of therapeutic interventions. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity

    PubMed Central

    Deota, Shaunak; Chattopadhyay, Tandrika; Ramachandran, Deepti; Armstrong, Eric; Camacho, Beatriz; Maniyadath, Babukrishna; Fulzele, Amit; Gonzalez-de-Peredo, Anne; Denu, John M.; Kolthur-Seetharam, Ullas

    2017-01-01

    Summary The conserved NAD+-dependent deacylase SIRT1 plays pivotal, sometimes contrasting, roles in diverse physiological and pathophysiological conditions. In this study, we uncover a tissue-restricted isoform of SIRT1 (SIRT1-ΔE2) that lacks exon 2 (E2). Candidate-based screening of SIRT1 substrates demonstrated that the domain encoded by this exon plays a key role in specifying SIRT1 protein-protein interactions. The E2 domain of SIRT1 was both necessary and sufficient for PGC1α binding, enhanced interaction with p53, and thus downstream functions. Since SIRT1-FL and SIRT1-ΔE2 were found to have similar intrinsic catalytic activities, we propose that the E2 domain tethers specific substrate proteins. Given the absence of SIRT1-ΔE2 in liver, our findings provide insight into the role of the E2 domain in specifying “metabolic functions” of SIRT1-FL. Identification of SIRT1-ΔE2 and the conserved specificity domain will enhance our understanding of SIRT1 and guide the development of therapeutic interventions. PMID:28355560

  7. Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences.

    PubMed Central

    Belogurov, A A; Delver, E P; Rodzevich, O V

    1993-01-01

    The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously. The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli. Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E. coli and only slightly affects the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system. Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively. The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins. We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids. It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating. Images PMID:8393008

  8. Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia.

    PubMed

    Caron, K M; Soo, S C; Wetsel, W C; Stocco, D M; Clark, B J; Parker, K L

    1997-10-14

    An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids-with loss of negative feedback regulation at hypothalamic-pituitary levels-whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR's essential roles in adrenocortical and gonadal steroidogenesis.

  9. A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila▿

    PubMed Central

    Tomazic, Mariela L.; Najle, Sebastián R.; Nusblat, Alejandro D.; Uttaro, Antonio D.; Nudel, Clara B.

    2011-01-01

    The gene TTHERM_00438800 (DES24) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known. PMID:21257793

  10. Regulation of the human MAT2B gene encoding the regulatory beta subunit of methionine adenosyltransferase, MAT II.

    PubMed

    LeGros, L; Halim, A B; Chamberlin, M E; Geller, A; Kotb, M

    2001-07-06

    Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5'-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between -4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position -115 provided full activity; this was attributed to a TATA at -32. The Sp1 site at position +9 was key for the formation of protein.DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

  11. ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

    PubMed Central

    Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

    2016-01-01

    Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. PMID:26497474

  12. Special relationship between sterols and oxygen: were sterols an adaptation to aerobic life?

    PubMed

    Galea, Anne M; Brown, Andrew J

    2009-09-15

    A fascinating link between sterols and molecular oxygen (O(2)) has been a common thread running through the fundamental work of Konrad Bloch, who elucidated the biosynthetic pathway for cholesterol, to recent work supporting a role of sterols in the sensing of O(2). Synthesis of sterols by eukaryotes is an O(2)-intensive process. In this review, we argue that increased levels of O(2) in the atmosphere not only made the evolution of sterols possible, but that these sterols may in turn have provided the eukaryote with an early defence mechanism against O(2). The idea that nature crafted sterols as a feedback loop to adapt to, or help protect against, the hazards of O(2) is novel and enticing. We marshal several lines of evidence to support this thesis: (1) coincidence of atmospheric O(2) and sterol evolution; (2) sterols regulate O(2) entry into eukaryotic cells and organelles; (3) sterols act as O(2) sensors across eukaryotic life; (4) sterols serve as a primitive cellular defence against O(2) (including reactive oxygen species). Therefore, sterols may have evolved in eukaryotes partially as an adaptive response to the rise of terrestrial O(2), rather than merely as a consequence of it.

  13. Bioorthogonal probes for imaging sterols in cells.

    PubMed

    Jao, Cindy Y; Nedelcu, Daniel; Lopez, Lyle V; Samarakoon, Thilani N; Welti, Ruth; Salic, Adrian

    2015-03-02

    Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Comparative molecular modelling of biologically active sterols

    NASA Astrophysics Data System (ADS)

    Baran, Mariusz; Mazerski, Jan

    2015-04-01

    Membrane sterols are targets for a clinically important antifungal agent - amphotericin B. The relatively specific antifungal action of the drug is based on a stronger interaction of amphotericin B with fungal ergosterol than with mammalian cholesterol. Conformational space occupied by six sterols has been defined using the molecular dynamics method to establish if the conformational features correspond to the preferential interaction of amphotericin B with ergosterol as compared with cholesterol. The compounds studied were chosen on the basis of structural features characteristic for cholesterol and ergosterol and on available experimental data on the ability to form complexes with the antibiotic. Statistical analysis of the data obtained has been performed. The results show similarity of the conformational spaces occupied by all the sterols tested. This suggests that the conformational differences of sterol molecules are not the major feature responsible for the differential sterol - drug affinity.

  15. Regulation of mRNAs encoding the steroidogenic acute regulatory protein and cholesterol side-chain cleavage enzyme in the elasmobranch interrenal gland.

    PubMed

    Evans, Andrew N; Nunez, B Scott

    2010-08-01

    The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes. Copyright 2010 Elsevier Inc. All rights reserved.

  16. A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators.

    PubMed Central

    Ueda, K; Miyake, K; Horinouchi, S; Beppu, T

    1993-01-01

    Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1. Images PMID:8458843

  17. Determination of plant sterol oxidation products in plant sterol enriched spreads, fat blends, and plant sterol concentrates.

    PubMed

    Louter, Arjan J H

    2004-01-01

    Plant sterols (PS) are very stable molecules but may undergo oxidation due to the presence of a double bond in the ring structure. In order to assess whether this occurs during heating and storage, an analytical procedure was developed for the determination of concentration levels and identity of PS oxidation products in functional food ingredients and products. The method is based on cold saponification, solvent extraction of unsaponifiables, isolation of sterol oxidation products by means of liquid chromatography, and final analysis by gas chromatography (GC) with flame ionization detection. Identification of the key PS oxidation products was performed by means of GC-mass spectrometry (GC-MS). Isotope dilution MS was used to verify the absence of the formation of potential artifacts by the method. The method described is applicable to spreads (containing 20-65% water), oils, sterol esters, pure sterols, and fat extracts from food. The between-day reproducibility of the total content of sterol oxidation products in control samples sample was 8%, and of individual sterol oxidation products, 6-15%. The recovery of sterol oxidation products was 91%. The limit of detection was 0.1 mg/kg.

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

    PubMed Central

    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

  19. Thresholds for sterol-limited growth of Daphnia magna: a comparative approach using 10 different sterols.

    PubMed

    Martin-Creuzburg, Dominik; Oexle, Sarah; Wacker, Alexander

    2014-09-01

    Arthropods are incapable of synthesizing sterols de novo and thus require a dietary source to cover their physiological demands. The most prominent sterol in animal tissues is cholesterol, which is an indispensable structural component of cell membranes and serves as precursor for steroid hormones. Instead of cholesterol, plants and algae contain a variety of different phytosterols. Consequently, herbivorous arthropods have to metabolize dietary phytosterols to cholesterol to meet their requirements for growth and reproduction. Here, we investigated sterol-limited growth responses of the freshwater herbivore Daphnia magna by supplementing a sterol-free diet with increasing amounts of 10 different phytosterols and comparing thresholds for sterol-limited growth. In addition, we analyzed the sterol composition of D. magna to explore sterol metabolic constraints and bioconversion capacities. We show that dietary phytosterols strongly differ in their potential to support somatic growth of D. magna. The dietary threshold concentrations obtained by supplementing the different sterols cover a wide range (3.5-34.4 μg mg C(-1)) and encompass the one for cholesterol (8.9 μg mg C(-1)), indicating that certain phytosterols are more efficient in supporting somatic growth than cholesterol (e.g., fucosterol, brassicasterol) while others are less efficient (e.g., dihydrocholesterol, lathosterol). The dietary sterol concentration gradients revealed that the poor quality of particular sterols can be alleviated partially by increasing dietary concentrations, and that qualitative differences among sterols are most pronounced at low to moderate dietary concentrations. We infer that the dietary sterol composition has to be considered in zooplankton nutritional ecology to accurately assess potential sterol limitations under field conditions.

  20. Sterols in spermatogenesis and sperm maturation

    PubMed Central

    Keber, Rok; Rozman, Damjana; Horvat, Simon

    2013-01-01

    Mammalian spermatogenesis is a complex developmental program in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. One intriguing aspect of sperm production is the dynamic change in membrane lipid composition that occurs throughout spermatogenesis. Cholesterol content, as well as its intermediates, differs vastly between the male reproductive system and nongonadal tissues. Accumulation of cholesterol precursors such as testis meiosis-activating sterol and desmosterol is observed in testes and spermatozoa from several mammalian species. Moreover, cholesterogenic genes, especially meiosis-activating sterol-producing enzyme cytochrome P450 lanosterol 14α-demethylase, display stage-specific expression patterns during spermatogenesis. Discrepancies in gene expression patterns suggest a complex temporal and cell-type specific regulation of sterol compounds during spermatogenesis, which also involves dynamic interactions between germ and Sertoli cells. The functional importance of sterol compounds in sperm production is further supported by the modulation of sterol composition in spermatozoal membranes during epididymal transit and in the female reproductive tract, which is a prerequisite for successful fertilization. However, the exact role of sterols in male reproduction is unknown. This review discusses sterol dynamics in sperm maturation and describes recent methodological advances that will help to illuminate the complexity of sperm formation and function. PMID:23093550

  1. Sterol chemotaxonomy of marine pelagophyte algae.

    PubMed

    Giner, José-Luis; Zhao, Hui; Boyer, Gregory L; Satchwell, Michael F; Andersen, Robert A

    2009-07-01

    Several marine algae of the class Pelagophyceae produce the unusual marine sterol 24-propylidenecholesterol, mainly as the (24E)-isomer. The (24Z)-isomer had previously been considered as a specific biomarker for Aureococcus anophagefferens, the 'brown tide' alga of the Northeast coast of the USA. To test this hypothesis and to generate chemotaxonomic information, the sterol compositions of 42 strains of pelagophyte algae including 17 strains of Aureococcus anophagefferens were determined by GC analysis. A more comprehensive sterol analysis by HPLC and (1)H-NMR was obtained for 17 selected pelagophyte strains. All strains analyzed contained 24-propylidenecholesterol. In all strains belonging to the order Sarcinochrysidales, this sterol was found only as the (E)-isomer, while all strains in the order Pelagomonadales contained the (Z)-isomer, either alone or together with the (E)-isomer. The occurrence of Delta(22) and 24alpha-sterols was limited to the Sarcinochrysidales. The first occurrence of Delta(22)-24-propylcholesterol in an alga, CCMP 1410, was reported. Traces of the rare sterol 26,26-dimethyl-24-methylenecholesterol were detected in Aureococcus anophagefferens, and the (25R)-configuration was proposed, based on biosynthetic considerations. Traces of a novel sterol, 24-propylidenecholesta-5,25-dien-3beta-ol, were detected in several species.

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

    PubMed Central

    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

  3. Sterol Regulation of Metabolism, Homeostasis and Development

    PubMed Central

    Wollam, Joshua; Antebi, Adam

    2014-01-01

    Sterol metabolites are critical signaling molecules that regulate metabolism, development, and homeostasis. Oxysterols, bile acids, and steroids work primarily through cognate sterol-responsive nuclear hormone receptors to control these processes through feed-forward and feedback mechanisms. These signaling pathways are conserved from simple invertebrates to mammals. Indeed, results from various model organisms have yielded fundamental insights into cholesterol and bile acid homeostasis, lipid and glucose metabolism, protective mechanisms, tissue differentiation, development, reproduction, and even aging. Here, we review how sterols act through evolutionarily ancient mechanisms to control these processes. PMID:21495846

  4. Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco1

    PubMed Central

    Devarenne, Timothy P.; Ghosh, Anirban; Chappell, Joe

    2002-01-01

    Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues. PMID:12114564

  5. The Sterol Methyltransferases SMT1, SMT2, and SMT3 Influence Arabidopsis Development through Nonbrassinosteroid Products1[W][OA

    PubMed Central

    Carland, Francine; Fujioka, Shozo; Nelson, Timothy

    2010-01-01

    Plant sterols are structural components of cell membranes that provide rigidity, permeability, and regional identity to membranes. Sterols are also the precursors to the brassinosteroid signaling molecules. Evidence is accumulating that specific sterols have roles in pattern formation during development. COTYLEDON VASCULAR PATTERNING1 (CVP1) encodes C-24 STEROL METHYLTRANSFERASE2 (SMT2), one of three SMTs in Arabidopsis (Arabidopsis thaliana). SMT2 and SMT3, which also encodes a C-24 SMT, catalyze the reaction that distinguishes the synthesis of structural sterols from signaling brassinosteroid derivatives and are highly regulated. The deficiency of SMT2 in the cvp1 mutant results in moderate developmental defects, including aberrant cotyledon vein patterning, serrated floral organs, and reduced stature, but plants are viable, suggesting that SMT3 activity can substitute for the loss of SMT2. To test the distinct developmental roles of SMT2 and SMT3, we identified a transcript null smt3 mutant. Although smt3 single mutants appear wild type, cvp1 smt3 double mutants show enhanced defects relative to cvp1 mutants, such as discontinuous cotyledon vein pattern, and produce novel phenotypes, including defective root growth, loss of apical dominance, sterility, and homeotic floral transformations. These phenotypes are correlated with major alterations in the profiles of specific sterols but without significant alterations to brassinosteroid profiles. The alterations to sterol profiles in cvp1 mutants affect auxin response, demonstrated by weak auxin insensitivity, enhanced axr1 auxin resistance, ectopically expressed DR5:β-glucuronidase in developing embryos, and defective response to auxin-inhibited PIN2-green fluorescent protein endocytosis. We discuss the developmental roles of sterols implied by these results. PMID:20421456

  6. Free sterol composition of species in the dinoflagellate genus Pyrocystis: a spectrum of sterol diversity.

    PubMed

    Dahmen, Jeremy L; Leblond, Jeffrey D

    2011-01-01

    The dinoflagellate genus Pyrocystis includes a small number of marine species, which spend the majority of their life cycles as nonmotile cells within a carbohydrate sheath, and which are found ubiquitously throughout the world's oceans. The biochemistry of this model dinoflagellate genus has been widely studied due to its ability to bioluminesce. However, Pyrocystis has been comparatively understudied with respect to its lipid biochemistry, in particular that of sterols. To date, examination of the sterols of Pyrocystis has focused primarily upon Pyrocystis lunula, which produces cholesterol and 4,24-dimethyl-5α-cholestan-3β-ol as its predominant sterols, while it lacks the common dinoflagellate sterol, dinosterol. We have examined the sterol composition of the two other commercially available species of Pyrocystis, Pyrocystis fusiformis and Pyrocystis noctiluca. Pyrocystis noctiluca possesses dinosterol as its most abundant sterol, while P. fusiformis possesses dinosterol and 4,24-dimethyl-5α-cholestan-3β-ol as the predominant sterols, placing it at an intermediate position between P. lunula and P. noctiluca, as based on sterol composition. The potential limitations of the dinoflagellate sterol biomarker dinosterol are also explored in this study due to its notable absence in P. lunula. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

  7. Mutations and Polymorphisms in the Gene Encoding Regulatory Subunit Type 1-alpha of Protein Kinase A (PRKAR1A): An Update

    PubMed Central

    Horvath, Anélia; Bertherat, Jérôme; Groussin, Lionel; Guillaud-Bataille, Marine; Tsang, Kitman; Cazabat, Laure; Libe, Rosella; Remmers, Elaine; René-Corail, Fernande; Faucz, Fabio Rueda; Clauser, Eric; Calender, Alain; Bertagna, Xavier; Carney, J Aidan; Stratakis, Constantine A.

    2010-01-01

    PRKAR1A encodes the regulatory subunit type 1-alpha (RIα), of the main mediator of the cAMP effects in the eukaryotic cells – cAMP dependant Protein Kinase A (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified. The majority of them are subject to non-sense mediated mRNA decay (NMD), leading to PRKAR1A haploinsufficiency and, as a result, activated cAMP signaling in the affected tissues. In recent years it became apparent that CNC may be caused not only by RIα haploinsufficiency, but also by the expression of altered PRKAR1A protein, as proven by analysis of the relatively small proportion of expressed mutations in the gene, consisting of aminoacid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered rather than missing protein has been analyzed – frame-shifts in the 3′end of the coding sequence that shift the stop codon downstream of the regular one and result in the expression of longer than the wild type protein. The PRKAR1A mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation negative CNC patients are characterized by significant phenotypic heterogeneity. In this paper, we present a comprehensive analysis of all 117 known to date pathogenic PRKAR1A sequence variations, and discuss their molecular context and clinical relevance. PMID:20358582

  8. Sterols from the Madagascar Sponge Fascaplysinopsis sp

    PubMed Central

    Aknin, Maurice; Gros, Emmanuelle; Vacelet, Jean; Kashman, Yoel; Gauvin-Bialecki, Anne

    2010-01-01

    The sponge Fascaplysinopsis sp. (order Dictyoceratida, Family Thorectidae) from the west coast of Madagascar (Indian Ocean) is a particularly rich source of bioactive nitrogenous macrolides. The previous studies on this organism led to the suggestion that the latter should originate from associated microsymbionts. In order to evaluate the influence of microsymbionts on lipid content, 10 samples of Fascaplysinopsis sp. were investigated for their sterol composition. Contrary to the secondary metabolites, the sterol patterns established were qualitatively and quantitatively stable: 14 sterols with different unsaturated nuclei, Δ5, Δ7 and Δ5,7, were identified; the last ones being the main sterols of the investigated sponges. The chemotaxonomic significance of these results for the order Dictyoceratida is also discussed in the context of the literature. The conjugated diene system in Δ5,7 sterols is known to be unstable and easily photo-oxidized during storage and/or experiments to produce 5α,8α-epidioxy sterols. However, in this study, no 5α,8α-epidioxysterols (or only trace amounts) were observed. Thus, it was supposed that photo-oxidation was avoided thanks to the natural antioxidants detected in Fascaplysinopsis sp. by both the DPPH and β-caroten bleaching assays. PMID:21339959

  9. Sterols from the Madagascar sponge Fascaplysinopsis sp.

    PubMed

    Aknin, Maurice; Gros, Emmanuelle; Vacelet, Jean; Kashman, Yoel; Gauvin-Bialecki, Anne

    2010-12-17

    The sponge Fascaplysinopsis sp. (order Dictyoceratida, Family Thorectidae) from the west coast of Madagascar (Indian Ocean) is a particularly rich source of bioactive nitrogenous macrolides. The previous studies on this organism led to the suggestion that the latter should originate from associated microsymbionts. In order to evaluate the influence of microsymbionts on lipid content, 10 samples of Fascaplysinopsis sp. were investigated for their sterol composition. Contrary to the secondary metabolites, the sterol patterns established were qualitatively and quantitatively stable: 14 sterols with different unsaturated nuclei, Δ(5), Δ(7) and Δ(5,7), were identified; the last ones being the main sterols of the investigated sponges. The chemotaxonomic significance of these results for the order Dictyoceratida is also discussed in the context of the literature. The conjugated diene system in Δ(5,7) sterols is known to be unstable and easily photo-oxidized during storage and/or experiments to produce 5α,8α-epidioxy sterols. However, in this study, no 5α,8α-epidioxysterols (or only trace amounts) were observed. Thus, it was supposed that photo-oxidation was avoided thanks to the natural antioxidants detected in Fascaplysinopsis sp. by both the DPPH and β-caroten bleaching assays.

  10. Purification, characterization and catalytic properties of human sterol 8-isomerase.

    PubMed Central

    Nes, W David; Zhou, Wenxu; Dennis, Allen L; Li, Haoxia; Jia, Zhonghua; Keith, Richard A; Piser, Timothy M; Furlong, Stephen T

    2002-01-01

    CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM) sterol formation in cholesterol synthesis. PMID:12133002

  11. Plant sterols in food: No consensus in guidelines

    SciTech Connect

    Weingärtner, Oliver; Baber, Ronny; Teupser, Daniel

    2014-04-11

    Highlights: • Plant sterols are used as food supplement to reduce serum cholesterol levels. • Reductions in serum cholesterol levels are achieved at the expense of increased plant sterol levels. • The potential atherogenicity of increased serum plant sterol levels is controversially debated. • This dispute is reflected by different guideline recommendations in regard to plant sterols. - Abstract: Plant sterols are supplemented in foods to reduce cardiovascular risk. Randomized controlled trials show 2 g of plant sterols a day reduce serum cholesterol by about 10%. This reduction in serum cholesterol levels is achieved at the expense of increased serum plant sterol levels. Findings in patients with phytosterolemia, in experimental studies and in clinical trials have lead to speculations that plant sterols might be atherogenic. In view of emerging safety issues the role of plant sterols in cardiovascular prevention has become controversial. This review reflects the ongoing controversial scientific debate and points out recent developments in guidelines of national and international societies.

  12. Influenza viral membrane fusion is sensitive to sterol concentration but surprisingly robust to sterol chemical identity

    PubMed Central

    Zawada, Katarzyna E.; Wrona, Dominik; Rawle, Robert J.; Kasson, Peter M.

    2016-01-01

    Influenza virions are enriched in cholesterol relative to the plasma membrane from which they bud. Previous work has shown that fusion between influenza virus and synthetic liposomes is sensitive to the amount of cholesterol in either the virus or the target membrane. Here, we test the chemical properties of cholesterol required to promote influenza fusion by replacing cholesterol with other sterols and assaying viral fusion kinetics. We find that influenza fusion with liposomes is surprisingly robust to sterol chemical identity, showing no significant dependence on sterol identity in target membranes for any of the sterols tested. In the viral membrane, lanosterol slowed fusion somewhat, while polar sterols produced a more pronounced slowing and inhibition of fusion. No other sterols tested showed a significant perturbation in fusion rates, including ones previously shown to alter membrane bending moduli or phase behavior. Although fusion rates depend on viral cholesterol, they thus do not require cholesterol’s ability to support liquid-liquid phase coexistence. Using electron cryo-microscopy, we further find that sterol-dependent changes to hemagglutinin spatial patterning in the viral membrane do not require liquid-liquid phase coexistence. We therefore speculate that local sterol-hemagglutinin interactions in the viral envelope may control the rate-limiting step of fusion. PMID:27431907

  13. Influenza viral membrane fusion is sensitive to sterol concentration but surprisingly robust to sterol chemical identity.

    PubMed

    Zawada, Katarzyna E; Wrona, Dominik; Rawle, Robert J; Kasson, Peter M

    2016-07-19

    Influenza virions are enriched in cholesterol relative to the plasma membrane from which they bud. Previous work has shown that fusion between influenza virus and synthetic liposomes is sensitive to the amount of cholesterol in either the virus or the target membrane. Here, we test the chemical properties of cholesterol required to promote influenza fusion by replacing cholesterol with other sterols and assaying viral fusion kinetics. We find that influenza fusion with liposomes is surprisingly robust to sterol chemical identity, showing no significant dependence on sterol identity in target membranes for any of the sterols tested. In the viral membrane, lanosterol slowed fusion somewhat, while polar sterols produced a more pronounced slowing and inhibition of fusion. No other sterols tested showed a significant perturbation in fusion rates, including ones previously shown to alter membrane bending moduli or phase behavior. Although fusion rates depend on viral cholesterol, they thus do not require cholesterol's ability to support liquid-liquid phase coexistence. Using electron cryo-microscopy, we further find that sterol-dependent changes to hemagglutinin spatial patterning in the viral membrane do not require liquid-liquid phase coexistence. We therefore speculate that local sterol-hemagglutinin interactions in the viral envelope may control the rate-limiting step of fusion.

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

    PubMed

    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.

  15. Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

    PubMed Central

    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-Δ5-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. PMID:25343375

  16. Effects of sterols on the development and aging of caenorhabditis elegans

    USDA-ARS?s Scientific Manuscript database

    Because Caenorhabditis elegans lacks several components of the de novo sterol biosynthesis pathway, it requires sterols as essential nutrients. Supplemented cholesterol undergoes extensive enzymatic modification in C. elegans to form other sterols of unknown function. Because sterol metabolism in ...

  17. Nuclear hormone receptors put immunity on sterols

    PubMed Central

    Santori, Fabio R.

    2015-01-01

    Nuclear hormone receptors (NHRs) are transcription factors regulated by small molecules. The functions of NHRs range from development of primary and secondary lymphoid organs, to regulation of differentiation and function of DCs, macrophages and T cells. The human genome has 48 classic (hormone and vitamin receptors) and non-classic (all others) NHRs; 17 non-classic receptors are orphans, meaning that the endogenous ligand is unknown. Understanding the function of orphan NHRs requires the identification of their natural ligands. The mevalonate pathway, including its sterol and non-sterol intermediates and derivatives, is a source of ligands for many classic and non-classic NHRs. For example, cholesterol biosynthetic intermediates (CBIs) are natural ligands for RORγ/γt. CBIs are universal endogenous metabolites in mammalian cells, and to study NHRs that bind CBIs requires ligand-free reporters system in sterol auxotroph cells. Furthermore, RORγ/γt shows broad specificity to sterol lipids, suggesting that RORγ/γt is either a general sterol sensor or specificity is defined by an abundant endogenous ligand. Unlike other NHRs, which regulate specific metabolic pathways, there is no connection between the genetic programs induced by RORγ/γt and ligand biosynthesis. In this review we summarize the roles of non-classic NHRs and their potential ligands in the immune system. PMID:26222181

  18. Aspergillus fumigatus C-5 Sterol Desaturases Erg3A and Erg3B: Role in Sterol Biosynthesis and Antifungal Drug Susceptibility

    PubMed Central

    Alcazar-Fuoli, Laura; Mellado, Emilia; Garcia-Effron, Guillermo; Buitrago, Maria J.; Lopez, Jordi F.; Grimalt, Joan O.; Cuenca-Estrella, J. Manuel; Rodriguez-Tudela, Juan L.

    2006-01-01

    Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A− erg3B−, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination. PMID:16436696

  19. Biofuels. Altered sterol composition renders yeast thermotolerant.

    PubMed

    Caspeta, Luis; Chen, Yun; Ghiaci, Payam; Feizi, Amir; Buskov, Steen; Hallström, Björn M; Petranovic, Dina; Nielsen, Jens

    2014-10-03

    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 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 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. Copyright © 2014, American Association for the Advancement of Science.

  20. Enzyme mechanisms for sterol C-methylations.

    PubMed

    Nes, W David

    2003-09-01

    The mechanisms by which sterol methyl transferases (SMT) transform olefins into structurally different C-methylated products are complex, prompting over 50 years of intense research. Recent enzymological studies, together with the latest discoveries in the fossil record, functional analyses and gene cloning, establish new insights into the enzymatic mechanisms of sterol C-methylation and form a basis for understanding regulation and evolution of the sterol pathway. These studies suggest that SMTs, originated shortly after life appeared on planet earth. SMTs, including those which ultimately give rise to 24 alpha- and 24 beta-alkyl sterols, align the si(beta)-face pi-electrons of the Delta(24)-double bond with the S-methyl group of AdoMet relative to a set of deprotonation bases in the active site. From the orientation of the conformationally flexible side chain in the SMT Michaelis complex, it has been found that either a single product is formed or cationic intermediates are partitioned into multiple olefins. The product structure and stereochemistry of SMT action is phylogenetically distinct and physiologically significant. SMTs control phytosterol homeostasis and their activity is subject to feedback regulation by specific sterol inserts in the membrane. A unified conceptual framework has been formulated in the steric-electric plug model that posits SMT substrate acceptability on the generation of single or double 24-alkylated side chains, which is the basis for binding order, stereospecificity and product diversity in this class of AdoMet-dependent methyl transferase enzymes. The focus of this review is the mechanism of the C-methylation process which, as discussed, can be altered by point mutations in the enzyme to direct the shape of sterol structure to optimize function.

  1. Biological removal of phyto-sterols in pulp mill effluents.

    PubMed

    Mahmood-Khan, Zahid; Hall, Eric R

    2013-12-15

    Phyto-sterols and extractives found in pulp mill effluents are suspected to cause endocrine abnormalities in receiving water fish. The control of sterols in pulp mill effluents through biological secondary wastewater treatment was studied using two lab-scale bioreactor systems. After achieving a stable performance, both bioreactor systems successfully removed (>90%) sterols and the estimated biodegradation was up to 80%. Reactor 1 system operating at 6.7 ± 0.2 pH effectively treated pulp mill effluent sterols spiked up to 4500 μg/L in 11 h HRT and 11 day SRT. However, Reactor 2 system operating at 7.6 ± 0.2 pH performed relatively poorly. Retention time reductions beyond critical values deteriorated the performance of treatment systems and quickly reduced the sterols biodegradation. The biodegradation loss was indicated by mixed liquor sterols content that started increasing. This biodegradation loss was compensated by the increased role of bio-adsorption and the overall sterols removal remained relatively high. Hence, a relatively small (20-30%) loss in the overall sterols removal efficiency did not fully reflect the associated major (60-70%) loss in the sterols biodegradation because the amount of sterols accumulated in the sludge due to adsorption increased so the estimate of sterols removal through adsorption increased from 30-40% to 70-80% keeping the overall sterols removal still high. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26898409

  3. Steroid and sterol 7-hydroxylation: ancient pathways.

    PubMed

    Lathe, Richard

    2002-11-01

    B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.

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

    PubMed Central

    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

  5. Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance.

    PubMed

    Lerksuthirat, Tassanee; Sangcakul, Areeporn; Lohnoo, Tassanee; Yingyong, Wanta; Rujirawat, Thidarat; Krajaejun, Theerapong

    2017-04-01

    Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

  6. Analysis of the activity and regulon of the two-component regulatory system encoded by Cjj1484 and Cjj1483 of Campylobacter jejuni

    USDA-ARS?s Scientific Manuscript database

    Campylobacter jejuni is a leading cause of bacterial diarrheal disease throughout the world and a frequent commensal in the intestinal tract of poultry and many other animals. For maintaining optimal growth and ability to colonize various hosts, C. jejuni depends upon two-component regulatory system...

  7. Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.

    PubMed

    Stasyk, Oleh V; Nazarko, Taras Y; Stasyk, Olena G; Krasovska, Olena S; Warnecke, Dirk; Nicaud, Jean-Marc; Cregg, James M; Sibirny, Andrei A

    2003-01-01

    Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.

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

    SciTech Connect

    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.

  9. The hypoxic regulator of sterol synthesis Nro1 is a nuclear import adaptor

    PubMed Central

    Yeh, Tzu-Lan; Lee, Chih-Yung S.; Amzel, L. Mario; Espenshade, Peter J.; Bianchet, Mario A.

    2011-01-01

    SUMMARY 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 Å resolution shows an all-α-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. PMID:21481773

  10. The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria.

    PubMed Central

    Contreras, A; Drummond, M; Bali, A; Blanco, G; Garcia, E; Bush, G; Kennedy, C; Merrick, M

    1991-01-01

    We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product. PMID:1683868

  11. Sterols from the Green Alga Ulva australis.

    PubMed

    Li, Guo-Liang; Guo, Wei-Jie; Wang, Guang-Bao; Wang, Rong-Rong; Hou, Yu-Xue; Liu, Kun; Liu, Yang; Wang, Wei

    2017-09-28

    Three new sterols, (24R)-5,28-stigmastadiene-3β,24-diol-7-one (1), (24S)-5,28-stigmastadiene-3β,24-diol-7-one (2), and 24R and 24S-vinylcholesta-3β,5α,6β,24-tetraol (3), together with three known sterols (4-6) were isolated from the green alga Ulva australis. The structures of the new compounds (1-3) were elucidated through 1D and 2D nuclear magnetic resonance spectroscopy as well as mass spectrometry. Compounds 4-6 were identified as isofucoterol (4), 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), and (24S)-stigmastadiene-3β,24-diol (6) on the basis of spectroscopic data analyses and comparison with those reported in the literature. Compounds 4-6 were isolated from U. australis for the first time. These compounds, together with the previously isolated secondary metabolites of this alga, were investigated for their inhibitory effects on human recombinant aldose reductase in vitro. Of the compounds, 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), 1-O-palmitoyl-3-O-(6'-sulfo-α-d-quinovopyranosyl) glycerol, (2S)-1-O-palmitoyl-3-O-[α-d-galactopyranosyl(1→2)β-d-galactopyranosyl] glycerol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and 8-hydroxy-(6E)-octenoic acid weakly inhibited the enzyme, while the three new sterols, 1-3, were almost inactive.

  12. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    PubMed

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  13. Insights into the mechanisms of sterol transport between organelles.

    PubMed

    Mesmin, Bruno; Antonny, Bruno; Drin, Guillaume

    2013-09-01

    In cells, the levels of sterol vary greatly among organelles. This uneven distribution depends largely on non-vesicular routes of transfer, which are mediated by soluble carriers called lipid-transfer proteins (LTPs). These proteins have a domain with a hydrophobic cavity that accommodates one sterol molecule. However, a demonstration of their role in sterol transport in cells remains difficult. Numerous LTPs also contain membrane-binding elements, but it is not clear how these LTPs couple their ability to target organelles with lipid transport activity. This issue appears critical, since many sterol transporters are thought to act at contact sites between two membrane-bound compartments. Here, we emphasize that biochemical and structural studies provide precious insights into the mode of action of sterol-binding proteins. Recent studies on START, Osh/ORP and NPC proteins suggest models on how these proteins could transport sterol between organelles and, thereby, influence cellular functions.

  14. SOME FACTORS AFFECTING STEROL FORMATION IN SACCHAROMYCES CEREVISIAE1

    PubMed Central

    Starr, Patricia R.; Parks, L. W.

    1962-01-01

    Starr, Patricia R. (Oregon State University, Corvallis) and L. W. Parks. Some factors affecting sterol formation in Saccharomyces cerevisiae. J. Bacteriol. 83:1042–1046. 1962.—A wild-type diploid strain of Saccharomyces cerevisiae was used in a study of factors that influence sterol synthesis. Maltose, glucose, sodium acetate, and ethanol were shown to be readily available for sterol synthesis in growing cultures of yeast. In cells grown anaerobically and then exposed to various substrates in aerobic resting-cell suspension, only glucose and ethanol stimulated ergosterol formation. Under these conditions, sterol synthesis was directly proportional to the amount of glucose provided. Sulfanilamide decreased the yield of sterol in growing cells, but had no effect on sterol synthesis by resting cultures. PMID:13916377

  15. [Plant sterols, cholesterol precursors and oxysterols: small amounts, big effects].

    PubMed

    Olkkonen, Vesa M; Gylling, Helena; Ikonen, Elina

    2015-01-01

    Noncholesterol sterols are present in the body in very low concentrations compared with cholesterol. Minor structural changes in sterols give them completely individual biological activities. Steroid hormones are the best known example of this. The knowledge of other relatives of cholesterol, particularly plant sterols, cholesterol precursors and oxysterols, their properties, physiological effects, significance in disease processes and diagnostic applications has recently undergone a rapid increase.

  16. Distribution of sterols in the fungi. I - Fungal spores

    NASA Technical Reports Server (NTRS)

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

    1974-01-01

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

  17. Distribution of sterols in the fungi. I - Fungal spores

    NASA Technical Reports Server (NTRS)

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

    1974-01-01

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

  18. The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

    PubMed Central

    Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

    2012-01-01

    Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

  19. Sterol and lipid composition of three Adriatic Sea sponges.

    PubMed

    De Rosa, Salvatore; Seizova, Katya; Kamenarska, Zornitsa; Petrova, Assia; Iodice, Carmine; Stefanov, Kamen; Popov, Simeon

    2006-01-01

    The sterol and fatty acid composition of three Adriatic Sea sponges (Geodia cydonium and two unidentified Tedania sp.), collected at the same time and same place, was established. Twenty-four sterols and forty fatty acids were identified. The identical ecological conditions, including the diet, allowed us to apply the results obtained for taxonomical conclusions, based on the biodiversity of the investigated sponges. On the basis of the sterol composition they can be separated into two groups: Tedania and Geodia sponges. The sterol and fatty acid composition indicates that the two investigated Tedania samples might be different species or subspecies.

  20. Non-cholesterol sterols and cholesterol metabolism in sitosterolemia.

    PubMed

    Othman, Rgia A; Myrie, Semone B; Jones, Peter J H

    2013-12-01

    Sitosterolemia (STSL) is a rare autosomal recessive disease, manifested by extremely elevated plant sterols (PS) in plasma and tissue, leading to xanthoma and premature atherosclerotic disease. Therapeutic approaches include limiting PS intake, interrupting enterohepatic circulation of bile acid using bile acid binding resins such as cholestyramine, and/or ileal bypass, and inhibiting intestinal sterol absorption by ezetimibe (EZE). The objective of this review is to evaluate sterol metabolism in STSL and the impact of the currently available treatments on sterol trafficking in this disease. The role of PS in initiation of xanthomas and premature atherosclerosis is also discussed. Blocking sterols absorption with EZE has revolutionized STSL patient treatment as it reduces circulating levels of non-cholesterol sterols in STSL. However, none of the available treatments including EZE have normalized plasma PS concentrations. Future studies are needed to: (i) explore where cholesterol and non-cholesterol sterols accumulate, (ii) assess to what extent these sterols in tissues can be mobilized after blocking their absorption, and (iii) define the factors governing sterol flux. Copyright © 2013. Published by Elsevier Ireland Ltd.

  1. Bioconversion and binding of sterols by thermophilic moulds.

    PubMed

    Satyanarayana, T; Chavant, L

    1987-01-01

    None of the fourteen thermophilic moulds was able to break down the aliphatic side chain of sterols, viz. cholesterol, lanosterol, sitosterol, and stigmasterol so as to yield 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione and progesterone. In Acremonium alabamensis and Talaromyces emersonii, cholestenone was detected as a product of fermentation of cholesterol whereas the former yielded stigmastadienone from stigmasterol and sitosterol. Lanosterol appeared to be resistant to fungal bioconversion. All the thermophilic moulds exhibited avidity for binding sterols to the mycelium, but the ability to bind sterol seemed to depend upon the nature of the organism and the sterol.

  2. Cholesterol homeostasis: How do cells sense sterol excess?

    PubMed

    Howe, Vicky; Sharpe, Laura J; Alexopoulos, Stephanie J; Kunze, Sarah V; Chua, Ngee Kiat; Li, Dianfan; Brown, Andrew J

    2016-09-01

    Cholesterol is vital in mammals, but toxic in excess. Consequently, elaborate molecular mechanisms have evolved to maintain this sterol within narrow limits. How cells sense excess cholesterol is an intriguing area of research. Cells sense cholesterol, and other related sterols such as oxysterols or cholesterol synthesis intermediates, and respond to changing levels through several elegant mechanisms of feedback regulation. Cholesterol sensing involves both direct binding of sterols to the homeostatic machinery located in the endoplasmic reticulum (ER), and indirect effects elicited by sterol-dependent alteration of the physical properties of membranes. Here, we examine the mechanisms employed by cells to maintain cholesterol homeostasis.

  3. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

    PubMed Central

    Li, Xiaochun; Roberti, Rita; Blobel, Günter

    2014-01-01

    Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Delta-14 sterol reductase (C14SR), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B Receptor2 (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Delta-14 sterol reductase (maSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z, a homolog of human C14SR, LBR, and DHCR7, with the cofactor NADPH. The enzyme contains 10 transmembrane segments (TM). Its catalytic domain comprises the C-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure3 suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves maSR1 can reduce the double bond of a cholesterol biosynthetic intermediate demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases. PMID:25307054

  4. Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Streptomyces sp. strain FRI-5.

    PubMed Central

    Waki, M; Nihira, T; Yamada, Y

    1997-01-01

    IM-2 is a butyrolactone autoregulator that controls production of blue pigment and nucleoside antibiotics in Streptomyces sp. strain FRI-5. An IM-2-specific receptor gene, farA, was cloned from strain FRI-5, and nucleotide sequencing revealed that the farA gene consists of 666 bp encoding a 221-amino-acid protein of 24.3 kDa with an NH2-terminal amino acid sequence identical to that of purified native receptor. Another gene, farX, encoding a homolog of AfsA of Streptomyces griseus, was present upstream of farA. The monocistronic nature of the farA transcript was shown by Northern blot hybridization, and the transcript level increased upon addition of IM-2. Recombinant FarA expressed in and purified from E. coli showed clear ligand specificity toward IM-2, with a dissociation constant (Kd) for [3H]IM-2-C5 of 18.2 nM. FarA showed high overall homology to BarA (virginiae butanolide receptor from S. virginiae) and ArpA (A-factor receptor from S. griseus). Sequence alignment of the three receptor proteins revealed that the NH2-terminal region containing a helix-turn-helix DNA binding motif was highly conserved. The DNA binding motif is common in procaryotic repressors of the TetR family, suggesting that all the Streptomyces autoregulator receptors may act as transcriptional repressors. PMID:9260956

  5. Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain.

    PubMed Central

    Minehart, P L; Magasanik, B

    1991-01-01

    The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability. Images PMID:1682800

  6. MtrR Control of a Transcriptional Regulatory Pathway in Neisseria meningitidis That Influences Expression of a Gene (nadA) Encoding a Vaccine Candidate

    PubMed Central

    Cloward, Jason M.; Shafer, William M.

    2013-01-01

    The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes. PMID:23409129

  7. MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate.

    PubMed

    Cloward, Jason M; Shafer, William M

    2013-01-01

    The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.

  8. ENCODE data at the ENCODE portal

    PubMed Central

    Sloan, Cricket A.; Chan, Esther T.; Davidson, Jean M.; Malladi, Venkat S.; Strattan, J. Seth; Hitz, Benjamin C.; Gabdank, Idan; Narayanan, Aditi K.; Ho, Marcus; Lee, Brian T.; Rowe, Laurence D.; Dreszer, Timothy R.; Roe, Greg; Podduturi, Nikhil R.; Tanaka, Forrest; Hong, Eurie L.; Cherry, J. Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  9. Exposure of a 23F Serotype Strain of Streptococcus pneumoniae to Cigarette Smoke Condensate Is Associated with Selective Upregulation of Genes Encoding the Two-Component Regulatory System 11 (TCS11)

    PubMed Central

    Herbert, Jenny A.; Mitchell, Timothy J.; Dix-Peek, Thérèse; Dickens, Caroline; Anderson, Ronald; Feldman, Charles

    2014-01-01

    Alterations in whole genome expression profiles following exposure of the pneumococcus (strain 172, serotype 23F) to cigarette smoke condensate (160 μg/mL) for 15 and 60 min have been determined using the TIGR4 DNA microarray chip. Exposure to CSC resulted in the significant (P < 0.014–0.0006) upregulation of the genes encoding the two-component regulatory system 11 (TCS11), consisting of the sensor kinase, hk11, and its cognate response regulator, rr11, in the setting of increased biofilm formation. These effects of cigarette smoke on the pneumococcus may contribute to colonization of the airways by this microbial pathogen. PMID:25013815

  10. Δγ₁134.5 herpes simplex viruses encoding human cytomegalovirus IRS1 or TRS1 induce interferon regulatory factor 3 phosphorylation and an interferon-stimulated gene response.

    PubMed

    Cassady, Kevin A; Saunders, Ute; Shimamura, Masako

    2012-01-01

    The chimeric herpes simplex viruses (HSV) are Δγ₁34.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ₁34.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ₁34.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ₁34.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).

  11. Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides.

    PubMed

    Valitova, Julia N; Minibayeva, Farida V; Kotlova, Ekaterina R; Novikov, Alexander V; Shavarda, Alexey L; Murtazina, Lyaisan I; Ryzhkina, Irina S

    2011-10-01

    Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

    PubMed

    Flores-Sánchez, Isvett J; Ortega-López, Jaime; del Carmen Montes-Horcasitas, María; Ramos-Valdivia, Ana C

    2002-12-01

    Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

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

    PubMed Central

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

    2015-01-01

    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-ERT2, 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. PMID:25896350

  14. Regulation pattern of fish irf4 (the gene encoding IFN regulatory factor 4) by STAT6, c-Rel and IRF4.

    PubMed

    Li, Shun; Guo, Xia; Lu, Long-Feng; Lu, Xiao-Bing; Wu, Nan; Zhang, Yong-An

    2015-07-01

    Interferon regulatory factor 4 (IRF4) plays pivotal roles in both innate and adaptive immune responses in mammals. In fish, there are two homologues of irf4, irf4a and irf4b. In this study, we examined the regulatory patterns of zebrafish irf4a and irf4b by STAT6 and c-Rel. Firstly, expression of irf4a and irf4b was monitored in several tissues at mRNA level. By infection with SVCV, irf4a and irf4b were upregulated in both kidney and spleen, and were immediately induced by treatment with poly I:C in ZF4 cells. Moreover, the activation of irf4a promoter was regulated by overexpression of stat6 and c-rel in a cooperation manner, which could be inhibited by mutation of the putative binding sites of STAT6 and c-Rel in irf4a promoter region. However, irf4b promoter was activated slightly only by STAT6 but not c-Rel. Furthermore, overexpression of irf4a inhibited the activation of its own promoter under induction of STAT6 and c-Rel, which was the result of that IRF4a bound to STAT6 and c-Rel directly. In addition, cellular location analysis showed that IRF4a was located only in nuclear region. These data indicate that fish irf4a can also be upregulated by STAT6 and c-Rel. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. STEROLS AS BIOMARKERS IN GYMNODINIUM BREVE DISTRIBUTION IN DINOFLAGELLATES

    EPA Science Inventory

    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 ...

  16. Composition of plant sterols and stanols in supplemented food products

    USDA-ARS?s Scientific Manuscript database

    All fruits, vegetables, grains and other plant materials contain small amounts of plant sterols, which are essential for the function of the biological membranes in living cells. The average human consumption of plant sterols has been estimated to be about 150-350 mg/day and trace amounts of stanol...

  17. Detailed sterol compositions of two pathogenic rust fungi.

    PubMed

    Giner, José-Luis; Zhao, Hui

    2004-08-01

    Teliospores of cedar-apple rust Gymnosporangium juniperi-virginianae were collected from the eastern red cedar Juniperus virginiana, and aeciospores of quince rust G. clavipes were collected from the fruit of English hawthorn Crataegus laevigata. The sterol fractions were separated by HPLC, and their identities were determined by 600 MHz 1H NMR. Twenty-six sterols were isolated from G. juniperi-virginianae and 18 sterols were isolated from G. clavipes. The principal sterol of both fungi was (Z)-stigmasta-7,24(28)-dien-3beta-ol. Other major sterols were (24S)-ergost-7-en-3beta-ol, (24S)-stigmast-7-en-3beta-ol, and (24S)-stigmasta-5,7-dien-3beta-ol. The sterols of the hosts were found to be very different from those of the fungi. The 24-alkyl sterols of the fungi had the 24alpha-configuration, whereas those of the hosts had the 24beta-configuration. Similarities to the sterol composition of the AIDS pneumonia fungus Pneumocystis carinii are discussed.

  18. Composition of Plant Sterols and Stanols in Supplemented Food Products.

    PubMed

    Moreau, Robert A

    2015-01-01

    All fruits, vegetables, grains and other plant materials contain small amounts of plant sterols, which are essential for the function of the biological membranes in living cells. The average human consumption of plant sterols has been estimated to be about 150-350 mg/day and trace amounts of stanols (which are defined as saturated sterols such as sitostanol), but this number varies regionally and is higher for vegetarians. When consumed in the diet, plant sterols reduce the levels of serum cholesterol. In 1995 the first functional food product, Benecol spread (enriched in plant stanol fatty acid esters), was developed by Raisio and marketed, first in Finland and then globally. Since then many other functional food products have been developed and are now available globally. In addition to stanol esters, other functional food products contain plant sterol esters and/or free (unesterified) plant sterols and stanols. In essentially all of the current functional foods that are enriched in sterols and stanols, the feedstock from which the sterols and stanols are obtained is either tall oil (a byproduct/coproduct of the pulping of pine wood) or vegetable oil deodorizer distillate (a byproduct/coproduct of the refining of vegetable oils).

  19. Sterol composition in larvae of the silkworm, Bombyx mori.

    PubMed

    Nagata, Shinji; Nagasawa, Hiromichi

    2011-01-01

    Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.

  20. Plant sterols in vegetables and fruits commonly consumed in Sweden.

    PubMed

    Normén, L; Johnsson, M; Andersson, H; van Gameren, Y; Dutta, P

    1999-04-01

    Plant sterols are known to have serum cholesterol lowering effects. A high dietary intake might therefore have a positive impact on health. All food items of vegetable origin contain some amount of plant sterols. The aim of this study was to analyse the plant sterol content of vegetables and fruits commonly consumed in Sweden, and to compare fresh and cooked samples of the same items. Altogether 20 different vegetables and 14 fruits were analysed. All vegetables and fruits were purchased in two shops in the city of Gothenburg, Sweden. Lyophilization was performed within one month of the items being purchased. The samples were frozen at -20 (C and analysed within six months, with a GLC method after acid hydrolysis, alkaline hydrolysis and silylation with tri-methylsilylether. The acid hydrolysis was done in order to detect the fraction of glycosylated plant sterols, which are split during boiling with HCl. The median plant sterol content of vegetables was 14 (3.8-50) mg/100 g edible portion. The highest concentrations were found in broccoli. Brussels sprouts, cauliflower and olives. The median plant sterol content of fruits was 16 (3-44) mg/100 g edible portion. The highest concentrations were found in oranges and passion fruits. The plant sterol concentrations were thus low in vegetables and fruits commonly consumed in Sweden. A serum cholesterol lowering effect attributed to the plant sterols in vegetables and fruits would therefore be of limited significance.

  1. STEROLS AS BIOMARKERS IN GYMNODINIUM BREVE DISTRIBUTION IN DINOFLAGELLATES

    EPA Science Inventory

    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 ...

  2. Identification of regulatory sequence elements within the transcription promoter region of NpABC1, a gene encoding a plant ABC transporter induced by diterpenes.

    PubMed

    Grec, Sébastien; Vanham, Delphine; de Ribaucourt, Jeoffrey Christyn; Purnelle, Bénédicte; Boutry, Marc

    2003-07-01

    Expression of NpABC1, a gene encoding a plasma membrane ATP binding cassette (ABC) transporter in Nicotiana plumbaginifolia, is induced by sclareol, an antifungal diterpene produced at the leaf surface, as well as by sclareolide, a close analog. A genomic fragment including the 1282-bp region upstream of the NpABC1 transcription start was fused to the reporter beta-glucuronidase (gus) gene and introduced into N. tabacum BY2 cells for stable transformation. A 25-fold increase in gus expression was observed when cells were treated with sclareolide and some other terpenes. The combined use of 5'-deletion promoter analysis, gel mobility shift assays, DNase I footprinting, and site-directed mutagenesis allowed us to identify three cis-elements (sclareol box 1 (SB1), SB2, and SB3) located, respectively, within nucleotides -827 to -802, -278 to -243, and -216 to -190 upstream of the NpABC1 transcription start. In vivo evaluation of these elements on sclareolide-induced expression showed that mutation of SB1 reduced expression by twofold, while that of SB2 had no effect. On the other hand, SB3 had a marked effect as it completely abolished sclareolide-mediated expression. NpABC1-gus expression was not induced by the stress signals, salicylic acid and ethylene, but was mediated, to some extent, by methyl jasmonate, which is known to promote diterpene synthesis.

  3. Characterization of various promoter regions of the human DNA helicase-encoding genes and identification of duplicated ets (GGAA) motifs as an essential transcription regulatory element.

    PubMed

    Uchiumi, Fumiaki; Watanabe, Takeshi; Tanuma, Sei-ichi

    2010-05-15

    DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells.

  4. Biallelic Truncating Mutations in FMN2, Encoding the Actin-Regulatory Protein Formin 2, Cause Nonsyndromic Autosomal-Recessive Intellectual Disability

    PubMed Central

    Law, Rosalind; Dixon-Salazar, Tracy; Jerber, Julie; Cai, Na; Abbasi, Ansar A.; Zaki, Maha S.; Mittal, Kirti; Gabriel, Stacey B.; Rafiq, Muhammad Arshad; Khan, Valeed; Nguyen, Maria; Ali, Ghazanfar; Copeland, Brett; Scott, Eric; Vasli, Nasim; Mikhailov, Anna; Khan, Muhammad Nasim; Andrade, Danielle M.; Ayaz, Muhammad; Ansar, Muhammad; Ayub, Muhammad; Vincent, John B.; Gleeson, Joseph G.

    2014-01-01

    Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density. PMID:25480035

  5. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): enzymes with multiple sterols as substrates and as activators

    PubMed Central

    Rogers, Maximillian A.; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C.Y.; Chang, Ta-Yuan

    2016-01-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the isooctyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  6. Sterols and Fatty Acids of the Harmful Dinoflagellate Cochlodinium polykrikoides.

    PubMed

    Giner, José-Luis; Ceballos, Harriette; Tang, Ying-Zhong; Gobler, Christopher J

    2016-02-01

    Sterol and fatty acid compositions were determined for Cochlodinium polykrikoides, a toxic, bloom-forming dinoflagellate of global significance. The major sterols were dinosterol (40% of total sterols), dihydrodinosterol (32%), and the rare 4α-methyl Δ(8(14)) sterol, amphisterol (23%). A minor sterol, 4α-methylergost-24(28)-enol was also detected (5.0%). The fatty acids had a high proportion of PUFAs (47%), consisting mainly of EPA (20%) and the relatively uncommon octadecapentaenoic acid (18 : 5, 22%). While unlikely to be responsible for toxicity to fish, these lipids may contribute to the deleterious effects of this alga to invertebrates. Copyright © 2016 Verlag Helvetica Chimica Acta AG, Zürich.

  7. Diversity of Sterol Composition in Tunisian Pistacia lentiscus Seed Oil.

    PubMed

    Mezni, Faten; Labidi, Arbia; Khouja, Mohamed Larbi; Martine, Lucy; Berdeaux, Olivier; Khaldi, Abdelhamid

    2016-05-01

    Pistacia lentiscus L. seed oil is used in some Mediterranean forest area for culinary and medicinal purposes. In this study, we aim to examine, for the first time, the effect of growing area on sterol content of Pistacia lentiscus seed oil. Fruits were harvested from 13 different sites located in northern and central Tunisia. Gas chromatography-flame-ionization detection (GC-FID) was used to quantify sterols and gas chromatography/mass spectrometry (GC/MS) was used to identify them. The major sterol identified was β-sitosterol with a value ranging from 854.12 to 1224.09 mg/kg of oil, thus making up more than 54% of the total sterols. The other two main sterols were cycloartenol (11%) and 24-methylene-cycloartenol (5%). Statistical results revealed that growing location significantly (P < 0.001) affected phytosterol levels in these oils.

  8. Profiling and Metabolism of Sterols in the Weaver Ant Genus Oecophylla.

    PubMed

    Vidkjær, Nanna H; Jensen, Karl-Martin V; Gislum, René; Fomsgaard, Inge S

    2016-01-01

    Sterols are essential to insects because they are vital for many biochemical processes, nevertheless insects cannot synthesize sterols but have to acquire them through their diet. Studies of sterols in ants are sparse and here the sterols of the weaver ant genus Oecophylla are identified for the first time. The sterol profile and the dietary sterols provided to a laboratory Oecophylla longinoda colony were analyzed. Most sterols originated from the diet, except one, which was probably formed via dealkylation in the ants and two sterols of fungal origin, which likely originate from hitherto unidentified endosymbionts responsible for supplying these two compounds. The sterol profile of a wild Oecophylla smaragdina colony was also investigated. Remarkable qualitative similarities were established between the two species despite the differences in diet, species, and origin. This may reflect a common sterol need/aversion in the weaver ants. Additionally, each individual caste of both species displayed unique sterol profiles.

  9. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene.

    PubMed

    Czub, Barbara; Shah, Amna Z; Alfano, Giovanna; Kruczek, Przemysław M; Chakarova, Christina F; Bhattacharya, Shomi S

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.

  10. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene

    PubMed Central

    Czub, Barbara; Shah, Amna Z.; Alfano, Giovanna; Kruczek, Przemysław M.; Chakarova, Christina F.; Bhattacharya, Shomi S.

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS. PMID:26872363

  11. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    PubMed

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  12. Sterol content analysis suggests altered eburicol 14alpha-demethylase (CYP51) activity in isolates of Mycosphaerella graminicola adapted to azole fungicides.

    PubMed

    Bean, Tim P; Cools, Hans J; Lucas, John A; Hawkins, Nathaniel D; Ward, Jane L; Shaw, Michael W; Fraaije, Bart A

    2009-06-01

    The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14alpha-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.

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

    NASA Astrophysics Data System (ADS)

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

    2004-12-01

    The biosynthesis of sterols is generally regarded as a eukaryotic process. The first enzymatic step in the production of sterols requires molecular oxygen. Therefore, both the origin of eukaryotes and the evolution of sterol biosynthesis were thought to postdate the rise of oxygen in earth's atmosphere, until Brocks et al. discovered steranes in rocks aged 2.7 Ga (1). Many prokaryotes produce hopanoids, sterol-like compounds that are synthesized from the common precursor squalene without the use of molecular oxygen. However, a few bacterial taxa are also known to produce sterols, suggesting this pathway could precede the rise of oxygen (2, 3). Recently, we discovered the shortest sterol-producing biosynthetic pathway known to date in the bacterium Gemmata obscuriglobus (4). Using genomic searches, we found that Gemmata has the enzymes necessary for synthesis of sterols, and lipid analyses showed that the sterols produced are lanosterol and its isomer parkeol. Gemmata is a member of the Planctomycetes, an unusual group of bacteria, all of the known species of which contain intracellular compartmentalization. Among the Planctomycetes, Gemmata uniquely is the only prokaryote known to contain a double-membrane-bounded nuclear body (5). Since sterols usually are found in eukaryotes, and Gemmata has a eukaryote-like nuclear organelle, we investigated the location of the sterols within Gemmata to postulate whether they play a role in stabilization of the nuclear membrane and control of genomic organization. We used the sterol-specific fluorescent dye Filipin III in conjunction with fluorescent dyes for internal and external cellular membranes in order to determine whether the sterols are located in the nuclear body membrane, external membrane, or both. We found that sterols in Gemmata are concentrated in the internal membrane, implying that they function in maintaining this unusual cellular component. It is notable that Gemmata also produce hopanoids, suggesting that they

  14. Regulatory RNAs in Planarians.

    PubMed

    Pawlicka, Kamila; Perrigue, Patrick M; Barciszewski, Jan

    2016-01-01

    The full scope of regulatory RNA evolution and function in epigenetic processes is still not well understood. The development of planarian flatworms to be used as a simple model organism for research has shown a great potential to address gaps in the knowledge in this field of study. The genomes of planarians encode a wide array of regulatory RNAs that function in gene regulation. Here, we review planarians as a suitable model organism for the identification and function of regulatory RNAs.

  15. Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

    PubMed

    Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

    2015-08-01

    In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG.

  16. Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase.

    PubMed

    Kaneshiro, Edna S; Johnston, Laura Q; Nkinin, Stephenson W; Romero, Becky I; Giner, José-Luis

    2015-01-01

    The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

  17. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification

    PubMed Central

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A.; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T.; Ruggles, Kelly V.; DeGiorgis, Joseph A.; Kohlwein, Sepp D.; Schon, Eric A.; Sturley, Stephen L.

    2015-01-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53–36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.—Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification. PMID:26220175

  18. Anatomical distribution of sterols in oysters (Crassostrea gigas).

    PubMed

    Gordon, D T; Collins, N

    1982-11-01

    Oysters (Crassostrea gigas) contain at least 8 predominant sterols as determined by gas liquid chromatography and a modified Liebermann-Burchard reaction. These sterols and the average amount found in mg/100 are: C26-sterol (22-trans-24-norcholesta-5, 22-diene-3 beta-ol), 19.1; 22-dehydrocholesterol, 15.1; cholesterol, 46.8; brassicasterol, 27.2; delta 5,7-sterols (i.e., 7-dehydrocholesterol) 22.5; 24-methylenecholesterol 29.1; 24-ethylcholesta-5,22-diene-3 beta-ol, 1.2; and 24-ethylcholesta-5-en-3 beta-ol, 12.7. The distribution of these sterols appears uniform (r2 = 0.938) between 5 major organs of the oyster. The percent body mass vs percent total sterols in these 5 organs are: mantle 44.1--41.4; visceral mass 30.3--36.7; gills 13.2--11.7; adductor muscle 8.3--3.7; and labial palps 4.2--6.5. The possible sources of these sterols are discussed.

  19. Sterols of a contemporary lacustrine sediment. [in English postglacial lake

    NASA Technical Reports Server (NTRS)

    Gaskell, S. J.; Eglinton, G.

    1976-01-01

    Results are reported for detailed sterol analyses of several depths (corresponding to between zero and about 150 yr in age) in a contemporary lacustrine sediment from a freshwater lake of postglacial origin in England. Delta 5-, delta 22-, and delta 5,22-sterols are identified along with 5 alpha- and 5 beta-stanols as well as a C26 stanol with a C7 side chain. Solvent extraction yields carbon number distributions for the 5 alpha- and 5 beta-stanol sediment constituents that parallel the corresponding delta 5-sterol distributions. The amounts of 5 alpha-stanols are found to exceed those of 5 beta-stanols in the sediment, and variations in the ratio of 5 alpha- to 5 beta-stanol between sediment samples from similar depths are shown to suggest an inhomogeneity of the sediment. It is found that the sterol composition of sediment cores varies markedly with depth, reflecting both the effects of a sterol hydrogenation process and a changing input to the sediment. It is concluded that C29 sterols, of probable higher-plant origin, predominate at lower sediment depths while C27 sterols, possibly derived from autochthonous sources, are more abundant in the surface sediment.

  20. Fecal Sterol and Runoff Analysis for Nonpoint Source Tracking.

    PubMed

    Fahrenfeld, N L; Del Monaco, N; Coates, J T; Elzerman, A W

    2016-01-01

    Fecal pollution source identification is needed to quantify risk, target installation of source controls, and assess performance of best management practices in impaired surface waters. Sterol analysis is a chemical method for fecal source tracking that allows for differentiation between several fecal pollution sources. The objectives of this study were to use these chemical tracers for quantifying human fecal inputs in a mixed-land-use watershed without point sources of pollution and to determine the relationship between land use and sterol ratios. Fecal sterol analysis was performed on bed and suspended sediment from impaired streams. Human fecal signatures were found at sites with sewer overflow and septic inputs. Different sterol ratios used to indicate human fecal pollution varied in their sensitivity. Next, geospatial data was used to determine the runoff volumes associated with each land-use category in the watersheds. Fecal sterol ratios were compared between sampling locations and correlations were tested between ratio values and percentage of runoff for a given land-use category. Correlation was not observed between percentage of runoff from developed land and any of the five tested human-indicating sterol ratios in streambed sediments, confirming that human fecal inputs were not evenly distributed across the urban landscape. Several practical considerations for adopting this chemical method for microbial source tracking in small watersheds are discussed. Results indicate that sterol analysis is useful for identifying the location of human fecal nonpoint-source inputs.

  1. Sterols of a contemporary lacustrine sediment. [in English postglacial lake

    NASA Technical Reports Server (NTRS)

    Gaskell, S. J.; Eglinton, G.

    1976-01-01

    Results are reported for detailed sterol analyses of several depths (corresponding to between zero and about 150 yr in age) in a contemporary lacustrine sediment from a freshwater lake of postglacial origin in England. Delta 5-, delta 22-, and delta 5,22-sterols are identified along with 5 alpha- and 5 beta-stanols as well as a C26 stanol with a C7 side chain. Solvent extraction yields carbon number distributions for the 5 alpha- and 5 beta-stanol sediment constituents that parallel the corresponding delta 5-sterol distributions. The amounts of 5 alpha-stanols are found to exceed those of 5 beta-stanols in the sediment, and variations in the ratio of 5 alpha- to 5 beta-stanol between sediment samples from similar depths are shown to suggest an inhomogeneity of the sediment. It is found that the sterol composition of sediment cores varies markedly with depth, reflecting both the effects of a sterol hydrogenation process and a changing input to the sediment. It is concluded that C29 sterols, of probable higher-plant origin, predominate at lower sediment depths while C27 sterols, possibly derived from autochthonous sources, are more abundant in the surface sediment.

  2. Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

    PubMed Central

    Wewer, Vera; Dombrink, Isabel; vom Dorp, Katharina; Dörmann, Peter

    2011-01-01

    Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers. PMID:21382968

  3. Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry.

    PubMed

    Wewer, Vera; Dombrink, Isabel; vom Dorp, Katharina; Dörmann, Peter

    2011-05-01

    Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

  4. Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

    PubMed

    Clay, Adam T; Lu, Peihua; Sharom, Frances J

    2015-11-03

    The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

  5. Paleoproterozoic sterol biosynthesis and the rise of oxygen

    NASA Astrophysics Data System (ADS)

    Gold, David A.; Caron, Abigail; Fournier, Gregory P.; Summons, Roger E.

    2017-03-01

    Natural products preserved in the geological record can function as ‘molecular fossils’, providing insight into organisms and physiologies that existed in the deep past. One important group of molecular fossils is the steroidal hydrocarbons (steranes), which are the diagenetic remains of sterol lipids. Complex sterols with modified side chains are unique to eukaryotes, although simpler sterols can also be synthesized by a few bacteria. Sterol biosynthesis is an oxygen-intensive process; thus, the presence of complex steranes in ancient rocks not only signals the presence of eukaryotes, but also aerobic metabolic processes. In 1999, steranes were reported in 2.7 billion year (Gyr)-old rocks from the Pilbara Craton in Australia, suggesting a long delay between photosynthetic oxygen production and its accumulation in the atmosphere (also known as the Great Oxidation Event) 2.45-2.32 Gyr ago. However, the recent reappraisal and rejection of these steranes as contaminants pushes the oldest reported steranes forward to around 1.64 Gyr ago (ref. 6). Here we use a molecular clock approach to improve constraints on the evolution of sterol biosynthesis. We infer that stem eukaryotes shared functionally modern sterol biosynthesis genes with bacteria via horizontal gene transfer. Comparing multiple molecular clock analyses, we find that the maximum marginal probability for the divergence time of bacterial and eukaryal sterol biosynthesis genes is around 2.31 Gyr ago, concurrent with the most recent geochemical evidence for the Great Oxidation Event. Our results therefore indicate that simple sterol biosynthesis existed well before the diversification of living eukaryotes, substantially predating the oldest detected sterane biomarkers (approximately 1.64 Gyr ago), and furthermore, that the evolutionary history of sterol biosynthesis is tied to the first widespread availability of molecular oxygen in the ocean-atmosphere system.

  6. Paleoproterozoic sterol biosynthesis and the rise of oxygen.

    PubMed

    Gold, David A; Caron, Abigail; Fournier, Gregory P; Summons, Roger E

    2017-03-16

    Natural products preserved in the geological record can function as 'molecular fossils', providing insight into organisms and physiologies that existed in the deep past. One important group of molecular fossils is the steroidal hydrocarbons (steranes), which are the diagenetic remains of sterol lipids. Complex sterols with modified side chains are unique to eukaryotes, although simpler sterols can also be synthesized by a few bacteria. Sterol biosynthesis is an oxygen-intensive process; thus, the presence of complex steranes in ancient rocks not only signals the presence of eukaryotes, but also aerobic metabolic processes. In 1999, steranes were reported in 2.7 billion year (Gyr)-old rocks from the Pilbara Craton in Australia, suggesting a long delay between photosynthetic oxygen production and its accumulation in the atmosphere (also known as the Great Oxidation Event) 2.45-2.32 Gyr ago. However, the recent reappraisal and rejection of these steranes as contaminants pushes the oldest reported steranes forward to around 1.64 Gyr ago (ref. 6). Here we use a molecular clock approach to improve constraints on the evolution of sterol biosynthesis. We infer that stem eukaryotes shared functionally modern sterol biosynthesis genes with bacteria via horizontal gene transfer. Comparing multiple molecular clock analyses, we find that the maximum marginal probability for the divergence time of bacterial and eukaryal sterol biosynthesis genes is around 2.31 Gyr ago, concurrent with the most recent geochemical evidence for the Great Oxidation Event. Our results therefore indicate that simple sterol biosynthesis existed well before the diversification of living eukaryotes, substantially predating the oldest detected sterane biomarkers (approximately 1.64 Gyr ago), and furthermore, that the evolutionary history of sterol biosynthesis is tied to the first widespread availability of molecular oxygen in the ocean-atmosphere system.

  7. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function

    PubMed Central

    Seedorf, Udo; Raabe, Martin; Ellinghaus, Peter; Kannenberg, Frank; Fobker, Manfred; Engel, Thomas; Denis, Simone; Wouters, Fred; Wirtz, Karel W.A.; Wanders, Ronald J.A.; Maeda, Nobuyo; Assmann, Gerd

    1998-01-01

    Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(−/−) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein. PMID:9553048

  8. Terpenoids and sterols from some Japanese mushrooms.

    PubMed

    Yaoita, Yasunori; Kikuchi, Masao; Machida, Koichi

    2014-03-01

    Over the past twenty years, our research group has been studying the chemical constituents of mushrooms. From nineteen species, namely, Amanita virgineoides Bas (Amanitaceae), Daedaleopsis tricolor (Bull.: Fr.) Bond. et Sing. (Polyporaceae), Grifolafrondosa (Fr.) S. F. Gray (Polyporaceae), Hericium erinaceum (Bull.: Fr.) Pers. (Hericiaceae), Hypsizigus marmoreus (Peck) Bigelow (Tricholomataceae), Lactarius piperatus (Scop.: Fr.) S. F. Gray (Russulaceae), Lentinula edodes (Berk.) Sing. (Pleurotaceae), Lyophyllyum connatum (Schum.: Fr.) Sing. (Tricholomataceae), Naematoloma sublateritium (Fr.) Karst. (Strophariaceae), Ompharia lapidescens Schroeter (Polyporaceae), Panellus serotinus (Pers.: Fr.) Kuhn. (Tricholomataceae), Pholiota nameko (T. Ito) S. Ito et Imai in Imai (Strophariaceae), Pleurotus eringii (DC.: Fr.) Quel. (Pleurotaceae), Polyporus umbellatus Fries (Polyporaceae), Russula delica Fr. (Russulaceae), Russula sanguinea (Bull.) Fr. (Russulaceae), Sarcodon aspratus (Berk.) S. Ito (Thelephoraceae), Tricholoma matsutake (S. Ito et Imai) Sing. (Tricholomataceae), and Tricholomaportentosum (Fr.) Quel. (Tricholomataceae), we isolated eight new sesquiterpenoids, six new meroterpenoids, three new triterpenoids, and twenty eight new sterols. In this review, structural features of these new compounds are discussed.

  9. Binding of tissue-specific forms of alpha A-CRYBP1 to their regulatory sequence in the mouse alpha A-crystallin-encoding gene: double-label immunoblotting of UV-crosslinked complexes.

    PubMed

    Kantorow, M; Becker, K; Sax, C M; Ozato, K; Piatigorsky, J

    1993-09-15

    The alpha A-CRYBP1 regulatory sequence (alpha A-CRYBP1RS), at nucleotides -66 to -57 of the mouse alpha A-crystallin-encoding gene (alpha A-CRY) promoter, is an important control element involved in the regulation of mouse alpha A-CRY expression. The gene encoding a protein (alpha A-CRYBP1) that specifically binds to the alpha A-CRYBP1RS sequence has been cloned from a cultured mouse lens cell line. In the present study, we have used an antibody (specific to the alpha A-CRYBP1 protein and made against a synthetic peptide) to directly identify UV-crosslinked protein-DNA complexes via a double-label immunoblotting technique. Multiple alpha A-CRYB1 antigenically related proteins interacted with alpha A-CRYBP1RS in nuclear extracts from both a cloned mouse lens cell line (alpha TN4-1) that expresses alpha A-CRY and a mouse fibroblast line (L929) that does not express the gene. Two sizes (50 kDa and 90 kDa) of proteins reacting with the alpha A-CRYBP1-specific Ab were detected in both cell lines and, in addition, a > 200-kDa protein reacting with the Ab was unique to the fibroblast line. Thus, alpha A-CRYBP1 antigenically related proteins interact with alpha A-CRYBP1RS regardless of alpha A-CRY expression. Moreover, differential processing of the alpha A-CRYBP1 protein and/or alternative splicing of the alpha A-CRY transcript may affect expression of alpha A-CRY.

  10. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

    PubMed Central

    Stockinger, Eric J.; Gilmour, Sarah J.; Thomashow, Michael F.

    1997-01-01

    Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis. PMID:9023378

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

    PubMed

    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.

  12. Suppressing a Putative Sterol Carrier Gene Reduces Plasmodesmal Permeability and Activates Sucrose Transporter Genes during Cotton Fiber Elongation.

    PubMed

    Zhang, Zhiyuan; Ruan, Yong-Ling; Zhou, Na; Wang, Fang; Guan, Xueying; Fang, Lei; Shang, Xiaoguang; Guo, Wangzhen; Zhu, Shuijin; Zhang, Tianzhen

    2017-08-01

    Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D, a putative sterol carrier protein gene from elongating cotton (Gossypium hirsutum) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D-suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D, which encodes a PD-targeting β-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs, leading to the switch from symplasmic to apoplasmic pathways. © 2017 American Society of Plant Biologists. All rights reserved.

  13. Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus

    PubMed Central

    Blosser, Sara J.; Merriman, Brittney; Grahl, Nora; Chung, Dawoon

    2014-01-01

    The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response. PMID:25107308

  14. Incomplete sterols and hopanoids pathways in ciliates: gene loss and acquisition during evolution as a source of biosynthetic genes.

    PubMed

    Tomazic, Mariela L; Poklepovich, Tomas J; Nudel, Clara B; Nusblat, Alejandro D

    2014-05-01

    Polycyclic triterpenoids, such as sterols and hopanoids, are essential components of plasmatic membrane in eukaryotic organisms. Although it is generally assumed that ciliates do not synthesize sterols, and many of them are indeed auxotrophic, a large set of annotated genomic sequences and experimental data from recently studied organisms indicate that they can carry putative genes and respond to the presence/absence of precursors in various ways. The pre-squalene pathway, for instance, is largely present in all sequenced ciliates except in Ichthyophthirius multifiliis; although Paramecium tetraurelia lacks the squalene synthase and Oxytricha trifallax the squalene hopene synthase, in addition to the former. On the other hand, the post-squalene pathway, requiring oxygen in several steps, is mostly incomplete in all ciliates analyzed. Nevertheless, a number of predicted genes, with high sequence similarity to C-4 methyl oxidase/s, C-14 demethylase, C-5 and C-7 desaturases and C-24 reductase of sterols are found in Tetrahymena and Paramecium, and scattered in other Stichotrichia ciliates. Moreover, several of these sequences are present in multiples paralogs, like the C-7 desaturase in Paramecium, that carries six versions of the only one present in Tetrahymena. The phylogenetic analyses suggest a mixed origin for the genes involved in the biosynthesis of sterols and surrogates in this phylum; while the genes encoding enzymes of the pre-squalene pathway are most likely of bacterial origin, those involved in the post-squalene pathway, including the processing of sterols obtained from the environment, may have been partially retained or acquired indistinctly from lower eukaryotes or prokaryotes. This particular combination of diverse gene/s acquisition patterns allows for survival in conditions of poor oxygen availability, in which tetrahymanol and other hopanoids may be advantageous, but also conditions of excess oxygen availability and abundant sterols, in which the

  15. Characteristics of a new sterol-nonrequiring Mycoplasma.

    PubMed

    Tully, J G; Razin, S

    1969-06-01

    Two Mycoplasma strains recovered from tissue culture environments were found to grow in complex media devoid of serum or serum fractions containing cholesterol and in a cholesterol-free synthetic medium. Neither strain was capable of synthesizing pigmented carotenoids, although these compounds are present in, and characteristic of, other sterol-nonrequiring mycoplasmas. Serological tests and an analysis of their cell protein patterns obtained by gel electrophoresis indicated that the isolates were similar to each other but distinct from other sterol-nonrequiring serotypes, Mycoplasma laidlawii and M. granularum, as well as from sterol-requiring species. The existence of Mycoplasma other than M. laidlawii and M. granularum without sterol requirements suggested the need for some taxonomic changes in this group of organisms.

  16. New Cytotoxic Oxygenated Sterols from the Marine Bryozoan Cryptosula pallasiana

    PubMed Central

    Tian, Xiang-Rong; Tang, Hai-Feng; Li, Yu-Shan; Lin, Hou-Wen; Chen, Xiao-Li; Ma, Ning; Yao, Min-Na; Zhang, Ping-Hu

    2011-01-01

    Six new sterols (1-6), together with seven known sterols (7-13), were isolated from the CCl4 extract of the marine bryozoan Cryptosula pallasiana, four (3-6) of which have already been reported as synthetic sterols. This is the first time that these compounds (3-6) are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC50 values from 14.73 to 22.11 µg/mL except for compounds 12 and 13. PMID:21566793

  17. New cytotoxic oxygenated sterols from the marine bryozoan Cryptosula pallasiana.

    PubMed

    Tian, Xiang-Rong; Tang, Hai-Feng; Li, Yu-Shan; Lin, Hou-Wen; Chen, Xiao-Li; Ma, Ning; Yao, Min-Na; Zhang, Ping-Hu

    2011-01-28

    Six new sterols (1-6), together with seven known sterols (7-13), were isolated from the CCl(4) extract of the marine bryozoan Cryptosula pallasiana, four (3-6) of which have already been reported as synthetic sterols. This is the first time that these compounds (3-6) are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC(50) values from 14.73 to 22.11 µg/mL except for compounds 12 and 13.

  18. Sterol Profile for Natural Juices Authentification by GC-MS

    SciTech Connect

    Culea, M.

    2007-04-23

    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, 0.25{mu}m 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.

  19. Inhibition of human polymorphonuclear leukocyte chemotaxis by oxygenated sterol compounds

    SciTech Connect

    Gordon, L.I.; Bass, J.; Yachnin, S.

    1980-07-01

    When preincubated with certain oxygenated sterol compounds in lipoprotein-depleted serum (20% (vol/vol)), human polymorphonuclear leukocytes show inhibition of chemotaxis toward the synthetic dipeptide N-formylmethionylphenylalinine without alteration of random movement or loss of cell viability. These effects can occur at sterol concentrations as low as 6.25 ..mu..M and after as little as 5 min of preincubation, but they are increased at higher concentrations and longer preincubation times. The inhibition can be almost completely reversed by preincubation in lipoprotein-replete serum (human AB serum, 20% (vol/vol)) and may be partially corrected by addition of free cholesterol (0.125 mM) to the medium. These effects are unlikely to be due to inhibition of cellular sterol synthesis, competition for chemotaxin membrane binding sites, or deactivation of the leukocytes but they may be a consequence of insertion of the sterol molecule into the leukocyte plasma membranes.

  20. Sterol Profile for Natural Juices Authentification by GC-MS

    NASA Astrophysics Data System (ADS)

    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.

  1. Digitonide precipitable sterols: a reevaluation with special attention to lanosterol

    SciTech Connect

    Cenedella, R.J.

    1982-06-01

    The ability of digitonin to precipitate lanosterol from prepared mixtures and biological sources was evaluated. Commercially available lanosterol was determined to be composed of about 60% lanosterol and 40% dihydrolanosterol. Both sterols were only partially precipitated by digitonin under all conditions examined. The presence of cholesterol increased the precipitation of lanosterol, but never to completion. About 40% of the lanosterols from saponified sheep's-wool fat was not precipitated by digitonin. Also /sup 14/C-labeled lanosterol recovered from rat brain following intracerebral injection of 2-(/sup 14/C)mevalonate was only 70% precipitated by digitonin. Steric hinderance by the methyl groups at carbon -4 is suggesed to explain the poor precipitability of this sterol. In conclusion, lanosterol can not be considered to be a digitonide-precipitable sterol equivalent to cholesterol. Caution should be exercised in situations where digitonin-precipitable sterols are being prepared from sources containing significant concentrations of lanosterol (i.e., mass and/or radiolabel).

  2. Free and glycosylated sterol bioaccumulation in developing Cycas micronesica seeds.

    PubMed

    Marler, Thomas E; Shaw, Christopher A

    2009-07-15

    The bioaccumulation of free and glycosylated forms of stigmasterol and β-sitosterol were determined from Cycas micronesica K.D. Hill seeds throughout seed ontogeny. Per-seed pool of the four compounds increased linearly from 2 to 24 months, indicating no developmental period elicited a major shift in the rate of bioaccumulation. The slopes were not homogeneous, signifying a change in relative sterol profile concomitant with seed maturation. This shift was in favour of the glucosides, as their rate of accumulation exceeded that of the free sterols. Stigmasterol content exceeded that of β-sitosterol, but ontogeny did not influence the ratio of these dominant sterols. The quantity and quality of sterol exposure during consumption of foods prepared from gametophytes by humans is strongly influenced by age of harvested seeds. Results are critical for a further understanding of the link between human neurodegenerative diseases and historical consumption of foods derived from the seed gametophyte tissue.

  3. Sterols in a unicellular relative of the metazoans

    PubMed Central

    Kodner, Robin B.; Summons, Roger E.; Pearson, Ann; King, Nicole; Knoll, Andrew H.

    2008-01-01

    Molecular clocks suggest that animals originated well before they first appear as macroscopic fossils, but geologic tests of these hypotheses have been elusive. A rare steroid hydrocarbon, 24-isopropylcholestane, has been hypothesized to be a biomarker for sponges or their immediate ancestors because of its relatively high abundance in pre-Ediacaran to Early Cambrian sedimentary rocks and oils. Biolipid precursors of this sterane have been reported to be prominent in several demosponges. Whether 24-isopropylcholestane can be interpreted as a sponge (and, hence, animal) biomarker, and so provide clues about early metazoan history, depends on an understanding of the distribution of sterol biosynthesis among animals and their protistan relatives. Accordingly, we characterized the sterol profile of the choanoflagellate Monosiga brevicollis, a representative of the unicellular sister group of animals. M. brevicollis does not produce a candidate sterol precursor for 24-isopropylcholestane under our experimental growth conditions. It does, however, produce a number of other sterols, and comparative genomics confirms its biosynthetic potential to produce the full suite of compounds recovered. Consistent with the phylogenetic position of choanoflagellates, the sterol profile and biosynthetic pathway of M. brevicollis display characteristics of both fungal and poriferan sterol biosynthesis. This is an example in which genomic and biochemical information have been used together to investigate the taxonomic specificity of a fossil biomarker. PMID:18632573

  4. Plant sterols: Friend or foe in CNS disorders?

    PubMed

    Vanmierlo, Tim; Bogie, Jeroen F J; Mailleux, Jo; Vanmol, Jasmine; Lütjohann, Dieter; Mulder, Monique; Hendriks, Jerome J A

    2015-04-01

    In mammals, the central nervous system (CNS) is the most cholesterol rich organ by weight. Cholesterol metabolism is tightly regulated in the CNS and all cholesterol available is synthesized in situ. Deficits in cholesterol homeostasis at the level of synthesis, transport, or catabolism result in severe disorders featured by neurological disability. Recent studies indicate that a disturbed cholesterol metabolism is involved in CNS disorders, such as Alzheimer's disease (AD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). In contrast to circulating cholesterol, dietary plant sterols, can cross the blood-brain barrier and accumulate in the membranes of CNS cells. Plant sterols are well-known for their ability to lower circulating cholesterol levels. The finding that they gain access to the CNS has fueled research focusing on the physiological roles of plant sterols in the healthy and diseased CNS. To date, both beneficial and detrimental effects of plant sterols on CNS disorders are defined. In this review, we discuss recent findings regarding the impact of plant sterols on homeostatic and pathogenic processes in the CNS, and elaborate on the therapeutic potential of plant sterols in CNS disorders.

  5. SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS, KARENIA MIKIMOTOI, AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE

    EPA Science Inventory

    The sterol composition of different marine microalgae was examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rare...

  6. SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS, KARENIA MIKIMOTOI, AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE

    EPA Science Inventory

    The sterol composition of different marine microalgae was examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rare...

  7. Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione.

    PubMed

    Yeh, Chin-Hsing; Kuo, Yung-Shun; Chang, Che-Ming; Liu, Wen-Hsiung; Sheu, Meei-Ling; Meng, Menghsiao

    2014-09-09

    The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). Because both ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from R. equi USA-18 may deserve further investigation for its application potential.

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

    PubMed Central

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

    2011-01-01

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

  9. Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga.

    PubMed Central

    Raederstorff, D; Rohmer, M

    1985-01-01

    The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells. PMID:4074326

  10. Sterol composition of phaeodactylum tricornutum as influenced by growth temperature and light spectral quality.

    PubMed

    Véron, B; Billard, C; Dauguet, J C; Hartmann, M A

    1996-09-01

    In a detailed sterol analysis of the marine diatom Phaeodactylum tricornutum, free sterols as well as esterified and glycosylated conjugates were found. When the alga was grown under standard conditions (i.e., at 13 degrees C under white light), 64% of total sterols were steryl glycosides. In all sterol classes, except steryl esters, (24S)-24-methylcholesta-5,22E-dien-3 beta-ol (epibrassicasterol) was the major (80 to 99%) sterol component. Eight other sterols were identified. Growth under different light spectral quality (red, blue, yellow, and green) at 13 and 23 degrees C was examined. At 23 degrees C, a dramatic decrease in total sterol content was observed, especially under blue light. The distribution of sterols between free and conjugated forms as well as sterol profile inside each class was found to be strongly dependent on the light spectral quality at both temperatures.

  11. Lanostane triterpenoids and sterols from Antrodia camphorata.

    PubMed

    Huang, Hui-Chi; Liaw, Chih-Chuang; Yang, Hsin-Ling; Hseu, You-Cheng; Kuo, Hsiou-Ting; Tsai, Yao-Ching; Chien, Shih-Chang; Amagaya, Sakae; Chen, Yu-Chang; Kuo, Yueh-Hsiung

    2012-12-01

    Four lanostane triterpenes, 3,7,11-trioxo-5α-lanosta-8,24(E)-dien-26-oic acid, methyl 11α-3,7-dioxo-5α-lanosta-8,24(E)-dien-26-oate, methyl 3,7,11,12,15,23-hexaoxo-5α-lanost-8-en-26-oate, and ethyl 3,7,11,12,15,23-hexaoxo-5α-lanost-8-en-26-oate, two sterols, (14α,22E)-14-hydroxyergosta-7,22-diene-3,6-dione and a steroid named as camphosterol A were isolated from a mixture of fruiting bodies and mycelia of solid cultures of Antrodia camphorata. The ¹H and ¹³C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, HMQC, HMBC and NOESY sequences. Six compounds were evaluated for cytotoxicity against several human tumor cell lines, all of which has moderate activity.

  12. Sterols of Pneumocystis carinii hominis Organisms Isolated from Human Lungs

    PubMed Central

    Kaneshiro, Edna S.; Amit, Zunika; Chandra, Jyotsna; Baughman, Robert P.; Contini, Carlo; Lundgren, Bettina

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C28 and C29 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently, pneumocysterol (C32), which is essentially lanosterol with a C-24 ethylidene group, was detected in lipids extracted from a formalin-fixed human P. carinii-infected lung, and its structures were elucidated by gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry 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 performed. Several of the same distinct sterols (e.g., fungisterol and methylcholest-7-ene-3β-ol) previously identified in P. carinii carinii were also present in organisms isolated from human specimens. Pneumocysterol was detected in only some of the samples. PMID:10548595

  13. Identifying avian sources of faecal contamination using sterol analysis.

    PubMed

    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.

  14. Plant sterol biosynthesis: identification of a NADPH dependent sterone reductase involved in sterol-4 demethylation.

    PubMed

    Pascal, S; Taton, M; Rahier, A

    1994-07-01

    Microsomes obtained from maize embryos were shown to catalyze the reduction of various sterones to produce stereoselectively the corresponding 3 beta-hydroxy derivatives. Enzymatic assay conditions have been developed to characterize this reduction step and the kinetics of the microsomal system has been established. Sterone reduction shows exclusive dependence on NADPH and is inactive with NADH. It is not sensitive to the azole inhibitors pyrifenox, ketoconazole, and itraconazole nor to phenobarbital nor pyrazole. Based on these coenzyme requirements and inhibitor susceptibility, and according to the common pattern of their classification, the maize microsomal sterone-reducing enzyme belongs to the family of ketone reductases. From a series of incubations with natural or synthetic sterones, the substrate specificity of the reduction at C-3 was determined. Our data indicate particularly that 4 alpha-methyl-9 beta,19-cyclo-C30-sterones and 4-desmethyl-delta 7-C27- or C30-sterones are preferentially reduced, while 4,4-dimethyl-C30- or C31-sterones react poorly. The results support the conclusion that the reductase activity identified is a constitutive component of the microsomal sterol 4-demethylation complex recently identified in photosynthetic organisms (S. Pascal et al., 1993, J. Biol. Chem. 268, 11639). They are consistent with the conclusion that 4 alpha-methylsterones are demethylation products of 4,4-gem-dimethylsterols rather than early intermediates in the 4 alpha-monomethyl-sterols-4-demethylation process.

  15. A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana.

    PubMed

    Nakashima, K; Kiyosue, T; Yamaguchi-Shinozaki, K; Shinozaki, K

    1997-10-01

    A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the beta-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.

  16. Herpes simplex virus 1-encoded tegument protein VP16 abrogates the production of beta interferon (IFN) by inhibiting NF-κB activation and blocking IFN regulatory factor 3 to recruit its coactivator CBP.

    PubMed

    Xing, Junji; Ni, Liwen; Wang, Shuai; Wang, Kezhen; Lin, Rongtuan; Zheng, Chunfu

    2013-09-01

    Host cells activate innate immune signaling pathways to defend against invading pathogens. To survive within an infected host, viruses have evolved intricate strategies to counteract host immune responses. Herpesviruses, including herpes simplex virus type 1 (HSV-1), have large genomes and therefore have the capacity to encode numerous proteins that modulate host innate immune responses. Here we define the contribution of HSV-1 tegument protein VP16 in the inhibition of beta interferon (IFN-β) production. VP16 was demonstrated to significantly inhibit Sendai virus (SeV)-induced IFN-β production, and its transcriptional activation domain was not responsible for this inhibition activity. Additionally, VP16 blocked the activation of the NF-κB promoter induced by SeV or tumor necrosis factor alpha treatment and expression of NF-κB-dependent genes through interaction with p65. Coexpression analysis revealed that VP16 selectively blocked IFN regulatory factor 3 (IRF-3)-mediated but not IRF-7-mediated transactivation. VP16 was able to bind to IRF-3 but not IRF-7 in vivo, based on coimmunoprecipitation analysis, but it did not affect IRF-3 dimerization, nuclear translocation, or DNA binding activity. Rather, VP16 interacted with the CREB binding protein (CBP) coactivator and efficiently inhibited the formation of the transcriptional complexes IRF-3-CBP in the context of HSV-1 infection. These results illustrate that VP16 is able to block the production of IFN-β by inhibiting NF-κB activation and interfering with IRF-3 to recruit its coactivator CBP, which may be important to the early events leading to HSV-1 infection.

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

    PubMed

    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

  18. Mechanisms Underlying the Health Benefits of Plant Sterol and Stanol Ester Consumption.

    PubMed

    Plat, Jogchum; Baumgartner, Sabine; Mensink, Ronald P

    2015-01-01

    The recent IMPROVE-IT trial clearly showed that lowering serum low-density lipoprotein cholesterol (LDL-C) concentrations via inhibiting intestinal cholesterol absorption through ezetimibe effectively lowered the number of new cardiovascular disease (CVD) events. This supports the use of other (dietary) interventions that lower serum LDL-C concentrations via comparable mechanisms such as described for plant sterol and stanol ester enriched functional foods. Therefore it is tempting to suggest that these compounds may have the same effects on CVD outcome, as described for ezetimibe in the IMPROVE-IT trial. This has however not been proven so far. A possible advantage of plant sterol and stanol ester enriched foods over ezetimibe-a typical single-target drug-is that these dietary compounds act on multiple targets, since they not only lower serum LDL-C concentrations, but also lower serum triacylglycerol (TAG) concentrations in subjects with elevated serum TAG concentrations. In addition, they might influence the functioning of our immune system via a changed activity of the regulatory T-cells. This combination of effects makes these compounds highly attractive to decrease CVD risk.

  19. Bioactivities of six sterols isolated from marine invertebrates.

    PubMed

    Zhou, Xuefeng; Sun, Jianfan; Ma, Wanlei; Fang, Wei; Chen, Zhefan; Yang, Bin; Liu, Yonghong

    2014-02-01

    Epidioxy sterols and sterols with special side chains, such as hydroperoxyl sterols, usually obtained from marine natural products, are attractive for bioactivities. To isolate and screen bioactive and special sterols from China Sea invertebrates. Two hydroperoxyl sterols (1 and 2) from the sponge Xestospongia testudinaria Lamarck (Petrosiidae), three epidioxy sterols (3-5) from the sea urchin Glyptocidaris crenularis A. Agassiz (Glyptocidaridae), sponge Mycale sp. (Mycalidae) and gorgonian Dichotella gemmacea Milne Edwards and Haime (Ellisellidae) and an unusual sterol with 25-acetoxy-19-oate (6) also from D. gemmacea were obtained and identified. Using high-throughput screening, their bioactivities were tested toward Forkhead box O 3a (Foxo3a), 3-hydroxy-3-methylglutaryl CoA reductase gene fluorescent protein (HMGCR-GFP), nuclear factor kappa B (NF-κB) luciferase, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α), protein-tyrosine phosphatase 1B (PTP1B), mitochondrial membrane permeabilization (MMP) and adenosine monophosphate-activated protein kinase. Their structures were determined by comparing their nuclear magnetic resonance data with those reported in the literature. Three epidioxy sterols (3-5) showed inhibitory activities toward Foxo3a, HMGCR-GFP and NF-κB-luciferase with the IC50 values 4.9-6.8 μg/mL. The hydroperoxyl sterol 29-hydroperoxystigmasta-5,24(28)-dien-3-ol (2) had diverse inhibitory activities against Foxo3a, HMGCR-GFP, NF-κB-luciferase, PGC-1α, PTP1B and MMP, with IC50 values of 3.8-19.1 μg/mL. The bioactivities of 3-5 showed that 5α,8α-epidioxy is the active group. Otherwise, the most plausible biosynthesis pathway for 1 and 2 in sponge involves the abstraction of an allylic proton by an activated oxygen, such as O2, along with migration of carbon-carbon double bond. Therefore, the bioactive and unstable steroid should be biosynthesized in sponge under a special ecological environment to act as a defensive

  20. Sterols indicate water quality and wastewater treatment efficiency.

    PubMed

    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

  1. Genetic variation in plant CYP51s confers resistance against voriconazole, a novel inhibitor of brassinosteroid-dependent sterol biosynthesis.

    PubMed

    Rozhon, Wilfried; Husar, Sigrid; Kalaivanan, Florian; Khan, Mamoona; Idlhammer, Markus; Shumilina, Daria; Lange, Theo; Hoffmann, Thomas; Schwab, Wilfried; Fujioka, Shozo; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed.

  2. Genetic Variation in Plant CYP51s Confers Resistance against Voriconazole, a Novel Inhibitor of Brassinosteroid-Dependent Sterol Biosynthesis

    PubMed Central

    Rozhon, Wilfried; Husar, Sigrid; Kalaivanan, Florian; Khan, Mamoona; Idlhammer, Markus; Shumilina, Daria; Lange, Theo; Hoffmann, Thomas; Schwab, Wilfried; Fujioka, Shozo; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed. PMID:23335967

  3. Effects of seaweed sterols fucosterol and desmosterol on lipid membranes.

    PubMed

    Mouritsen, Ole G; Bagatolli, Luis A; Duelund, Lars; Garvik, Olav; Ipsen, John H; Simonsen, Adam Cohen

    2017-03-30

    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 in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

  4. Origin assessment of EV olive oils by esterified sterols analysis.

    PubMed

    Giacalone, Rosa; Giuliano, Salvatore; Gulotta, Eleonora; Monfreda, Maria; Presti, Giovanni

    2015-12-01

    In this study extra virgin olive oils of Italian and non-Italian origin (from Spain, Tunisia and blends of EU origin) were differentiated by GC-FID analysis of sterols and esterified sterols followed by chemometric tools. PCA allowed to highlight the high significance of esterified sterols to characterise extra virgin olive oils in relation to their origin. SIMCA provided a sensitivity and specificity of 94.39% and 91.59% respectively; furthermore, an external set of 54 extra virgin olive oils bearing a designation of Italian origin on the labelling was tested by SIMCA. Prediction results were also compared with organoleptic assessment. Finally, the poor correlation found between ethylesters and esterified sterols allowed to hazard the guess, worthy of further investigations, that esterified sterols may prove to be promising in studies of geographical discrimination: indeed they appear to be independent of those factors causing the formation of ethyl esters and related to olive oil production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Multicolor bleach-rate imaging enlightens in vivo sterol transport

    PubMed Central

    Sage, Daniel

    2010-01-01

    Elucidation of in vivo cholesterol transport and its aberrations in cardiovascular diseases requires suitable model organisms and the development of appropriate monitoring technology. We recently presented a new approach to visualize transport of the intrinsically fluorescent sterol, 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 position, which can be used as a fingerprint to distinguish rapidly bleaching DHE from slowly bleaching autofluorescence in the animals. Here, we introduce multicolor bleach-rate sterol imaging. By this method, we demonstrate that some DHE is targeted to a population of basolateral recycling endosomes (RE) labelled with GFP-tagged RME-1 (GFP-RME-1) in the intestine of both, wild-type nematodes and mutant animals lacking intestinal gut granules (glo1-mutants). DHE-enriched intestinal organelles of glo1-mutants were decorated with GFPrme8, a marker for early endosomes. No co-localization was found with a lysosomal marker, GFP-LMP1. Our new methods hold great promise for further studies on endosomal sterol transport in C. elegans. PMID:20798830

  6. Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum.

    PubMed

    Fabris, Michele; Matthijs, Michiel; Carbonelle, Sophie; Moses, Tessa; Pollier, Jacob; Dasseville, Renaat; Baart, Gino J E; Vyverman, Wim; Goossens, Alain

    2014-11-01

    Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified. Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches. Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme. The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals.

  7. An unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the C. elegans putative aminophospholipid translocases.

    PubMed

    Lyssenko, Nicholas N; Miteva, Yana; Gilroy, Simon; Hanna-Rose, Wendy; Schlegel, Robert A

    2008-10-02

    P-type ATPases in subfamily IV are exclusively eukaryotic transmembrane proteins that have been proposed to directly translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine from the exofacial to the cytofacial monolayer of the plasma membrane. Eukaryotic genomes contain many genes encoding members of this subfamily. At present it is unclear why there are so many genes of this kind per organism or what individual roles these genes perform in organism development. We have systematically investigated expression and developmental function of the six, tat-1 through 6, subfamily IV P-type ATPase genes encoded in the Caenorhabditis elegans genome. tat-5 is the only ubiquitously-expressed essential gene in the group. tat-6 is a poorly-transcribed recent duplicate of tat-5. tat-2 through 4 exhibit tissue-specific developmentally-regulated expression patterns. Strong expression of both tat-2 and tat-4 occurs in the intestine and certain other cells of the alimentary system. The two are also expressed in the uterus, during spermatogenesis and in the fully-formed spermatheca. tat-2 alone is expressed in the pharyngeal gland cells, the excretory system and a few cells of the developing vulva. The expression pattern of tat-3 is almost completely different from those of tat-2 and tat-4. tat-3 expression is detectable in the steroidogenic tissues: the hypodermis and the XXX cells, as well as in most cells of the pharynx (except gland), various tissues of the reproductive system (except uterus and spermatheca) and seam cells. Deletion of tat-1 through 4 individually interferes little or not at all with the regular progression of organism growth and development under normal conditions. However, tat-2 through 4 become essential for reproductive growth during sterol starvation. tat-5 likely encodes a housekeeping protein that performs the proposed aminophospholipid translocase function routinely. Although individually dispensable, tat-1 through 4 seem to be

  8. Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice

    PubMed Central

    Aqul, Amal; Lopez, Adam M.; Posey, Kenneth S.; Taylor, Anna M.; Repa, Joyce J.; Burns, Dennis K.

    2014-01-01

    Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal−/− and matching lal+/+ mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal−/− mice sequestered cholesterol at an average rate of 3.2 mg·day−1·animal−1. The proportion of the body sterol pool contained in the liver of the lal−/− mice was 64 vs. 6.3% in their lal+/+ controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal−/− mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal−/− mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal+/+ controls. The rate of cholesterol synthesis in the lal−/− mice exceeded that in the lal+/+ controls by 3.7 mg·day−1·animal−1. Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal−/− mice, but their rate of neutral sterol excretion was 59% higher than in their lal+/+ controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal−/− mice was 355 days. PMID:25147230

  9. Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice.

    PubMed

    Aqul, Amal; Lopez, Adam M; Posey, Kenneth S; Taylor, Anna M; Repa, Joyce J; Burns, Dennis K; Turley, Stephen D

    2014-10-15

    Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal(-/-) and matching lal(+/+) mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal(-/-) mice sequestered cholesterol at an average rate of 3.2 mg·day(-1)·animal(-1). The proportion of the body sterol pool contained in the liver of the lal(-/-) mice was 64 vs. 6.3% in their lal(+/+) controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal(-/-) mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal(-/-) mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal(+/+) controls. The rate of cholesterol synthesis in the lal(-/-) mice exceeded that in the lal(+/+) controls by 3.7 mg·day(-1)·animal(-1). Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal(-/-) mice, but their rate of neutral sterol excretion was 59% higher than in their lal(+/+) controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal(-/-) mice was 355 days.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Comparative seasonal sterol profiles in edible parts of Mediterranean fish and shellfish species.

    PubMed

    Ozyurt, Gülsün; Kuley, Esmeray; Etyemez, Miray; Ozoğul, Fatih

    2013-06-01

    The effect of different seasons on sterol content of seafoods was investigated. There were four sterols (cholesterol, sitosterol, desmosterol and stigmasterol) identified, with cholesterol being the predominant sterol. Stigmasterol was a minor component in fish muscle, whilst sitosterol was one of the main phytosterols found in fish muscle. Cholesterol content of fish consisted of 38-100% of total sterols in fish and 54-80% of total sterols in shellfish. The highest cholesterol content of fish muscle was found in summer and the lowest in autumn, whereas season did not have any effect on cholesterol level of green tiger prawn and speckled shrimp. Total sterol content of fish muscle ranged from 49 to 110 mg/100 g, although the range of total sterols in shrimp muscle was between 62 and 91 mg/100 g. The result of the study showed that total sterols in fish were generally found at lower levels in winter compared with other seasons.

  12. Sterols and sphingolipids: Dynamic duo or partners in crime?

    PubMed Central

    Gulati, Sonia; Liu, Ying; Munkacsi, Andrew B.; Wilcox, Lisa; Sturley, Stephen L.

    2010-01-01

    One manner in which eukaryotic cells respond to their environments is by optimizing the composition and proportions of sterols and sphingolipids in membranes. The physical association of the planar ring of sterols with the acyl chains of phospholipids, particularly sphingolipids, produces membrane micro-heterogeneity that is exploited to coordinate several crucial pathways. We hypothesize that these lipid molecules play an integrated role in human disease; when one of the partners is mis-regulated, pathology frequently ensues. Sterols and sphingolipid levels are not coordinated by the action of a single master regulator, however the cross talk between their metabolic pathways is considerable. We describe our perspectives on the key components of synthesis, catabolism and transport of these lipid partners with an emphasis on evolutionarily conserved reactions that produce disease states when defective. PMID:20362613

  13. New Marine Sterols from a Gorgonian Pinnigorgia sp.

    PubMed

    Chang, Yu-Chia; Hwang, Tsong-Long; Chao, Chih-Hua; Sung, Ping-Jyun

    2017-03-03

    Continuous chemical investigation of the gorgonian coral Pinnigorgia sp. resulted in the isolation of two new sterols, 5α,6α-epoxy-(22E,24R)-3β,11-dihydroxy-9,11-secoergosta-7-en-9-one (1) and (22R)-acetoxy-(24ξ)-ergosta-5-en-3β,25-diol (2). The structures of sterols 1 and 2 were elucidated using spectroscopic methods. Sterol 1 displayed inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils with IC50 values of 8.65 and 5.86 μM, respectively. The structure of a known metabolite, pubinernoid A (3), is revised as (+)-loliolide (4).

  14. Sterol and genomic analyses validate the sponge biomarker hypothesis

    PubMed Central

    Gold, David A.; Grabenstatter, Jonathan; de Mendoza, Alex; Riesgo, Ana; Ruiz-Trillo, Iñaki

    2016-01-01

    Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650–540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago. PMID:26903629

  15. Sterol and genomic analyses validate the sponge biomarker hypothesis.

    PubMed

    Gold, David A; Grabenstatter, Jonathan; de Mendoza, Alex; Riesgo, Ana; Ruiz-Trillo, Iñaki; Summons, Roger E

    2016-03-08

    Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650-540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago.

  16. Properties, structure, and applications of microbial sterol esterases.

    PubMed

    Vaquero, Maria Eugenia; Barriuso, Jorge; Martínez, María Jesús; Prieto, Alicia

    2016-03-01

    According to their substrate preferences, carboxylic ester hydrolases are organized in smaller clusters. Among them, sterol esterases (EC 3.1.1.13), also known as cholesterol esterases, act on fatty acid esters of cholesterol and other sterols in aqueous media, and are also able to catalyze synthesis by esterification or transesterification in the presence of organic solvents. Mammalian cholesterol esterases are intracellular enzymes that have been extensively studied since they are essential in lipid metabolism and cholesterol absorption, and the natural role of some microbial sterol esterases is supposed to be similar. However, besides these intracellular enzymes, a number of microbes produce extracellular sterol esterases, which show broad stability, selectivity, or wide substrate specificity, making them interesting for the industry. In spite of this, there is little information about microbial sterol esterases, and only a small amount of them have been characterized. Some of the most commercially exploited cholesterol esterases are produced by Pseudomonas species and by Candida rugosa, although in the last case they are usually described and named as "high substrate versatility lipases." From a structural point of view, most of them belong to the α/β-hydrolase superfamily and have a conserved "catalytic triad" formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. In this review, the information available on microbial sterol esterases has been gathered, taking into account their origin, production and purification, heterologous expression, structure, stability, or substrate specificity, which are the main properties that make them attractive for different applications. Moreover, a comprehensive phylogenetic analysis on available sequences of cholesterol esterases has been done, including putative sequences deduced from public genomes.

  17. Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

    PubMed Central

    Ness, Frédérique; Achstetter, Tilman; Duport, Catherine; Karst, Francis; Spagnoli, Roberto; Degryse, Eric

    1998-01-01

    The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs. PMID:9537392

  18. Non-cholesterol sterols in serum, lipoproteins, and red cells in statin-treated FH subjects off and on plant stanol and sterol ester spreads.

    PubMed

    Ketomäki, Anna; Gylling, Helena; Miettinen, Tatu A

    2005-03-01

    Serum plant sterol levels are increased by consumption of statins and dietary plant sterols, and decreased by dietary plant stanols, but little is known about combination therapy of statin and plant sterols. We measured plant sterols in serum, lipoproteins, and red cells in subjects with familial hypercholesterolemia (FH) (n=18) treated with variable doses of statins off and on plant stanol (STA) and sterol ester (STE) spreads. STA and STE spreads lowered LDL cholesterol approximately 15%. Plant sterols were decreased in serum, lipoproteins, and red cells by approximately 25% with STA and increased from 37% to 80% with STE, especially with high statin doses. The changes in serum were related to those in red cells. The baseline levels of serum plant sterols were negatively (r-range -0.639 to -0.935) and positively (r-range 0.526 to 0.598) correlated with the respective changes evoked by the STA and STE spreads. STE reduces LDL cholesterol, but increases serum, lipoprotein, and red cell plant sterol levels in statin-treated FH subjects, while all the respective values are decreased with STA. Recent predictions that elevated serum plant sterols pose an increased coronary risk suggest that increases of serum plant sterol levels should be avoided, especially in atherosclerosis-prone individuals, such as subjects with FH.

  19. Protein Mediators of Sterol Transport Across Intestinal Brush Border Membrane

    PubMed Central

    Brown, J. Mark; Yu, Liqing

    2012-01-01

    Dysregulation of cholesterol balance contributes significantly to atherosclerotic cardiovascular disease (ASCVD), the leading cause of death in the United States. The intestine has the unique capability to act as a gatekeeper for entry of cholesterol into the body, and inhibition of intestinal cholesterol absorption is now widely regarded as an attractive non-statin therapeutic strategy for ASCVD prevention. In this chapter we discuss the current state of knowledge regarding sterol transport across the intestinal brush border membrane. The purpose of this work is to summarize substantial progress made in the last decade in regards to protein-mediated sterol trafficking, and to discuss this in the context of human disease. PMID:20213550

  20. Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

    PubMed Central

    de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2009-01-01

    Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14α-demethylase, and (f) azasterols, which inhibit Δ24(25)-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take place in

  1. 4-Methyl Sterols Regulate Fission Yeast SREBP-Scap under Low Oxygen and Cell Stress*

    PubMed Central

    Hughes, Adam L.; Lee, Chih-Yung S.; Bien, Clara M.; Espenshade, Peter J.

    2008-01-01

    In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterize the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol. PMID:17595166

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

    Treesearch

    Rachel A. Stong; Eli Kolodny; Rick G. Kelsey; M.P. Gonzalez-Hernandez; Jorge M. Vivanco; Daniel K. Manter

    2013-01-01

    Elicitin-mediated acquisition of plant sterols is required for growth and sporulation of Phytophthora spp. This study examined the interactions between elicitins, sterols, and tannins. Ground leaf tissue, sterols, and tannin-enriched extracts were obtained from three different plant species (California bay laurel, California black oak, and Oregon...

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

    PubMed

    Piepho, Maike; Martin-Creuzburg, Dominik; Wacker, Alexander

    2010-12-31

    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.

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

    PubMed

    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.

  5. Plant Sterol Metabolism. Δ7-Sterol-C5-Desaturase (STE1/DWARF7), Δ5,7-Sterol-Δ7-Reductase (DWARF5) and Δ24-Sterol-Δ24-Reductase (DIMINUTO/DWARF1) Show Multiple Subcellular Localizations in Arabidopsis thaliana (Heynh) L

    PubMed Central

    Silvestro, Daniele; Andersen, Tonni Grube; Schaller, Hubert; Jensen, Poul Erik

    2013-01-01

    Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ7-sterol-C5-desaturase (STE1/DWARF7), Δ24-sterol-Δ24-reductase (DIMINUTO/DWARF1) and Δ5,7-sterol-Δ7-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into Arabidopsis thaliana mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ5,7-sterol-Δ7-reductase and Δ24-sterol-Δ24-reductase are in addition localized to the plasma membrane, whereas Δ7-sterol-C5-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants. PMID:23409184

  6. Plant sterol metabolism. Δ(7)-Sterol-C5-desaturase (STE1/DWARF7), Δ(5,7)-sterol-Δ(7)-reductase (DWARF5) and Δ(24)-sterol-Δ(24)-reductase (DIMINUTO/DWARF1) show multiple subcellular localizations in Arabidopsis thaliana (Heynh) L.

    PubMed

    Silvestro, Daniele; Andersen, Tonni Grube; Schaller, Hubert; Jensen, Poul Erik

    2013-01-01

    Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ(7)-sterol-C(5)-desaturase (STE1/DWARF7), Δ(24)-sterol-Δ(24)-reductase (DIMINUTO/DWARF1) and Δ(5,7)-sterol-Δ(7)-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into Arabidopsis thaliana mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ(5,7)-sterol-Δ(7)-reductase and Δ(24)-sterol-Δ(24)-reductase are in addition localized to the plasma membrane, whereas Δ(7)-sterol-C(5)-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.

  7. Transcript profiling of two potato cultivars during glycoalkaloid-inducing treatments shows differential expression of genes in sterol and glycoalkaloid metabolism

    PubMed Central

    Nahar, Nurun; Westerberg, Erik; Arif, Usman; Huchelmann, Alexandre; Olarte Guasca, Alexandra; Beste, Lisa; Dalman, Kerstin; Dutta, Paresh C.; Jonsson, Lisbeth; Sitbon, Folke

    2017-01-01

    Steroidal glycoalkaloids (SGA) are sterol-derived neurotoxic defence substances present in several members of the Solanaceae. In the potato (Solanum tuberosum), high SGA levels may render tubers harmful for consumption. Tuber SGA levels depend on genetic factors, and can increase as a response to certain stresses and environmental conditions. To identify genes underlying the cultivar variation in tuber SGA levels, we investigated two potato cultivars differing in their SGA accumulation during wounding or light exposure; two known SGA-inducing treatments. Using microarray analysis coupled to sterol and SGA quantifications, we identified a small number of differentially expressed genes that were associated with increased SGA levels. Two of these genes, encoding distinct types of sterol Δ24-reductases, were by sense/antisense expression in transgenic potato plants shown to have differing roles in sterol and SGA metabolism. The results show that an increased SGA level in potato tubers during both wounding and light exposure is mediated by coordinated expression of a set of key genes in isoprenoid and steroid metabolism, and suggest that differences in this expression underlie cultivar variations in SGA levels. These results may find use within potato breeding and quality assessment. PMID:28256633

  8. LM cell growth and membrane lipid adaptation to sterol structure.

    PubMed

    Rujanavech, C; Silbert, D F

    1986-06-05

    Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.

  9. The counterflow transport of sterols and PI4P.

    PubMed

    Mesmin, Bruno; Antonny, Bruno

    2016-08-01

    Cholesterol levels in intracellular membranes are constantly adjusted to match with specific organelle functions. Cholesterol is kept high in the plasma membrane (PM) because it is essential for its barrier function, while low levels are found in the endoplasmic reticulum (ER) where cholesterol mediates feedback control of its own synthesis by sterol-sensor proteins. The ER→Golgi→PM concentration gradient of cholesterol in mammalian cells, and ergosterol in yeast, appears to be sustained by specific intracellular transport processes, which are mostly mediated by lipid transfer proteins (LTPs). Here we review a recently described function of two LTPs, OSBP and its yeast homolog Osh4p, which consists in creating a sterol gradient between membranes by vectorial transport. OSBP also contributes to the formation of ER/Golgi membrane contact sites, which are important hubs for the transfer of several lipid species. OSBP and Osh4p organize a counterflow transport of lipids whereby sterols are exchanged for the phosphoinositide PI4P, which is used as a fuel to drive sterol transport. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

  10. Insect molting hormone and sterol biosynthesis in spinach

    SciTech Connect

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

    1990-05-01

    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 (24{alpha}-ethyl-5{alpha}-cholesta-7,24(28)-dien-3{beta}-ol), spinasterol (24{alpha}-ethyl-5{alpha}-cholesta-7,22-dien-3{beta}-ol), and 22-dihydrospinasterol (24{alpha}-ethyl-5{alpha}-cholest-7-en-3{beta}-ol). The major ecdysteroids biosynthesized are ecdysterone (2{beta},3{beta},14{alpha},20R,22R,25-hexahydroxy-5{beta}-cholest-7-en-6-one) and polypodine B (2{beta},3{beta},5{beta},14{alpha},20R,22R,25-heptahycroxycholest-7-en-6-one) and polypodine B (2{beta},3{beta},5{beta},14{alpha},20R,22R,25-heptahydroxycholest-7-en-6-one). When labeled 2-{sup 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 {mu}g 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.

  11. Sterols and squalene in apricot (Prunus armeniaca L.) kernel oils: the variety as a key factor.

    PubMed

    Rudzińska, Magdalena; Górnaś, Paweł; Raczyk, Marianna; Soliven, Arianne

    2017-01-01

    The profile of sterols and squalene content in oils recovered from the kernels of 15 apricot (Prunus armeniaca L.) varieties were investigated. Nine sterols (campesterol, β-sitosterol, Δ5-avenasterol, 24-methylene-cycloartanol, cholesterol, gramisterol, Δ7-stigmasterol, Δ7-avenasterol and citrostadienol) were identified in apricot kernel oils. The β-sitosterol was the predominant sterol in each cultivar and consisted of 76-86% of the total detected sterols. The content of total sterols and squalene were significantly affected by the variety and ranged between 215.7-973.6 and 12.6-43.9 mg/100 g of oil, respectively.

  12. Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971.

    PubMed Central

    Weeks, O B; Francesconi, M D

    1978-01-01

    The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%. Images PMID:101527

  13. Red cell and plasma plant sterols are related during consumption of plant stanol and sterol ester spreads in children with hypercholesterolemia.

    PubMed

    Ketomäki, Anna M; Gylling, Helena; Antikainen, Marjatta; Siimes, Martti A; Miettinen, Tatu A

    2003-05-01

    To show whether the ratios of squalene and cholesterol precursor sterols to cholesterol and cholestanol and plant sterols to cholesterol change differently in plasma and especially in the red cells of hypercholesterolemic children during consumption of plant stanol and sterol ester spreads. In a randomized, double-blind, crossover study, hypercholesterolemic children (n = 23) consumed low-fat plant stanol and sterol ester spreads for 5-week periods separated by a 5-week washout period. Plasma and red cell lipids, squalene, and noncholesterol sterols were measured before and at the end of each period. The plant stanol and sterol ester spreads lowered plasma total (-9% and -6%, respectively) and low-density lipoprotein (-12% and -9%) cholesterol but had no effect on red cell cholesterol, high-density lipoprotein cholesterol, or plasma triglycerides. The ratios of plasma and red cell sitosterol and campesterol to cholesterol decreased by 32% to 36% (P <.001) with the plant stanol ester and increased by 40% to 52% (P <.001) with the sterol ester spread. Consumption of plant sterols increases and consumption of plant stanols decreases the ratios of plant sterols to cholesterol in red cells of hypercholesterolemic children proportionately to the respective changes in plasma.

  14. Free Fatty acids and sterols in swine manure.

    PubMed

    Loughrin, John H; Szogi, Ariel A

    2006-01-01

    Free fatty acids and sterols were assessed in fresh manure and anaerobic lagoon sludge from swine production facilities in North Carolina. Eight free fatty acids and five sterols were identified and quantified in both manure and sludge samples. Compound identification was performed by gas chromatography/mass spectroscopy (GC-MS), and compound quantities were determined by gas chromatography after solid phase extraction with a 50:50 mixture of diethyl ether and hexane. The free fatty acids occurring in greatest abundance in both fresh manure and lagoon sludge were palmitic, oleic, and stearic. Free fatty acid content in fresh manure ranged from approximately 3 microg g(-1) dry weight (dw) to over 45 microg g(-1) dw. In lagoon sludge, free fatty acid content ranged from about 0.8 microg g(-1) dw to nearly 4 microg g(-1) dw. Coprostanol and epicoprostanol were the sterols in largest concentrations in fresh manure and lagoon sludge samples. Total sterol content ranged from approximately 0.5 microg g(-1) dw to around 11 microg g(-1) dw in fresh manure and from 3.5 microg g(-1) dw to almost 9 microg g(-1) dw in lagoon sludge. Fresh manure and lagoon sludge both had high levels of inorganic cations (e.g., Ca, Mg, Fe) capable of binding free fatty acids and forming insoluble complexes, thereby potentially reducing fatty acid biodegradation. In anaerobic lagoons, sterols are an organic fraction of sludge that are resistant to bacterial degradation. In the case of fresh manure, fatty acids could represent a potential source of energy via the manufacture of biodiesel fuel, if efficient means for their extraction and transesterification can be devised.

  15. An SREBP Responsive micro-RNA Operon Contributes to a Regulatory Loop for Intracellular Lipid Homeostasis

    PubMed Central

    Jeon, Tae-Il; Esquejo, Ryan M.; Roqueta-Rivera, Manuel; Phelan, Peter E.; Moon, Young-Ah; Govindarajan, Subramaniam S.; Esau, Christine C.; Osborne, Timothy F.

    2013-01-01

    Sterol regulatory element binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic micro-RNA locus that is activated directly by SREBP-2. Two of the encoded miRs, miR-182 and miR-96, negatively regulate expression of Fbxw7 and Insig-2 respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miR pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a new mechanism for regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRs that are expressed from the same SREBP-2 regulated miR locus and each targets a different protein of the multi-step pathway that regulates SREBP function. These studies reveal a miR “operon” analogous to the classic model for genetic control in bacterial regulatory systems. PMID:23823476

  16. Current and new insights on phytosterol oxides in plant sterol-enriched food.

    PubMed

    García-Llatas, Guadalupe; Rodríguez-Estrada, María Teresa

    2011-09-01

    Over the past 15 years, plant sterol-enriched foods have faced a great increase in the market, due to the asserted cholesterol-lowering effect of plant sterols. However, owing to their chemical structures, plant sterols can oxidize and produce a wide variety of oxidation products with controversial biological effects. Although oxyphytosterols can derive from dietary sources and endogenous formation, their single contribution should be better defined. The following review provides an overall and critical picture on the current knowledge and future perspectives of plant sterols-enriched food, particularly focused on occurrence of plant sterol oxidation products and their biological effects. The final objective of this overview is to evince the different aspects of plant sterols-enriched food that require further research, for a better understanding of the influence of plant sterols and their oxides on consumers' health.

  17. Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

    PubMed

    Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

    2015-12-01

    Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides

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

    PubMed Central

    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

  19. Higher sterol content regulated by CYP51 with concomitant lower phospholipid content in membranes is a common strategy for aluminium tolerance in several plant species

    PubMed Central

    Wagatsuma, Tadao; Khan, Md. Shahadat Hossain; Watanabe, Toshihiro; Maejima, Eriko; Sekimoto, Hitoshi; Yokota, Takao; Nakano, Takeshi; Toyomasu, Tomonobu; Tawaraya, Keitaro; Koyama, Hiroyuki; Uemura, Matsuo; Ishikawa, Satoru; Ikka, Takashi; Ishikawa, Akifumi; Kawamura, Takeshi; Murakami, Satoshi; Ueki, Nozomi; Umetsu, Asami; Kannari, Takayuki

    2015-01-01

    Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al3+ ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species. PMID:25416794

  20. Higher sterol content regulated by CYP51 with concomitant lower phospholipid content in membranes is a common strategy for aluminium tolerance in several plant species.

    PubMed

    Wagatsuma, Tadao; Khan, Md Shahadat Hossain; Watanabe, Toshihiro; Maejima, Eriko; Sekimoto, Hitoshi; Yokota, Takao; Nakano, Takeshi; Toyomasu, Tomonobu; Tawaraya, Keitaro; Koyama, Hiroyuki; Uemura, Matsuo; Ishikawa, Satoru; Ikka, Takashi; Ishikawa, Akifumi; Kawamura, Takeshi; Murakami, Satoshi; Ueki, Nozomi; Umetsu, Asami; Kannari, Takayuki

    2015-02-01

    Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Distribution of sterols and the sources of pollution in surface sediments of Ulungur lake, Xinjiang.

    PubMed

    Yao, Xiaorui; Lu, Jianjiang; Liu, Zilong; Ran, Dan; Huang, Yating

    2013-01-01

    Domestic sewage discharged into lakes brings great pressure to the ecological environment. This study selected sediment from an inland lake as a research object to evaluate pollution of the environment. Eight sterols were used to evaluate the content of pollutants, while the ratios of sterols were used as the index to analyze the sources of pollution. The correlations were analyzed between sterols and total organic carbon (TOC), salinity and particle size. The distribution and composition of sterol compounds were determined in 12 surface sediment samples collected from Ulungur lake. The total concentrations of detected sterols in the sediments ranged from 1.3 to 36.3 μg/g.dw. The most abundant sterol detected was β-sitosterol (STI) with average concentrations of 2.6 μg/g.dw, followed by cholesterol (CHOE), stigmasterol (STIG) and stigmastanol (STAN). The concentration of coprostanol (COP) was between 0.03 and 1.66 μg/g.dw. Through correlation analysis, it was found that there was a significant correlation between fecal sterols and plant sterols. So the plant sterols shall not be neglected in evaluating the sources of pollution for their impact to identify the fecal sources. The study suggests that the composition and distribution of sterols in surface sediment provide useful information for environmental contamination monitoring and assessment in the inland lake.

  2. Sterol Biosynthesis Is Required for Heat Resistance but Not Extracellular Survival in Leishmania

    PubMed Central

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

    2014-01-01

    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. PMID:25340392

  3. Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane

    PubMed Central

    Iaea, David B.; Mao, Shu; Lund, Frederik W.; Maxfield, Frederick R.

    2017-01-01

    Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12–15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. PMID:28209730

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

    PubMed Central

    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. PMID:28152045

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

    PubMed

    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.

  6. Tracing origins of sewage and organic matter using dissolved sterols in Masan and Haengam Bay, Korea

    NASA Astrophysics Data System (ADS)

    Lee, Hyo Jin; Hong, Sang Hee; Kim, Moonkoo; Ha, Sung Yong; An, Soon Mo; Shim, Won Joon

    2011-06-01

    Masan and Haengam Bays in Korea are highly polluted and semi-enclosed. Domestic and industrial effluents are directly or indirectly discharged into the bays through sewage treatment plants (STP) and creeks. In this study, 15 dissolved sterol compounds were determined in order to understand their sources and relative contribution. Freshwater samples were taken from 13 creeks and at two STP sites on a monthly basis. Total dissolved sterol concentrations ranged from 993 to 4158 ng/L. The concentrations of sterols in winter were higher than in summer. Among the sterols analyzed, cholesterol, β-sitosterol, coprostanol and cholestanone were major compounds in creek water. Seawater samples were concurrently collected at 21 stations in Masan Bay. Total sterol concentrations ranged 118-6,956 ng/L. Inner bay showed high concentrations of sterols in summer, while outer bay showed high sterol concentrations in winter. Among the sterols, cholesterol, β-sitosterol and brassicasterol were major compounds in seawater. In order to examine the contribution of urban sewage, the concentration of coprostanol and fecal sterol ratios were calculated. Most of the creek water, inner bay and near STP outlet samples were affected by sewage. Terrestrial organic matters accounted for a high proportion of dissolved organic matter origin. Fecal origins were relatively high in the inner bay areas and in the STP outlet, while sterols of marine origin were high in the outer bay areas.

  7. Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane.

    PubMed

    Iaea, David B; Mao, Shu; Lund, Frederik W; Maxfield, Frederick R

    2017-02-16

    Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and non-vesicular sterol transport processes. Using the fluorescent cholesterol analog, dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 of 12-15 minutes. Approximately 70% of sterol transport is ATP-independent and, therefore, non-vesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. We found that a soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of non-vesicular sterol transport between the plasma membrane and ERC. This study shows that non-vesicular sterol transport mechanisms, and STARD4 in particular, account for a large fraction of sterol transport between the plasma membrane and the ERC.

  8. Effects of a diet high in plant sterols, vegetable proteins, and viscous fibers (dietary portfolio) on circulating sterol levels and red cell fragility in hypercholesterolemic subjects.

    PubMed

    Jones, Peter J; Raeini-Sarjaz, Mahmoud; Jenkins, David J A; Kendall, Cyril W C; Vidgen, Edward; Trautwein, Elke A; Lapsley, Karen G; Marchie, Augustine; Cunnane, Stephen C; Connelly, Philip W

    2005-02-01

    Plant sterols, soy proteins, viscous fibers, and nuts are advised for cholesterol reduction, but their combined effect on plant sterol absorption has never been tested. We assessed their combined action on serum sterols in hyperlipidemic subjects who were following low-saturated fat diets before starting the study and who returned to these diets post-test. The 1-mon test (combination) diet was high in plant sterols (1 g/1,000 kcal), soy protein (23 g/1,000 kcal), viscous fiber (9 g/1,000 kcal), and almonds (14 g/1000 kcal). Fasting blood was obtained for serum lipids and sterols, and erythrocytes were obtained for fragility prior to and at 2-wk intervals during the study. The combination diet raised serum campesterol concentrations by 50% and beta-sitosterol by 27%, although these changes were not significant after Bonferroni correction; near-maximal rises were found by the end of the first week, but no change was found in red cell fragility despite a 29% reduction in the LDL cholesterol level. No significant associations were observed between changes in red cell fragility and blood lipids or sterols. We conclude that plant sterols had a minimal impact on serum sterol concentrations or red cell fragility in hyperlipidemic subjects on diets that greatly reduced their serum lipids.

  9. The effect of variations in phospholipid and sterol structure on the nature of lipid-sterol interactions in lipid bilayer model membranes.

    PubMed

    Mannock, David A; Lewis, Ruthven N A H; McMullen, Todd P W; McElhaney, Ronald N

    2010-06-01

    This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  10. Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

    PubMed Central

    Hossack, J A; Rose, A H

    1976-01-01

    Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8. PMID:776948

  11. StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

    PubMed Central

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

    2014-01-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

  12. StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2014-02-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

  13. New anti-inflammatory sterols from a gorgonian Pinnigorgia sp.

    PubMed

    Su, Yin-Di; Cheng, Ching-Hsiao; Wen, Zhi-Hong; Wu, Yang-Chang; Sung, Ping-Jyun

    2016-07-01

    Chemical investigation on the EtOAc-soluble fraction from the MeOH/DCM extract of a gorgonian Pinnigorgia sp. afforded two new sterols, 11-acetoxy-24S-methyl-3β,5α,6α-trihydroxy-9,11-secocholest-7-en-9-one (1) and 5β,6β-epoxy-(22E,24R)-ergosta-8,22-diene-3β,7β-diol (2). The structures of sterols 1 and 2 were elucidated on the basis of spectroscopic analysis and by comparison of their spectroscopic data with those of related analogues. Both 1 and 2 were shown to significantly inhibit the accumulation of the pro-inflammatory iNOS and COX-2 protein in LPS-stimulated RAW264.7 macrophage cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. [Sterol extracts from Begonia Sinensis Rhizome against respiratory inflammation].

    PubMed

    Yao, Yong; Jiang, Wei; Li, Yu-shan

    2015-08-01

    The acute and chronic respiratory tract inflammation models were made to investigate the effect and mechanism of sterol extracts from Begonia Sinensis Rhizome (BSR). The first model of acute lung injury was made with Kunming mice by inhaling cigarette smoke, then the mice were treated with different concentrations of BSR sterol extracts. Lung tissue morphology was detected by HE staining, TNF-alpha/MPO were detected by Elisa, and cPLA2 protein were, detected by Western blotting respectively. Results showed that in model group, lung sheet became real, alveolar space shrank or disappeared, alveolar septum was thickened, plenty of inflammatory cells were infiltrated, capillary blood vessels were congestive and the expression of TNF-α, MPO, cPLA2 increased; after administration, a small amount of inflammatory cells were infiltrated, alveolar septum became obvious, capillary congestion status was significantly relieved and the expression of TNF-α, MPO, cPLA2 decreased (P < 0.05). The second model of chronic respiratory tract inflammation in BALB/c mice with bronchial asthma was induced by OVA, then the mice were treated with different concentrations of BSR sterol extracts. Lung tissue morphology was detected by HE staining, indexes such as IL-4, IL-5, IL-13 were detected by Elisa, and the cPLA2 protein expression was detected by Western blotting respectively. Results showed that in model group, a lot of inflammatory cells around lung vessels and bronchi exuded, bronchial goblet cells proliferated and the expression of IL-4, IL-5, IL-13, cPLA2 increased; after administration, inflammatory and goblet cell hyperplasia reduced, the expression of IL-4, IL-5, IL-13, cPLA2 also decreased (P < 0.05). The above results showed BSR sterol extracts could resist against respiratory inflammation by inhibiting cPLA2 in a dose-dependent manner.

  15. A lanostane triterpenoid and three cholestane sterols from Tilia kiusiana.

    PubMed

    Shimada, Marie; Ozawa, Masaaki; Iwamoto, Kojiro; Fukuyama, Yoshiyasu; Kishida, Akio; Ohsaki, Ayumi

    2014-01-01

    Kiusianins A-D (1-4) were isolated from the leaves of a Japanese endemic plant, Tilia kiusiana, together with 14 known compounds. The structures of a new lanostane-type triterpenoid 1 and three new cholestane-type sterols 2-4 were elucidated by spectroscopic methods, including two dimensional (2D) NMR. All the compounds isolated were evaluated for their cytotoxicity against two human cancer cell lines, HeLa and HL-60.

  16. Attenuation of Leishmania infantum chagasi metacyclic promastigotes by sterol depletion.

    PubMed

    Yao, Chaoqun; Gaur Dixit, Upasna; Barker, Jason H; Teesch, Lynn M; Love-Homan, Laurie; Donelson, John E; Wilson, Mary E

    2013-07-01

    The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.

  17. Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    PubMed Central

    Gaur Dixit, Upasna; Barker, Jason H.; Teesch, Lynn M.; Love-Homan, Laurie; Donelson, John E.; Wilson, Mary E.

    2013-01-01

    The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. PMID:23630964

  18. Distribution of free and glycosylated sterols within Cycas micronesica plants.

    PubMed

    Marler, Thomas E; Shaw, Christopher A

    2010-02-02

    Flour derived from Cycas micronesica seeds was once the dominant source of starch for Guam's residents. Cycad consumption has been linked to high incidence of human neurodegenerative diseases. We determined the distribution of the sterols stigmasterol and β-sitosterol and their derived glucosides stigmasterol β-d-glucoside and β-sitosterol β-d-glucoside among various plant parts because they have been identified in cycad flour and have been shown to elicit neurodegenerative outcomes. All four compounds were common in seeds, sporophylls, pollen, leaves, stems, and roots. Roots contained the greatest concentration of both free sterols, and photosynthetic leaflet tissue contained the greatest concentration of both steryl glucosides. Concentration within the three stem tissue categories was low compared to other organs. Reproductive sporophyll tissue contained free sterols similar to seeds, but greater concentration of steryl glucosides than seeds. One of the glucosides was absent from pollen. Concentration in young seeds was higher than old seeds as reported earlier, but concentration did not differ among age categories of leaf, sporophyll, or vascular tissue. The profile differences among the various tissues within these organs may help clarify the physiological role of these compounds.

  19. Targeting Trypanosoma cruzi Sterol 14α-Demethylase (CYP51)

    PubMed Central

    Lepesheva, Galina I.; Villalta, Fernando; Waterman, Michael R.

    2012-01-01

    There are at least two obvious features that must be considered upon targeting specific metabolic pathways/enzymes for drug development: the pathway must be essential and the enzyme must allow the design of pharmacologically useful inhibitors. Here, we describe Trypanosoma cruzi sterol 14α-demethylase as a promising target for anti-Chagasic chemotherapy. The use of anti-fungal azoles, which block sterol biosynthesis and therefore membrane formation in fungi, against the protozoan parasite has turned out to be highly successful: a broad spectrum anti-fungal drug, the triazole compound posaconazole, is now entering phase II clinical trials for treatment of Chagas disease. This review summarizes comparative information on anti-fungal azoles and novel inhibitory scaffolds selective for Trypanosomatidae sterol 14α-demethylase through the lens of recent structure/functional characterization of the target enzyme. We believe our studies open wide opportunities for rational design of novel, pathogen-specific and therefore more potent and efficient anti-trypanosomal drugs. PMID:21820552

  20. Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

    PubMed

    Makushok, Tatyana; Alves, Paulo; Huisman, Stephen Michiel; Kijowski, Adam Rafal; Brunner, Damian

    2016-05-19

    Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.

  1. Dietary phosphilipids and sterols protective against peptic ulceration.

    PubMed

    Tovey, F I; Bardhan, K D; Hobsley, M

    2013-09-01

    The prevalence of duodenal ulceration in regions of developing countries with a stable diet is related to the staple food(s) in that diet. A higher prevalence occurs in areas where the diet is principally milled rice, refined wheat or maize, yams, cassava, sweet potato or green bananas, and a lower prevalence in areas where the staple diet is based on unrefined wheat or maize, soya, certain millets or certain pulses. Experiments using animal peptic ulcer models showed that the lipid fraction in foods from the staple diets of low prevalence areas gave protection against both gastric and duodenal ulceration, including ulceration due to non-steroidal anti-inflammatory drugs (NSAIDs), and also promoted healing of ulceration. The protective activity was found to lie in the phospholipid, sterol and sterol ester fractions of the lipid. Amongst individual phospholipids present in the phospholipid fraction, phosphatidyl ethanolamine (cephalin) and phosphatidyl choline (Lecithin) predominated. The sterol fraction showing activity contained β-sitosterol, stigmasterol and an unidentified isomer of β-sitosterol. The evidence shows that dietary phytosterols and phospholipids, both individually and in combination, have a protective effect on gastroduodenal mucosa. These findings may prove to be important in the prevention and management of duodenal and gastric ulceration including ulceration due to NSAIDs.

  2. Building Synthetic Sterols Computationally – Unlocking the Secrets of Evolution?

    PubMed Central

    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 cholesterol. Practical applications of cholesterol include its use in liposomes in drug delivery and cosmetics, cholesterol-based detergents in membrane protein crystallography, its fluorescent analogs in studies of cholesterol transport in cells and tissues, etc. 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 non-existent 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. PMID:26347865

  3. Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase (SAM:SMT)

    PubMed Central

    Kaneshiro, Edna S.; Johnston, Laura Q.; Nkinin, Stephenson W.; Romero, Becky I.; Giner, José-Luis

    2014-01-01

    The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The P. carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance spectroscopy (1H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ24(28)-sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii. PMID:25230683

  4. Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve.

    PubMed

    Basen, Timo; Rothhaupt, Karl-Otto; Martin-Creuzburg, Dominik

    2012-09-01

    The accumulation of cyanobacterial biomass may severely affect the performance of aquatic consumers. Here, we investigated the role of sterols in determining the food quality of cyanobacteria for the invasive clam Corbicula fluminea, which has become a common benthic invertebrate in many freshwater ecosystems throughout the world. In standardized growth experiments, juvenile clams were fed mixtures of different cyanobacteria (Anabaena variabilis, Aphanothece clathrata, Synechococcus elongatus) or sterol-containing eukaryotic algae (Cryptomonas sp., Nannochloropsis limnetica, Scenedesmus obliquus). In addition, the cyanobacterial food was supplemented with different sterols. We provide evidence that somatic growth of C. fluminea on cyanobacterial diets is constrained by the absence of sterols, as indicated by a growth-enhancing effect of sterol supplementation. Thus, our findings contribute to our understanding of the consequences of cyanobacterial mass developments for benthic consumers and highlight the importance of considering sterols as potentially limiting nutrients in aquatic food webs.

  5. Bilayer Interactions among Unsaturated Phospholipids, Sterols, and Ceramide.

    PubMed

    Slotte, J Peter; Yasuda, Tomokazu; Engberg, Oskar; Al Sazzad, Md Abdullah; Hautala, Victor; Nyholm, Thomas K M; Murata, Michio

    2017-04-25

    Using differential scanning calorimetry and lifetime analysis of trans-parinaric acid fluorescence, we have examined how cholesterol and cholesteryl phosphocholine (CholPC) affect gel-phase properties of palmitoyl ceramide (PCer) in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC) bilayers. By (2)H NMR, we also measured fluid-phase interactions among these lipids using deuterated analogs of POPC, PCer, and cholesterol. The PCer-rich gel phase in POPC bilayers (9:1 molar ratio of POPC to PCer) was partially and similarly dissolved (and thermostability decreased) by both cholesterol and CholPC (sterol was present equimolar to PCer, or in fourfold excess). In DOPC bilayers (4:1 DOPC/PCer molar ratio), CholPC was much more efficient in dissolving the PCer-rich gel phase when compared to cholesterol. This can be interpreted as indicating that PCer interaction with POPC was stronger than PCer interaction with DOPC. PCer-CholPC interactions were also more favored in DOPC bilayers compared to POPC bilayers. In the fluid POPC-rich phase, cholesterol increased the order of the acyl chain of d2-PCer much more than did CholPC. In DOPC-rich fluid bilayers, both cholesterol and CholPC increased d2-PCer acyl chain order, and the ordering induced by CholPC was more efficient in DOPC than in POPC bilayers. In fluid POPC bilayers, the ordering of 3-d1-cholesterol by PCer was weak. In summary, we found that in the gel phase, sterol effects on the PCer-rich gel phase were markedly influenced by the acyl chain composition of the fluid PC. The same was true for fluid-phase interactions involving the sterols. Our results further suggest that PCer did not display high affinity toward either of the sterols used. We conclude that the nature of unsaturated phospholipids (POPC versus DOPC) in bilayers has major effects on the properties of ceramide gel phases and on sterol-ceramide-phospholipid interactions in such complex bilayers

  6. Sterol O-Acyltransferase 2-Driven Cholesterol Esterification Opposes Liver X Receptor-Stimulated Fecal Neutral Sterol Loss

    PubMed Central

    Warrier, Manya; Zhang, Jun; Bura, Kanwardeep; Kelley, Kathryn; Wilson, Martha D.; Rudel, Lawrence L.; Brown, J. Mark

    2016-01-01

    Statin drugs have proven a successful and relatively safe therapy for the treatment of atherosclerotic cardiovascular disease (CVD). However, even with the substantial low-density lipoprotein (LDL) cholesterol lowering achieved with statin treatment, CVD remains the top cause of death in developed countries. Selective inhibitors of the cholesterol esterifying enzyme sterol-O acyltransferase 2 (SOAT2) hold great promise as effective CVD therapeutics. In mouse models, previous work has demonstrated that either antisense oligonucleotide (ASO) or small molecule inhibitors of SOAT2 can effectively reduce CVD progression, and even promote regression of established CVD. Although it is well known that SOAT2-driven cholesterol esterification can alter both the packaging and retention of atherogenic apoB-containing lipoproteins, here we set out to determine whether SOAT2-driven cholesterol esterification can also impact basal and liver X receptor (LXR)-stimulated fecal neutral sterol loss. These studies demonstrate that SOAT2 is a negative regulator of LXR-stimulated fecal neutral sterol loss in mice. PMID:26729489

  7. Sterol O-Acyltransferase 2-Driven Cholesterol Esterification Opposes Liver X Receptor-Stimulated Fecal Neutral Sterol Loss.

    PubMed

    Warrier, Manya; Zhang, Jun; Bura, Kanwardeep; Kelley, Kathryn; Wilson, Martha D; Rudel, Lawrence L; Brown, J Mark

    2016-02-01

    Statin drugs have proven a successful and relatively safe therapy for the treatment of atherosclerotic cardiovascular disease (CVD). However, even with the substantial low-density lipoprotein (LDL) cholesterol lowering achieved with statin treatment, CVD remains the top cause of death in developed countries. Selective inhibitors of the cholesterol esterifying enzyme sterol-O acyltransferase 2 (SOAT2) hold great promise as effective CVD therapeutics. In mouse models, previous work has demonstrated that either antisense oligonucleotide (ASO) or small molecule inhibitors of SOAT2 can effectively reduce CVD progression, and even promote regression of established CVD. Although it is well known that SOAT2-driven cholesterol esterification can alter both the packaging and retention of atherogenic apoB-containing lipoproteins, here we set out to determine whether SOAT2-driven cholesterol esterification can also impact basal and liver X receptor (LXR)-stimulated fecal neutral sterol loss. These studies demonstrate that SOAT2 is a negative regulator of LXR-stimulated fecal neutral sterol loss in mice.

  8. The physiology of sterol nutrition in the pea aphid Acyrthosiphon pisum.

    PubMed

    Bouvaine, Sophie; T Behmer, Spencer; Lin, George G; Faure, Marie-Line; Grebenok, Robert J; Douglas, Angela E

    2012-11-01

    The phloem sap of fava bean (Vicia faba) plants utilized by the pea aphid Acyrthosiphon pisum contains three sterols, cholesterol, stigmasterol and sitosterol, in a 2:2:1 ratio. To investigate the nutritional value of these sterols, pea aphids were reared on chemically-defined diets containing each sterol at 0.1, 1 and 10μgml(-1) with a sterol-free diet as control. Larval growth rate and aphid lifespan did not vary significantly across the diets, indicating that sterol reserves can buffer some performance indices against a shortfall in dietary sterol over at least one generation. However, lifetime reproductive output was depressed in aphids on diets containing stigmasterol or no sterol, relative to diets supplemented with cholesterol or sitosterol. The cholesterol density of embryos in teneral adults was significantly higher than in the total body; and the number and biomass of embryos in aphids on diets with stigmasterol and no sterols were reduced relative to diets with cholesterol or sitosterol, indicating that the reproductive output of the pea aphid can be limited by the amount and composition of dietary sterol. In a complementary RNA-seq analysis of pea aphids reared on plants and diets with different sterol contents, 7.6% of the 17,417 detected gene transcripts were differentially expressed. Transcript abundance of genes with annotated function in sterol utilization did not vary significantly among treatments, suggesting that the metabolic response to dietary sterol may be mediated primarily at the level of enzyme function or metabolite concentration. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Factors affecting intestinal absorption of cholesterol and plant sterols and stanols.

    PubMed

    Ikeda, Ikuo

    2015-01-01

    Various factors affect intestinal absorption of cholesterol and plant sterols and stanols. Plant sterols and stanols are generally less absorptive than cholesterol. Differential absorption rates among various plant sterols and stanols have been also reported. Although it was suggested that differential absorption among cholesterol and various plant sterols was determined by difference in excretion rates of sterols and stanols through ATP-binding cassette transporter (ABC) G5/ABCG8 of intestinal cells, our study suggests that affinity for and solubility in bile salt micelles can be important determinants for differential absorption of plant sterols and stanols. It was also suggested that plant sterols were transiently incorporated into intestinal cells and then excreted to intestinal lumen through ABCG5/ABCG8. However, in a rat study, transient incorporation of sitosterol into intestinal cells was not observed, suggesting that sitosterol is differentiated from cholesterol at the incorporation site of intestinal cells. It is well established that plant sterols inhibit intestinal absorption of cholesterol and exert a hypocholesterolemic activity. Plant sterols are solubilized in bile salt micelles as cholesterol. Our study clearly showed that because the sterol-solubilizing capacity of bile salt micelles was limited, plant sterols solubilized in micelles reduced the solubility of cholesterol. This can be the major cause of inhibition of cholesterol absorption by plant sterols. Pancreatic cholesterol esterase accelerates intestinal absorption of unesterified cholesterol. Although it was suggested that cholesterol esterase accelerated esterification of cholesterol incorporated into intestinal cells and acted as a transporter at the surface of intestinal cells, our research revealed that the accelerated cholesterol absorption was caused by hydrolysis of phosphatidylcholine in bile salt micelles. It is thought that hydrolysis of phosphatidylcholine reduces the affinity of

  10. Bioactive sterols from marine resources and their potential benefits for human health.

    PubMed

    Kim, Se-Kwon; Van Ta, Quang

    2012-01-01

    Bioactive agents from marine resources have shown their valuable health beneficial effects. Therefore, increase knowledge on novel functional ingredients with biological activities from marine animal and microbe has gained much attention. Sterols are recognized as potential in development functional food ingredients and pharmaceutical agents. Marine resources, with a great diversity, can be a very interesting natural resource of sterols. This chapter focuses on biological activities of marine animal and microbe sterols with potential health beneficial applications in functional foods and pharmaceuticals.

  11. Integrating sequence, evolution and functional genomics in regulatory genomics

    PubMed Central

    Vingron, Martin; Brazma, Alvis; Coulson, Richard; van Helden, Jacques; Manke, Thomas; Palin, Kimmo; Sand, Olivier; Ukkonen, Esko

    2009-01-01

    With genome analysis expanding from the study of genes to the study of gene regulation, 'regulatory genomics' utilizes sequence information, evolution and functional genomics measurements to unravel how regulatory information is encoded in the genome. PMID:19226437

  12. Plant sterols, cholesterol precursors and oxysterols: Minute concentrations-Major physiological effects.

    PubMed

    Olkkonen, Vesa M; Gylling, Helena; Ikonen, Elina

    2017-05-01

    Non-cholesterol sterols are present in our body at very low concentrations as compared to cholesterol. Small changes in the structure of sterol molecules confer them highly distinct biological activities. The best-known example are steroid hormones derived from cholesterol. During the past decade, our knowledge of also other biomolecules related to or derived from cholesterol, particularly plant sterols, biosynthetic precursors of cholesterol, and oxysterols, has expanded rapidly. In this review article we recapitulate the latest insights into the properties and physiological activities of these non-cholesterol sterols, as well as their importance in disease processes and potential as diagnostic biomarkers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Progress and prospective of plant sterol and plant stanol research: report of the Maastricht meeting.

    PubMed

    Plat, J; Mackay, D; Baumgartner, S; Clifton, P M; Gylling, H; Jones, P J H

    2012-12-01

    Abundant evidence over past decades shows that foods with added plant sterols and plant stanols lower serum LDL cholesterol concentrations. However, despite the overwhelming data, numerous scientific questions still remain. The objective of this paper is to summarize the considerations of 60 academic and industrial experts who participated in the scientific meeting in Maastricht, the Netherlands, on issues related to the health effects of plant sterols and plant stanols. The meeting participants discussed issues including efficacy profiling, heterogeneity in responsiveness, effects beyond LDL-C lowering, and food formulation aspects of plant sterol and stanol consumption. Furthermore, aspects related to the potential atherogenicity of elevated circulatory plant sterol concentrations were discussed. Until the potential atherogenicity of plant sterols is resolved, based on the results >200 clinical trials, the risk to benefit of plant sterol use is favorable. Evidence on these topics in plant sterol and plant stanol research was presented and used to reach consensus where possible. It was concluded that endpoint studies looking at plant sterol and plant stanol efficacy are needed, however, there was no clear opinion on the best marker and best design for such a study. Based on the current scientific evidence, plant sterols and plant stanols are recommended for use as dietary options to lower serum cholesterol. Copyright © 2012. Published by Elsevier Ireland Ltd.. All rights reserved.

  14. [Sources, Migration and Conversion of Dissolved Sterols in Qingmuguan Underground River].

    PubMed

    Liang, Zuo-bing; Shen, Li-cheng; Sun, Yu-chuan; Wang, Zun-bo; Jiang, Ze-li; Zhang Mei; LIAO, Yu; Xie, Zheng-lan; Zhang, Yuan-zhu

    2015-11-01

    Water samples were collected from the Qinmuguan underground river from July to November in 2013. By gas chromatography-mass spectrometer (GC-MS), dissolved sterols were quantitatively analyzed. The results show that the average variation content of dissolved sterols ranges from 415 to 629 ng x L(-1), with the increasing migration distance of dissolved sterols in underground river, its contents are decreased. Between the inlet and outlet of Qingmuguan underground river, the average variation contents of dissolved sterol are between 724 and 374 ng x L(-1), and the average variation ratios of the content of stigmasterol with cholesterol range from 0.29 to 0.12. In short, their values are decreased accompanied by the increasing migration distance of underground river. The composing component in dissolved sterols varied differently between July to December, and the main component of dissolved sterols is cholesterin, the ratios of the content of dissolved sterols with cholesterin to the total dissolved sterols range from 37.30% to 94.85%. In addition, the ratios of the content of dissolved sterols with coprostanol to cholesterin, coprostanol to cholesterin are below 0.2 respectively, indicating the water quality of underground river is not contaminated by domestic sewage, but with the passage of time water quality tends to deterioration.

  15. An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.

    PubMed

    Jeon, Tae-Il; Esquejo, Ryan M; Roqueta-Rivera, Manuel; Phelan, Peter E; Moon, Young-Ah; Govindarajan, Subramaniam S; Esau, Christine C; Osborne, Timothy F

    2013-07-02

    Sterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA "operon" analogous to the classic model for genetic control in bacterial regulatory systems. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Cloning and functional identification of C-4 methyl sterol oxidase genes from the penicillin-producing fungus Penicillium chrysogenum.

    PubMed

    Wang, Fu-Qiang; Zhao, Ying; Dai, Meng; Liu, Jing; Zheng, Gui-Zhen; Ren, Zhi-Hong; He, Jian-Gong

    2008-10-01

    Two C-4 methyl sterol oxidase genes (Pcerg25A and Pcerg25B) that are involved in ergosterol biosynthesis have been cloned from the penicillin-producing fungus Penicillium chrysogenum. cDNAs of both Pcerg25A and Pcerg25B have an ORF 885 bp in length, encoding a peptide of 295 residues. The deduced amino acid sequences of PcErg25A and PcErg25B show 86% identity, and have high identities to the characterized C-4 methyl sterol oxidases from Candida albicans and Saccharomyces cerevisiae. The function of Pcerg25A and Pcerg25B was identified by complementation of a yeast erg25-deficient strain. Pcerg25A is located in the DNA region containing the penicillin gene cluster, and thus its copy number is dependent on the patterns of the cluster region. Up to eight copies of Pcerg25A were found in the high-productivity strain NCPC 10086. By contrast, Pcerg25B was present in just a single copy in all tested P. chrysogenum genomes. Differences in the transcript level of either Pcerg25A or Pcerg25B were observed in different P. chrysogenum strains by real-time quantitative reverse transcriptase PCR analysis.

  17. SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata

    PubMed Central

    Podduturi, Nikhil R.; Glick, David I.; Baymuradov, Ulugbek K.; Malladi, Venkat S.; Chan, Esther T.; Davidson, Jean M.; Gabdank, Idan; Narayana, Aditi K.; Onate, Kathrina C.; Hilton, Jason; Ho, Marcus C.; Lee, Brian T.; Miyasato, Stuart R.; Dreszer, Timothy R.; Sloan, Cricket A.; Strattan, J. Seth; Tanaka, Forrest Y.; Hong, Eurie L.; Cherry, J. Michael

    2017-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package. PMID:28403240

  18. SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

    PubMed

    Hitz, Benjamin C; Rowe, Laurence D; Podduturi, Nikhil R; Glick, David I; Baymuradov, Ulugbek K; Malladi, Venkat S; Chan, Esther T; Davidson, Jean M; Gabdank, Idan; Narayana, Aditi K; Onate, Kathrina C; Hilton, Jason; Ho, Marcus C; Lee, Brian T; Miyasato, Stuart R; Dreszer, Timothy R; Sloan, Cricket A; Strattan, J Seth; Tanaka, Forrest Y; Hong, Eurie L; Cherry, J Michael

    2017-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.

  19. GLUCAN SYNTHASE-LIKE8 and STEROL METHYLTRANSFERASE2 Are Required for Ploidy Consistency of the Sexual Reproduction System in Arabidopsis[C][W][OA

    PubMed Central

    De Storme, Nico; De Schrijver, Joachim; Van Criekinge, Wim; Wewer, Vera; Dörmann, Peter; Geelen, Danny

    2013-01-01

    In sexually reproducing plants, the meiocyte-producing archesporal cell lineage is maintained at the diploid state to consolidate the formation of haploid gametes. In search of molecular factors that regulate this ploidy consistency, we isolated an Arabidopsis thaliana mutant, called enlarged tetrad2 (et2), which produces tetraploid meiocytes through the stochastic occurrence of premeiotic endomitosis. Endomitotic polyploidization events were induced by alterations in cell wall formation, and similar cytokinetic defects were sporadically observed in other tissues, including cotyledons and leaves. ET2 encodes GLUCAN SYNTHASE-LIKE8 (GSL8), a callose synthase that mediates the deposition of callose at developing cell plates, root hairs, and plasmodesmata. Unlike other gsl8 mutants, in which defects in cell plate formation are seedling lethal, cytokinetic defects in et2 predominantly occur in flowers and have little effect on vegetative growth and development. Similarly, mutations in STEROL METHYLTRANSFERASE2 (SMT2), a major sterol biosynthesis enzyme, also lead to weak cytokinetic defects, primarily in the flowers. In addition, SMT2 allelic mutants also generate tetraploid meiocytes through the ectopic induction of premeiotic endomitosis. These observations demonstrate that appropriate callose and sterol biosynthesis are required for maintaining the ploidy level of the premeiotic germ lineage and that subtle defects in cytokinesis may lead to diploid gametes and polyploid offspring. PMID:23404886

  20. Molecular cloning and biochemical characterization of a recombinant sterol 3-O-glucosyltransferase from Gymnema sylvestre R.Br. catalyzing biosynthesis of steryl glucosides.

    PubMed

    Tiwari, Pragya; Sangwan, Rajender Singh; Asha; Mishra, B N; Sabir, Farzana; Sangwan, Neelam S

    2014-01-01

    Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

  1. The yeast ABC transporter Pdr18 (ORF YNR070w) controls plasma membrane sterol composition, playing a role in multidrug resistance

    PubMed Central

    Cabrito, Tânia R.; Teixeira, Miguel C.; Singh, Ashutosh; Prasad, Rajendra; Sá-Correia, Isabel

    2011-01-01

    The action of multidrug efflux pumps in MDR (multidrug resistance) acquisition has been proposed to partially depend on the transport of physiological substrates which may indirectly affect drug partition and transport across cell membranes. In the present study, the PDR18 gene [ORF (open reading frame) YNR070w], encoding a putative PDR (pleiotropic drug resistance) transporter of the ATP-binding cassette superfamily, was found to mediate plasma membrane sterol incorporation in yeast. The physiological role of Pdr18 is demonstrated to affect plasma membrane potential and is proposed to underlie its action as a MDR determinant, conferring resistance to the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). The action of Pdr18 in yeast tolerance to 2,4-D, which was found to contribute to reduce [14C]2,4-D intracellular accumulation, may be indirect, given the observation that 2,4-D exposure deeply affects the sterol plasma membrane composition, this effect being much stronger in a Δpdr18 background. PDR18 activation under 2,4-D stress is regulated by the transcription factors Nrg1, controlling carbon source availability and the stress response, and, less significantly, Yap1, involved in oxidative stress and MDR, and Pdr3, a key regulator of the yeast PDR network, consistent with a broad role in stress defence. Taken together, the results of the present study suggest that Pdr18 plays a role in plasma membrane sterol incorporation, this physiological trait contributing to an MDR phenotype. PMID:21831043

  2. Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides

    PubMed Central

    Sangwan, Rajender Singh; Asha; Mishra, B. N.; Sangwan, Neelam S.

    2014-01-01

    Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants. PMID:25250339

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

    NASA Astrophysics Data System (ADS)

    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)

  4. Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

    PubMed Central

    Nyström, Joel H.; Lönnfors, Max; Nyholm, Thomas K.M.

    2010-01-01

    Abstract Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins

  5. Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-Delta24-reductase-deficient cell line.

    PubMed

    Rodríguez-Acebes, Sara; de la Cueva, Paloma; Fernández-Hernando, Carlos; Ferruelo, Antonio J; Lasunción, Miguel A; Rawson, Robert B; Martínez-Botas, Javier; Gómez-Coronado, Diego

    2009-05-13

    Cholesterol homoeostasis is critical for cell viability and proliferation. The SREBP (sterol regulatory element-binding protein) pathway is crucial for the maintenance of cholesterol homoeostasis. This pathway is controlled by cholesterol and cholesterol-derived oxysterols. J774 cells cannot convert desmosterol into cholesterol, a defect resulting from the absence of mRNA for sterol-Delta24-reductase. Using J774 cells, we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway. J774 cells were able to grow indefinitely after the virtually total replacement of cholesterol by desmosterol (J774-D cells). Inhibition of sterol biosynthesis with lovastatin suppressed J774-D cell proliferation. Desmosterol prevented this effect, but its analogue, cholest-5,22-trans-dien-3beta-ol, did not. Addition of desmosterol inhibited processing of SREBP-1 and -2 and also reduced the expression of SREBP-targeted genes. As occurs in cholesterol-containing cells, 25-hydroxycholesterol was more potent than desmosterol or cholesterol in suppressing these processes. Moreover, desmosterol addition enhanced the expression of Abca1 and Srebf1c, two LXR (liver X receptor)-targeted genes. To test the ability of endogenously produced desmosterol to regulate gene expression, J774-D cells were pretreated with lovastatin to inhibit sterol biosynthesis. After removal of the inhibitor the expression of SREBP-targeted genes decreased and that of an LXR-targeted gene increased, reaching control levels. Our results demonstrate that the virtually complete replacement of cholesterol by desmosterol is compatible with cell growth and the functioning of the SREBP pathway. In these cells, desmosterol suppresses SREBP processing and targeted gene expression, and it is especially effective activating LXR-targeted genes.

  6. Effect of Sterol Structure on Chain Ordering of an Unsaturated Phospholipid: A 2H-NMR Study of POPC/Sterol Membranes

    NASA Astrophysics Data System (ADS)

    Shaghaghi, Mehran; Thewalt, Jenifer; Zuckermann, Martin

    2012-10-01

    The physical properties of biological membranes are considerably altered by the presence of sterols. In particular, sterols help to maintain the integrity of the cell by adjusting the fluidity of the plasma membrane. Cholesterol is in addition an important component of lipid rafts which are hypothesized to compartmentalize the cell membrane surface thereby making it possible for certain proteins to function. Using 2H-NMR spectroscopy, we studied the effect of a series of different sterols on the chain ordering of POPC, an unsaturated phospholipid present in eukaryotic cell membranes. We were able to assigned specific roles to the structural differences between the sterols by comparing the manner in which they affect the average lipid chain conformation of POPC.

  7. Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

    PubMed Central

    Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

  8. Expression of a fungal sterol desaturase improves tomato drought tolerance, pathogen resistance and nutritional quality

    PubMed Central

    Kamthan, Ayushi; Kamthan, Mohan; Azam, Mohammad; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2012-01-01

    Crop genetic engineering mostly aims at improving environmental stress (biotic and abiotic) tolerance as well as nutritional quality. Empowering a single crop with multiple traits is highly demanding and requires manipulation of more than one gene. However, we report improved drought tolerance and fungal resistance along with the increased iron and polyunsaturated fatty acid content in tomato by expressing a single gene encoding C-5 sterol desaturase (FvC5SD) from an edible fungus Flammulina velutipes. FvC5SD is an iron binding protein involved in ergosterol biosynthesis. Morphological and biochemical analyses indicated ≈23% more epicuticular wax deposition in leaves of transgenic plants that provides an effective waterproof barrier resulting in improved protection from drought and infection by phytopathogenic fungus Sclerotinia sclerotiorum. Furthermore, the transgenic fruits have improved nutritional value attributed to enhanced level of beneficial PUFA and 2-3 fold increase in total iron content. This strategy can be extended to other economically important crops. PMID:23230516

  9. Role of Aspergillus lentulus 14-α sterol demethylase (Cyp51A) in azole drug susceptibility.

    PubMed

    Mellado, Emilia; Alcazar-Fuoli, Laura; Cuenca-Estrella, Manuel; Rodriguez-Tudela, Juan L

    2011-12-01

    Recent studies have demonstrated that some morphologically atypical Aspergillus fumigatus strains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentulus cytochrome P450 14-α sterol demethylases, encoded by A. lentulus cyp51A (Alcyp51A) and Alcyp51B genes, were identified. Targeted cyp51A gene knockout in A. lentulus showed that the intrinsic azole resistance of this species is cyp51A dependent. The Δcyp51A strain was morphologically indistinguishable from the A. lentulus wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentulus cyp51A was performed in an A. fumigatus cyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.

  10. Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone

    PubMed Central

    Torres-Santos, Eduardo Caio; Sampaio-Santos, Maria Isabel; Buckner, Frederick S.; Yokoyama, Kohei; Gelb, Michael; Urbina, Julio A.; Rossi-Bergmann, Bartira

    2009-01-01

    Objectives The effects of the antileishmanial chalcone 2′,6′-dihydroxy-4′-methoxychalcone (DMC) on Leishmania amazonensis sterol composition and biosynthesis were investigated to obtain information about the mechanism of growth inhibition by DMC on this parasite. Methods The interference of sterol biosynthesis by DMC was studied in drug-treated promastigotes by two different methods. (i) Newly synthesized sterols from parasites grown in the presence of [3H]mevalonate were analysed by thin layer chromatography (TLC)/fluorography. (ii) Total sterols extracted from the parasites grown with or without DMC were characterized by gas chromatography coupled to mass spectroscopy (GC/MS). Results TLC and GC/MS analyses of sterols extracted from DMC-treated promastigotes revealed the accumulation of early precursors and a reduction in the levels of C-14 demethylated and C-24 alkylated sterols, as well as a reduction in exogenous cholesterol uptake. Conclusions This study demonstrates that the natural chalcone DMC alters the sterol composition of L. amazonensis and suggests that the parasite target is different from other known sterol inhibitors. PMID:19176591

  11. Sterols as biomarkers in the surface microlayer of the estuarine areas.

    PubMed

    Alsalahi, Murad Ali; Latif, Mohd Talib; Ali, Masni Mohd; Dominick, Doreena; Khan, Md Firoz; Mustaffa, Nur Ili Hamizah; Nadzir, Mohd Shahrul Mohd; Nasher, Essam; Zakaria, Mohamad Pauzi

    2015-04-15

    This study aims to determine the concentration of sterols used as biomarkers in the surface microlayer (SML) in estuarine areas of the Selangor River, Malaysia. Samples were collected during different seasons through the use of a rotation drum. The analysis of sterols was performed using gas chromatography equipped with a flame ionisation detector (GC-FID). The results showed that the concentrations of total sterols in the SML ranged from 107.06 to 505.55 ng L(-1). The total sterol concentration was found to be higher in the wet season. Cholesterol was found to be the most abundant sterols component in the SML. The diagnostic ratios of sterols show the influence of natural sources and waste on the contribution of sterols in the SML. Further analysis, using principal component analysis (PCA), showed distinct inputs of sterols derived from human activity (40.58%), terrigenous and plant inputs (22.59%) as well as phytoplankton and marine inputs (17.35%). Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols

    PubMed Central

    Tint, G. S.; Yu, Hongwei; Shang, Quan; Xu, Guorong; Patel, Shailendra B.

    2006-01-01

    Mice with a targeted mutation of 3β-hydroxysterol Δ7-reductase (Dhcr7) that cannot convert 7-dehydrocholesterol to cholesterol were used to identify the origin of fetal sterols. Because their heterozygous mothers synthesize cholesterol normally, virtually all sterols found in a Dhcr7 knockout fetus having a Δ7 or a Δ8 double bond must have been synthesized by the fetus itself but any cholesterol had to have come from the mother. Early in gestation, most fetal sterols were of maternal origin, but at approximately E13–14, in situ synthesis became increasingly important, and by birth, 55–60% of liver and lung sterols had been made by the fetus. In contrast, at E10–11, upon formation of the blood-brain barrier, the brain rapidly became the source of almost all of its own sterols (90% at birth). New, rapid, de novo sterol synthesis in brain was confirmed by the observation that concentrations of C24,25-unsaturated sterols were low in the brains of all very young fetuses but increased rapidly beginning at approximately E11–12. Reduced activity of sterol C24,25-reductase (Dhcr24) in brain, suggested by the abundance of C24,25-unsaturated compounds, seems to be the result of suppressed Dhcr24 expression. The early fetal brain also appears to conserve cholesterol by keeping cholesterol 24-hydroxylase expression low until approximately E18. PMID:16651660

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

    USDA-ARS?s Scientific Manuscript database

    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...

  14. Sterols of the green-pigmented, aberrant plastid dinoflagellate, Lepidodinium chlorophorum (Dinophyceae).

    PubMed

    Leblond, Jeffrey D; Lasiter, Andrew D

    2012-01-01

    Lepidodinium chlorophorum is a green-pigmented dinoflagellate with an aberrant, tertiary plastid of chlorophyte ancestry rather than the typical red algal, secondary endosymbiont found in the vast majority of photosynthetic dinoflagellates. To date, only one published study exists on the galactolipids of L. chlorophorum, with nothing known about other lipid classes, including sterols. Our objectives were to examine the sterol composition of L. chlorophorum to determine if it produces any unique sterols with the potential to serve as biomarkers, and to compare it to members of the Chlorophyceae to determine if it has inherited any signature green algal sterols from its chlorophyte-derived endosymbiont. We have found that L. chlorophorum produces 6 sterols, all with a 4α-methyl substituent and none of which are known to occur in the Chlorophyceae. Rather, the sterols produced by L. chlorophorum place it within a group of dinoflagellates that have the common dinoflagellate sterols, dinosterol and dinostanol, as part of their sterol composition. Copyright © 2011 Elsevier GmbH. All rights reserved.

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

    PubMed

    Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D; Andersen, Tonni G; Pomorski, Thomas G

    2014-12-01

    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 implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans. © 2014 The Authors. FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  16. Applying Clustering and Phylogeny Analysis to Study Dinoflagellates based on Sterol Composition.

    USDA-ARS?s Scientific Manuscript database

    Sterol compositions of dinoflagellates have been studied for several decades as a means of assessing whether certain species possess unique chemical biomarkers. However, no attempt has been made to compile the results from numerous studies to examine how sterol compositions may relate to the phylog...

  17. Processes of recovering fatty acids and sterols from tall oil pitch

    SciTech Connect

    Hughes, R. E.

    1985-06-18

    An improved process of enhancing the recovery of fatty acids from tall oil pitch is disclosed. The process includes a hydrolysis step for increasing the free fatty acid available for recovery from tall oil pitch during the distillation process. The hydrolysis step also enables the recovery of sterols where the tall oil pitch is of the type which is rich in sterol esters.

  18. Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

    PubMed

    Le Fur, Y; Maume, G; Feuillat, M; Maume, B F

    1999-07-01

    Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

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

    SciTech Connect

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

    1988-08-01

    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-/sup 14/C)cholesterol or beta-(4-/sup 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.

  20. Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts.

    PubMed

    Sharma, Aabha I; Olson, Cheryl L; Mamede, João I; Gazos-Lopes, Felipe; Epting, Conrad L; Almeida, Igor C; Engman, David M

    2017-08-22

    Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.

  1. A potential biochemical mechanism underlying the influence of sterol deprivation stress on Caenorhabditis elegans longevity

    USDA-ARS?s Scientific Manuscript database

    To investigate the biochemical mechanism for sterol-mediated alteration in aging in Caenorhabditis elegans, we established sterol depletion conditions by treating worms with azacoprostane, which reduced mean lifespan of adult C. elegans by 35%. Proteomic analyses of egg proteins from treated and un...

  2. Surfactants and Sterols Concentrations in the Surface Microlayer of the Estuarine Areas of Selangor River, Malaysia

    NASA Astrophysics Data System (ADS)

    Latif, M. T.; Alsalahi, M. A.; Ali, M. M.; Dominick, D.; Khan, M. F.; Wahid, N. B. A.; Mustaffa, N. I. H.

    2016-02-01

    This study aims to determine the concentration of surfactant and sterols as biomarkers in the surface microlayer (SML) in estuarine areas of the Selangor River, Malaysia. SML samples were collected during different seasons using a rotation drum method. The compositions of surfactants were determined as methylene blue active substances (MBAS) and disulphine blue active substances (DBAS) as anionic and cationic surfactants respectively. The concentration of sterols was determined using a gas chromatography equipped with a flame ionization detector (GC-FID). The results show that the concentrations of surfactants around the estuarine area were dominated by anionic surfactants (MBAS) with average concentrations of 0.39 µmol L-1. .The concentrations of total sterols in the SML ranged from 107.06 to 505.55 ng L-1. The surfactants and total sterol concentrations were found to be higher in the wet season compare to dry season. Cholesterol was found to be the most abundant sterols component in the SML of the Selangor River. The diagnostic ratios of sterols show the influence of natural sources and waste on the contribution of sterols in the SML. Further analysis, using principal component analysis (PCA), showed distinct inputs of sterols derived from human activity (40.58%), terrigenous and plant inputs (22.59%) as well as phytoplankton and marine inputs (17.35%).

  3. Transport of resistance-inducing sterols in phloem sap of barley.

    PubMed

    Lehrer, A T; Dugassa-Gobena, D; Vidal, S; Seifert, K

    2000-01-01

    After root application of [7alpha-3H]-7beta-hydroxysitosterol and [3alpha,6beta-3H2]-6alpha-hydroxylathosterol these sterols could be detected in the leaves and phloem sap feeding aphids. These results imply that the phloem sap is a sterol transport system in barley plants.

  4. Effect of sterol esters on lipid composition and antioxidant status of erythrocyte membrane of hypercholesterolemic rats.

    PubMed

    Sengupta, Avery; Ghosh, Mahua

    2014-01-01

    Hypercholesterolemia is a major cause of coronary heart disease. Erythrocyte membrane is affected during hypercholesterolemia. The effect of EPA-DHA rich sterol ester and ALA rich sterol ester on erythrocyte membrane composition, osmotic fragility in normal and hypercholesterolemic rats and changes in antioxidant status of erythrocyte membrane were studied. Erythrocyte membrane composition, osmotic fragility of the membrane and antioxidant enzyme activities was analyzed. Osmotic fragility data suggested that the erythrocyte membrane of hypercholesterolemia was relatively more fragile than that of the normal rats' membrane which could be reversed with the addition of sterol esters in the diet. The increased plasma cholesterol in hypercholesterolemic rats could also be lowered by the sterol ester administration. There was also marked changes in the antioxidant enzyme activities of the erythrocyte membrane. Antioxidant enzyme levels decreased in the membrane of the hypercholesterolemic subjects were increased with the treatment of the sterol esters. The antioxidative activity of ALA rich sterol ester was better in comparison to EPA-DHA rich sterol ester. In conclusion, rat erythrocytes appear to be deformed and became more fragile in cholesterol rich blood. This deformity and fragility was partially reversed by sterol esters by virtue of their ability to lower the extent of hypercholesterolemia.

  5. Sterol ratios as a tool for sewage pollution assessment of river sediments in Serbia.

    PubMed

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

    2016-06-01

    In this work, source pollution tracing of the sediments of the Danube River and its tributaries in Serbia was performed using sterol ratios. Improved liquid chromatography-tandem mass spectrometry method, which enabled complete chromatographic separation of four analytes with identical fragmentation reactions (epicoprostanol, coprostanol, epicholestanol and cholestanol), was applied for the determination of steroid compounds (hormones, human/animal and plant sterols). A widespread occurrence of sterols was identified in all analyzed samples, whereas the only detected hormones were mestranol and 17α-estradiol. A human-sourced sewage marker coprostanol was detected at the highest concentration (up to 1939 ng g(-1)). The ratios between the key sterol biomarkers, as well as the percentage of coprostanol relative to the total sterol amount, were applied with the aim of selecting the most reliable for distinction between human-sourced pollution and the sterols originated from the natural sources in river sediments. The coprostanol/(cholesterol + cholestanol) and coprostanol/epicoprostanol ratios do not distinguish between human and natural sources of sterols in the river sediments in Serbia. The most reliable sterol ratios for the sewage pollution assessment of river sediments in the studied area were found to be coprostanol/(coprostanol + cholestanol), coprostanol/cholesterol and epicoprostanol/coprostanol. For the majority of sediments, human-derived pollution was determined. Two sediment samples were identified as influenced by a combination of human and natural biogenic sources. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Regulation of Sterol Content in Membranes by Subcellular Compartmentation of Steryl-Esters Accumulating in a Sterol-Overproducing Tobacco Mutant.

    PubMed Central

    Gondet, L.; Bronner, R.; Benveniste, P.

    1994-01-01

    The study of sterol overproduction in tissues of LAB 1-4 mutant tobacco (Nicotiana tabacum L. cv Xanthi) (P. Maillot-Vernier, H. Schaller, P. Benveniste, G. Belliard [1989] Biochem Biophys Res Commun 165: 125-130) over several generations showed that the overproduction phenotype is stable in calli, with a 10-fold stimulation of sterol content when compared with wild-type calli. However, leaves of LAB 1-4 plants obtained after two steps of self-fertilization were characterized by a mere 3-fold stimulation, whereas calli obtained from these plants retained a typical sterol-overproducing mutant phenotype (i.e. a 10-fold increase of sterol content). These results suggest that the expression of the LAB 1-4 phenotype is dependent on the differentiation state of cells. Most of the sterols accumulating in the mutant tissues were present as steryl-esters, which were minor species in wild-type tissues. Subcellular fractionation showed that in both mutant and wild-type tissues, free sterols were associated mainly with microsomal membranes. In contrast, the bulk of steryl-esters present in mutant tissues was found in the soluble fraction of cells. Numerous lipid droplets were detected in the hyaloplasm of LAB 1-4 cells by cytochemical and cytological techniques. After isolation, these lipid granules were shown to contain steryl-esters. These results show that the overproduced sterols of mutant tissues accumulate as steryl-esters in hyaloplasmic bodies. The esterification process thus allows regulation of the amount of free sterols in membranes by subcellular compartmentation. PMID:12232218

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

    PubMed

    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, p<0.001) and increased markers of cholesterol synthesis 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.

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

    PubMed

    Hammann, Simon; Vetter, Walter

    2016-05-04

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

  9. Plant sterols and stanols as cholesterol-lowering ingredients in functional foods.

    PubMed

    Kamal-Eldin, Afaf; Moazzami, Ali

    2009-01-01

    This article reviews developments related to the use of plant sterols and stanols as cholesterol-lowering ingredients in foods and nutraceuticals preparations. Plant sterols and stanols are extracted from the deodorizer distillates of vegetable oil refining and from tall oil, a by-product of paper pulping industry. Plant sterols/stanols inhibit cholesterol absorption possibly by competitively inhibiting its incorporation into the mixed micelles in the small intestine although other mechanisms can not be excluded. Daily consumption of 1-2 grams of plant sterols or stanols was shown to cause 10-20% reduction in low-density lipoprotein cholesterol (LDL cholesterol). Combinations of plant sterols/stanols with certain lipid-lowering ingredients were shown to potentate their cholesterol-lowering effects and, in some cases, add triacylglycerol-lowering effects. In this article, patents based information is also discussed.

  10. Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania.

    PubMed

    Manzano, José Ignacio; Carvalho, Luis; García-Hernández, Raquel; Poveda, José Antonio; Ferragut, José Antonio; Castanys, Santiago; Gamarro, Francisco

    2011-11-01

    The present study was designed to elucidate the mechanism of tafenoquine uptake in Leishmania and its sterol dependence. Because tafenoquine is a fluorescent compound, spectrofluorimetric analysis allowed us to monitor its uptake by Leishmania promastigotes and intracellular amastigotes, and to evaluate the effect of temperature, energy and H+ gradient on drug entry. The influence of sterols on tafenoquine uptake in Leishmania parasites was determined in experiments using sterol-depleting agents such as methyl-β-cyclodextrin or cholesterol oxidase. Tafenoquine exhibited fast entry kinetics into Leishmania in an energy-independent, but pH- and temperature-dependent, non-saturable process. Furthermore, sterol depletion decreased tafenoquine uptake. These findings suggest that Leishmania takes up tafenoquine by a diffusion process and that decreases in membrane sterol content may induce a decrease in drug uptake.

  11. Evaluation of bioactive potential of an Aloe vera sterol extract.

    PubMed

    Bawankar, Raksha; Deepti, V C; Singh, Pooja; Subashkumar, Rathinasamy; Vivekanandhan, Govindasamy; Babu, Subramanian

    2013-06-01

    We prepared a crude gel material from Aloe vera succulent leaf tissues. The ethanolic extract of lyophilized A. vera gel was used for the GC-MS analysis. Hexadecanoic acid (22.22%) was identified as major compound. Sitosterol and stigmasterol were found to be 2.89% and 2.1% in the extract. HPLC analysis was carried out to confirm the presence of stigmasterol. The concentration of sterol extract needed to scavenge DPPH free radical by 50% was calculated as 5.2 mg mL(-1). In the FRAP assay, the sterol extract showed significant hydroxyl radical scavenging in a dose-dependent manner (IC50 value 1.17 µg mL(-1)). Concentration of the sample required to reduce lipid peroxidation was found to be 4.18 µg mL(-1), and the extract also possessed acetylcholinesterase activity (IC50 - 5.26 µg mL(-1)). Catalase activity was 0.196 μM H2 O2 decomposed min(-1) µg(-1) protein, whereas the peroxidase activity was 17.01 μM of pyragallol oxidized min(-1) µg(-1) protein. The extract recorded higher activity against growth of S. greseus and C. albicans in the experiments carried out to determine antibacterial and antifungal activity, respectively. Copyright © 2012 John Wiley & Sons, Ltd.

  12. Serum lipid and antioxidant responses in hypercholesterolemic men and women receiving plant sterol esters vary by apolipoprotein E genotype

    USDA-ARS?s Scientific Manuscript database

    Plant sterol esters reduce serum total cholesterol (TC) and LDL cholesterol (LDL-C), but with striking inter-individual variability. In this randomized, double-blind, controlled study, serum lipid, plant sterol, fat-soluble vitamin, and carotenoid responses to plant sterols were studied according to...

  13. Cholesterol-lowering effect of spreads enriched with microcrystalline plant sterols in hypercholesterolemic subjects.

    PubMed

    Christiansen, L I; Lähteenmäki, P L; Mannelin, M R; Seppänen-Laakso, T E; Hiltunen, R V; Yliruusi, J K

    2001-04-01

    Plant sterols have been shown to reduce serum lipid concentrations. The effectiveness is highly dependent on the physical state of the plant sterols. By means of a new crystallizing method, plant sterols can be added into dietary fats and oils homogeneously. In this fat ingredient, plant sterols are in a microcrystalline form. We investigated the cholesterol-lowering effect and possible side effects of vegetable oil-based spreads fortified with two different doses of microcrystalline plant sterols. This double-blind randomized, placebo-controlled study consisted of a 6-wk run-in and a 6-month experimental period. During the run-in period, all 155 hypercholesterolemic subjects received rapeseed oil-based control spread. In the beginning of the experimental period subjects were randomly assigned into one of three experimental groups. The control group continued to use control spread, and the two test groups used spreads with added plant sterols of either 1.5 g/d or 3.0 g/d. The subjects consumed test spreads as a part of their normal diet without any restrictions in lifestyle and diet. Plasma total- and LDL-cholesterol concentrations were significantly reduced by 7.5-11.6% (0.46-0.62 mmol/1) in groups consuming margarine enriched with free plant sterols, compared with the control group. The effects were similar between the two groups consuming either 1.5g or 3.0 g plant sterols per day. No effect on HDL-cholesterol or triacylglycerol concentrations occurred. The test spreads did not induce any adverse effects in blood clinical chemistry, hematology or decreases in serum concentrations of lipid soluble vitamins. Microcrystalline plant sterols are effective in lowering serum total- and LDL-cholesterol concentrations without obvious side effects. The daily dose of 1.5 g plant sterols is enough to reach the maximum effect.

  14. Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

    PubMed Central

    Tsukamoto, T; Toyama, R; Itoh, H; Kozasa, T; Matsuoka, M; Kaziro, Y

    1991-01-01

    Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain. Images PMID:1901650

  15. Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk

    PubMed Central

    Calandra, Sebastiano; Tarugi, Patrizia; Speedy, Helen E.; Dean, Andrew F.; Bertolini, Stefano; Shoulders, Carol C.

    2011-01-01

    This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means. PMID:21862702

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

    PubMed

    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

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

    2014-01-01

    Background 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. Methods 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. Results 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. Conclusions Serum non-cholesterol sterols are valid markers of cholesterol absorption

  20. Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

    PubMed Central

    Daimiel, Lidia A.; Fernández-Suárez, María E.; Rodríguez-Acebes, Sara; Crespo, Lorena; Lasunción, Miguel A.; Gómez-Coronado, Diego; Martínez-Botas, Javier

    2012-01-01

    DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression. PMID:23050906

  1. Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis.

    PubMed

    Pereira, Maristela; Song, Zhihong; Santos-Silva, Ludier Kesser; Richards, Mathew H; Nguyen, Thi Thuy Minh; Liu, JiaLin; de Almeida Soares, Celia Maria; da Silva Cruz, Aline Helena; Ganapathy, Kulothungan; Nes, W David

    2010-10-01

    The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K(mapp) of 38microM and k(catapp) of 0.14min(-1). Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-(13)C]- or [24-(2)H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of (1)H and (13)CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Delta(24(25))-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K(i) 14nM) and 26,27-dehydrolanosterol (K(i) 54muM and k(inact) of 0.24min(-1)) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC(50), 30nM) promoting lanosterol accumulation and 24

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

    PubMed

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

    2014-05-01

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

  3. An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae

    PubMed Central

    Tiwari, Rashi; Köffel, René; Schneiter, Roger

    2007-01-01

    Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved. PMID:18034159

  4. Evidence for metabolic and functional discrimination of sterols by Phytophthora cactorum

    PubMed Central

    Nes, W. David; Stafford, Allen E.

    1983-01-01

    When fed 10 ppm of one of the following sterols: cholesterol (cholest-5-en-3β-ol), wingsterol (21-isopentylcholesterol), desmosterol [cholesta-5,24(25)-dien-3β-ol], 24-methylenecholesterol [ergosta-5,24(28)-dien-3β-ol], or fucosterol [stigmasta-5,24(28)-dien-3β-ol], the pathogenic fungus Phytophthora cactorum, which is naturally unable to epoxidize squalene, accumulated each of the test compounds to similar levels. Fucosterol, the only sterol metabolized, was reduced to yield 24-ethylcholesterol. All the sterols tested induced the formation of sex structures. Fertilization and subsequent maturation of oospores capable of germination occurred only with the naturally occurring sterols. Wingsterol treatments resulted in aborted oospores. None of the sterols tested was inhibitory to growth, measured as changes in the 21-day mycelial dry weight. The results are consistent with the view that the accumulated sterol functions to regulate the life cycle of P. cactorum. However, the metabolism and kinds of recognition of the sterol molecule, in terms of uptake and effects on growth and induction of the various sexual events, contrast sharply with what is known for other oomycetous fungi such as Achlya and Saprolegnia. This implies that the evolutionary histories of the Oomycetes may be different. Images PMID:16593322

  5. Evidence for similarities and differences in the biosynthesis of fungal sterols.

    PubMed

    Nes, W D; Xu, S H; Haddon, W F

    1989-01-01

    The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.

  6. Effects of plant sterols and stanols on intestinal cholesterol metabolism: suggested mechanisms from past to present.

    PubMed

    De Smet, Els; Mensink, Ronald P; Plat, Jogchum

    2012-07-01

    Plant sterols and stanols are natural food ingredients found in plants. It was already shown in 1950 that they lower serum low-density lipoprotein cholesterol (LDL-C) concentrations. Meta-analysis has reported that a daily intake of 2.5 g plant sterols/stanols reduced serum LDL-C concentrations up to 10%. Despite many studies, the underlying mechanism remains to be elucidated. Therefore, the proposed mechanisms that have been presented over the past decades will be described and discussed in the context of the current knowledge. In the early days, it was suggested that plant sterols/stanols compete with intestinal cholesterol for incorporation into mixed micelles as well as into chylomicrons. Next, the focus shifted toward cellular processes. In particular, a role for sterol transporters localized in the membranes of enterocytes was suggested. All these processes ultimately lowered intestinal cholesterol absorption. More recently, the existence of a direct secretion of cholesterol from the circulation into the intestinal lumen was described. First results in animal studies suggested that plant sterols/stanols activate this pathway, which also explains the increased fecal neutral sterol content and as such could explain the cholesterol-lowering activity of plant sterols/stanols.

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

    PubMed

    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.

  8. Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants▿

    PubMed Central

    Sharma, Monika; Sasvari, Zsuzsanna; Nagy, Peter D.

    2010-01-01

    The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3- to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92pol viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication. PMID:20015981

  9. Plant sterol-fortified orange juice effectively lowers cholesterol levels in mildly hypercholesterolemic healthy individuals.

    PubMed

    Devaraj, Sridevi; Jialal, Ishwarlal; Vega-López, Sonia

    2004-03-01

    Hypercholesterolemia is a major risk factor for coronary artery disease. Therapeutic lifestyle changes include dietary modifications such as inclusion of phytosterols, which effectively lowers low-density lipoprotein (LDL) cholesterol in margarines and other fats. Their effectiveness in nonfat moieties is not yet established. The aim of this study was to examine if phytosterols alter the plasma lipoprotein profile when incorporated into nonfat orange juice. After a 2-week run-in phase with orange juice, 72 mildly hypercholesterolemic healthy subjects were randomized to receive either placebo orange juice (placebo OJ) or plant sterol-fortified orange juice (sterol OJ) (2g/d) for 8 weeks. Fasting blood was obtained at baseline, after 2 weeks of OJ, and after 8 weeks of placebo/sterol-OJ supplementation. Sterol OJ supplementation significantly decreased total (7.2%), LDL (12.4%), and non-high-density lipoprotein (HDL) cholesterol (7.8%) compared with baseline and compared with placebo OJ (P<0.01). Apolipoprotein B levels were significantly decreased (9.5%) with sterol OJ. There were no significant changes in HDL cholesterol or triglycerides with the sterol OJ. While folate and B12 levels significantly increased, homocysteine levels were unchanged. Orange juice fortified with plant sterols are effective in reducing LDL cholesterol and could easily be incorporated into the therapeutic lifestyle changes dietary regimen.

  10. Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin’s Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

    PubMed Central

    Baumforth, Karl R.N.; Birgersdotter, Anna; Reynolds, Gary M.; Wei, Wenbin; Kapatai, Georgia; Flavell, Joanne R.; Kalk, Emma; Piper, Karen; Lee, Steve; Machado, Lee; Hadley, Kerry; Sundblad, Anne; Sjoberg, Jan; Bjorkholm, Magnus; Porwit, Anna A.; Yap, Lee-Fah; Teo, Soohwang; Grundy, Richard G.; Young, Lawrence S.; Ernberg, Ingemar; Woodman, Ciaran B.J.; Murray, Paul G.

    2008-01-01

    In ∼50% of patients with Hodgkin’s lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4+ lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4+ T cells that inhibit effector CD4+ and CD8+ T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity. PMID:18502823

  11. The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages

    PubMed Central

    Girard, Emmanuelle; Paul, Jean Louis; Fournier, Natalie; Beaune, Philippe; Johannes, Ludger

    2011-01-01

    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. PMID:22205993

  12. Reduction in cholesterol absorption is enhanced by stearate-enriched plant sterol esters in hamsters.

    PubMed

    Rasmussen, Heather E; Guderian, David M; Wray, Curtis A; Dussault, Patrick H; Schlegel, Vicki L; Carr, Timothy P

    2006-11-01

    Consumption of plant sterol esters reduces plasma LDL cholesterol concentration by inhibiting intestinal cholesterol absorption. Commercially available plant sterol esters are prepared by esterifying free sterols to fatty acids from edible plant oils such as canola, soybean, and sunflower. To determine the influence of the fatty acid moiety on cholesterol metabolism, plant sterol esters were made with fatty acids from soybean oil (SO), beef tallow (BT), or purified stearic acid (SA) and fed to male hamsters for 4 wk. A control group fed no plant sterol esters was also included. Hamsters fed BT and SA had significantly lower cholesterol absorption and decreased concentrations of plasma non-HDL cholesterol and liver esterified cholesterol, and significantly greater fecal sterol excretion than SO and control hamsters. Cholesterol absorption was lowest in hamsters fed SA (7.5%), whereas it was 72.9% in control hamsters. Cholesterol absorption was correlated with fecal sterol excretion (r = -0.72, P < 0.001), liver cholesterol concentration (r = 0.88, P < 0.001), and plasma non-HDL cholesterol concentration (r = 0.85, P < 0.001). A multiple regression model that included each sterol ester type vs. cholesterol absorption indicated that intake of steryl stearate was the only dietary component that contributed significantly to the model (R2 = -0.75, P < 0.001). Therefore, our results demonstrate that BT and SA are more effective than SO in reducing cholesterol absorption, liver cholesterol, and plasma non-HDL cholesterol concentration, suggesting that cardioprotective benefits can be achieved by consuming stearate-enriched plant sterol esters.

  13. Sterols of the green-pigmented, freshwater raphidophyte, Gonyostomum semen, from Scandinavian lakes.

    PubMed

    Leblond, Jeffrey D; Dahmen, Aaron S; Lebret, Karen; Rengefors, Karin

    2013-01-01

    Sterols are a class of membrane-reinforcing, ringed lipids which have a long history of examination in algae as a means of deriving chemotaxonomic relationships and as potential lipidic biomarkers. The Raphidophyceae represent a class of harmful, bloom-forming, marine and freshwater algae. To date, there have been four published examinations of their sterol composition, focusing primarily on brown-pigmented, marine species within the genera, Chattonella, Fibrocapsa, and Heterosigma. Lacking in these examinations has been the species Gonyostomum semen Ehrenb., which is a green-pigmented, freshwater raphidophyte with a worldwide distribution. The goal of this study was to examine the sterol composition of this nuisance alga, determine the potential of using its sterol profile as a biomarker, and finally to determine if there is any intraspecific variability between isolates. We have examined 21 isolates of G. semen from a number of Scandinavian lakes, and all were found to produce two major sterols, 24-ethylcholesta-5,22E-dien-3β-ol and 24-ethylcholest-5-en-3β-ol, and 24-methylcholest-5-en-3β-ol as a minor sterol; the presence of 24-ethylcholesta-5,22E-dien-3β-ol differentiates G. semen from brown-pigmented, marine raphidophytes which generally lack it. The results of this study indicate that isolates of G. semen from geographically separate lakes across Finland and Scandinavia have the same sterol biosynthetic pathway, and that there is no evolutionary divergence between the isolates with regard to sterol composition. The sterols of G. semen are not considered to be useful biomarkers for this particular organism because they are commonly found in other algae and plants. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  14. Contributions of other sterols to the estimation of cholesterol.

    PubMed

    Munster, D J; Lever, M; Carrell, R W

    1976-04-15

    The responses of 5alpha-cholestan-3beta-ol, 5alpha-cholest-7-ene-3beta-ol and cholesta-5,7-dien-3beta-ol, normally found in human serum, were examined by: (1) the Liebermann-Burchard reaction, (2) the Zak (ferric chloride) reaction, (3) an enzymatic cholesterol method monitored by estimating the amount of hydrogen peroxide produced, (4) an enzymatic cholesterol method monitored by observing the change in absorbance at 240 nm, and (5) gas chromatography. The results show that none of these methods is specific for cholesterol; contributions from the sterols examined range from zero to more than 150% relative to cholesterol. For the first four methods contributions depend on the conditions under which each test is performed.

  15. Bioactive polyhydroxylated sterols from the marine sponge Haliclona crassiloba.

    PubMed

    Cheng, Zhong-Bin; Xiao, Han; Fan, Cheng-Qi; Lu, Ya-Nan; Zhang, Ge; Yin, Sheng

    2013-12-20

    Four new polyhydroxylated sterols, named halicrasterols A-D (1-4), together with six known analogs (5-10) were isolated from the marine sponge Haliclona crassiloba. Compounds 1 and 2 represented rare examples of steroids featuring 17(20)E-double bonds. The structures of 1-10 were elucidated by spectroscopic analysis and comparison with reported data. This is the first report of a steroid profile for this species. The antimicrobial activities of 1-10 were evaluated against a panel of bacterial and fungal strains in vitro, and compounds 4 and 9 showed moderate activity against some of the Gram-positive strains with MICs ranging from 4 to 32 μg/mL. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Sterols and triterpenes in cell culture of Hyssopus officinalis L.

    PubMed

    Skrzypek, Zuzanna; Wysokińska, Halina

    2003-01-01

    Cell suspension cultures from hypocotyl-derived callus of Hyssopus officinalis were found to produce two sterols i. e. beta-sitosterol (1) and stigmasterol (2), as well as several known pentacyclic triterpenes with an oleanene and ursene skeleton. The triterpenes were identified as oleanolic acid (3), ursolic acid (4), 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (5), 2alpha,3beta-dihydroxyurs-12-en-28-oic acid (6), 2alpha,3beta,24-trihydroxyolean-12-en-28-oic acid (7), and 2alpha,3beta,24-trihydroxyurs-12-en-28-oic acid (8). Compounds 5-8 were isolated as their acetates (6, 8) or bromolactone acetates (5, 7).

  17. Cell-free transfer of sterols by plant fractions

    SciTech Connect

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

    1990-05-01

    Microsomes from etiolated hypocotyls of soybean or leaves of light-grown spinach radiolabeled in vivo with ({sup 3}H)acetate or in vitro with ({sup 3}H)squalene or ({sup 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.

  18. Sterol composition and phytosterol utilization and metabolism in the milkweed bug.

    PubMed

    Svoboda, J A; Dutky, S R; Robbins, W E; Kaplanis, J N

    1977-03-01

    Analysis of the sterols of the milkweed bug, Oncopeltus fasciatus (Dallas) and dietary sunflowerseeds revealed that there is little, if any, conversion of dietary C28 OR C29 phytosterols to cholesterol in this phytophagous insect. The dietary sterols are apparently utilized with little alteration both during development to the adult stage and egg production, and cholesterol comprises less than 1% of the sterols in either adult males and females or in the eggs. The significance of these findings are discussed in light of the recent discovery that the C28-ecdysone, makisterone A, is the predominant molting hormone inthe embryonated egg of the milkweed bug.

  19. Gas chromatography-mass spectrometry study of sterols from Pinus elliotti tissues.

    NASA Technical Reports Server (NTRS)

    Laseter, J. L.; Evans, R.; Weete, J. D.; Walkinshaw, C. H.

    1973-01-01

    A comparative study of the sterol components of slash pine (Pinus elliotti) callus tissue cultures, seeds, and seedlings was carried out using GC-MS techniques. Cholesterol, desmosterol, campesterol, stigmasterol, sitosterol and cycloeucalenol were identified in all tissues while lophenol and 24-methylenelophenol were identified in only the seed and seedlings. 24-Ethylidenelophenol was detected in trace concentrations in only the seedlings. Sitosterol was the predominant sterol component, i.e., 80.8, 38.1 and 47.8% of the tissue culture, seed and seedling sterols, respectively.

  20. Distribution and factors affecting adsorption of sterols in the surface sediments of Bosten Lake and Manas Lake, Xinjiang.

    PubMed

    Liu, Jiang; Yao, Xiaorui; Lu, Jianjiang; Qiao, Xiuwen; Liu, Zilong; Li, Shanman

    2016-03-01

    This study investigated the concentrations and distribution of eight sterol compounds in the surface sediments of Bosten Lake and Manas Lake, Xinjiang, China. The ratios of sterols as diagnostic indices were used to identify pollution sources. The sediment of the two lakes was selected as an adsorbent to investigate the adsorption behaviour of sterols. Results showed that the sterols were widely distributed in the sediments of the lakes in the study areas. The total concentrations of the detected sterols in Bosten Lake and in Manas Lake were 1.584-27.897 and 2.048-18.373 μg g(-1)∙dw, respectively. In all of the sampling sites, the amount of faecal sterols was less than that of plant sterols. β-sitosterol was the dominant plant sterol with a mean concentration of 2.378 ± 2.234 μg g(-1)∙dw; cholesterol was the most abundant faecal sterol with a mean concentration of 1.060 ± 1.402 μg g(-1)∙dw. The pollution level was higher in Bosten Lake than in Manas Lake. Majority of the ratios clearly demonstrated that the contamination by human faecal sources was occurring at stations which are adjacent to residential areas and water inlets. The adsorption behaviour of sterols to sediment suggested that the sterol adsorption coefficients were reduced as temperature increased. As salinity increased, the adsorption quantity also increased. As pH increased, the sediment adsorption of sterol slightly increased because the strong alkaline solution is not conducive to the adsorption of sterols. The ratios between sterols did not change largely with the change in external factors.

  1. Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.

    PubMed

    Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X

    2013-12-01

    During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.

  2. Mutations in UDP-Glucose:sterol glucosyltransferase in Arabidopsis cause transparent testa phenotype and suberization defect in seeds.

    PubMed

    DeBolt, Seth; Scheible, Wolf-Rüdiger; Schrick, Kathrin; Auer, Manfred; Beisson, Fred; Bischoff, Volker; Bouvier-Navé, Pierrette; Carroll, Andrew; Hematy, Kian; Li, Yonghua; Milne, Jennifer; Nair, Meera; Schaller, Hubert; Zemla, Marcin; Somerville, Chris

    2009-09-01

    In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.

  3. 7-Dehydrocholesterol metabolites produced by sterol 27-hydroxylase (CYP27A1) modulate liver X receptor activity.

    PubMed

    Endo-Umeda, Kaori; Yasuda, Kaori; Sugita, Kazuyuki; Honda, Akira; Ohta, Miho; Ishikawa, Minoru; Hashimoto, Yuichi; Sakaki, Toshiyuki; Makishima, Makoto

    2014-03-01

    7-Dehydrocholesterol (7-DHC) is a common precursor of vitamin D3 and cholesterol. Although various oxysterols, oxygenated cholesterol derivatives, have been implicated in cellular signaling pathways, 7-DHC metabolism and potential functions of its metabolites remain poorly understood. We examined 7-DHC metabolism by various P450 enzymes and detected three metabolites produced by sterol 27-hydroxylase (CYP27A1) using high-performance liquid chromatography. Two were further identified as 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC. These 7-DHC metabolites were detected in serum of a patient with Smith-Lemli-Opitz syndrome. Luciferase reporter assays showed that 25-hydroxy-7-DHC activates liver X receptor (LXR) α, LXRβ and vitamin D receptor and that 26/27-hydroxy-7-DHC induces activation of LXRα and LXRβ, although the activities of both compounds on LXRs were weak. In a mammalian two-hybrid assay, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC induced interaction between LXRα and a coactivator fragment less efficiently than a natural LXR agonist, 22(R)-hydroxycholesterol. These 7-DHC metabolites did not oppose agonist-induced LXR activation and interacted directly to LXRα in a manner distinct from a potent agonist. These findings indicate that the 7-DHC metabolites are partial LXR activators. Interestingly, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC suppressed mRNA expression of sterol regulatory element-binding protein 1c, an LXR target gene, in HepG2 cells and HaCaT cells, while they weakly increased mRNA levels of ATP-binding cassette transporter A1, another LXR target, in HaCaT cells. Thus, 7-DHC is catabolized by CYP27A1 to metabolites that act as selective LXR modulators.

  4. Non-cholesterol Sterols in the Diagnosis and Treatment of Dyslipidemias: A Review.

    PubMed

    Baila-Rueda, Lucía; Cenarro, Ana; Civeira, Fernando

    2016-01-01

    Non-cholesterol sterols have been used as markers of cholesterol intestinal absorption and hepatic synthesis, leading to a better understanding of cholesterol homeostasis in humans. This review discusses the main noncholesterol sterols that are clinically useful, different methods to quantify the factors associated with blood concentration, and the potential role of non-cholesterol sterols in the diagnosis and treatment of different types of dyslipidemia. The main indication is the use of non-cholesterol sterols for the diagnosis of rare diseases associated with defects in cholesterol synthesis or anomalies in the absorption and/or elimination of phytosterols. However, other potential uses, including the diagnosis of certain hypercholesterolemias and the individualization of lipid-lowering therapies, are promising as they could help treat a wider population.

  5. Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans.

    PubMed

    Ikekawa, Nobuo; Fujimoto, Yoshinori; Ishiguro, Masaji

    2013-01-01

    Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970-1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids.

  6. Plant sterols as dietary adjuvants in the reduction of cardiovascular risk: theory and evidence.

    PubMed

    Patch, Craig S; Tapsell, Linda C; Williams, Peter G; Gordon, Michelle

    2006-01-01

    Plant sterol-enriched foods are an effective dietary adjuvant in reducing cardiovascular risk by lowering total cholesterol and low density lipoprotein-cholesterol (LDL-C) in serum by up to approximately 15%. The mechanism of action of plant sterols is different from those of 3-hydroxy-3-methylglutaryl coenzyme A inhibitors (statins) and thus their effect is additive. Combining plant sterols with other dietary components known to reduce cholesterol in a portfolio approach has proven to be most effective for reduction of hypercholesterolemia and provide an alternative treatment option for clinicians. Plant sterol-enriched foods provides clinicians with a relatively cheap, safe, and effective way to help patients manage their cardiovascular risk.

  7. Isolation of a biodegradable sterol-rich fraction from industrial wastes.

    PubMed

    Dias, A C P; Fernandes, P; Cabral, J M S; Pinheiro, H M

    2002-05-01

    Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96-98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.

  8. Investigation of the Sterol Composition and Azole Resistance in Field Isolates of Septoria tritici

    PubMed Central

    Joseph-Horne, T.; Hollomon, D.; Manning, N.; Kelly, S. L.

    1996-01-01

    We report here a biochemical study of resistance to azole antifungal agents in a field isolate (S-27) of a fungal phytopathogen. Isolates of Septoria tritici were compared in vitro, and their responses reflected that observed in the field, with S-27 exhibiting resistance relative to RL2. In untreated cultures, both RL2 and S-27 contained isomers of ergosterol and ergosta-5,7-dienol, although in differing concentrations. Under azole treatment, this phytopathogen exhibited a response similar to that of other pathogenic fungi, with a reduction in desmethyl sterols and an accumulation of 14(alpha)-methyl sterols, indicative of inhibition of the P450-mediating sterol 14(alpha)-demethylase. Growth arrest was attributed to the reduction of ergosterol combined with an accumulation of nonutilizable sterols. Strain S-27 exhibited an azole-resistant phenotype which was correlated with decreased cellular content of azole. PMID:16535210

  9. Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Koukkou, A I; Tsoukatos, D; Drainas, C

    1993-08-01

    Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squalene precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.

  10. Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans

    PubMed Central

    IKEKAWA, Nobuo; FUJIMOTO, Yoshinori; ISHIGURO, Masaji

    2013-01-01

    Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970–1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids. PMID:24126284

  11. Plant Sterols as Dietary Adjuvants in the Reduction of Cardiovascular Risk: Theory and Evidence

    PubMed Central

    Patch, Craig S; Tapsell, Linda C; Williams, Peter G; Gordon, Michelle

    2006-01-01

    Plant sterol-enriched foods are an effective dietary adjuvant in reducing cardiovascular risk by lowering total cholesterol and low density lipoprotein-cholesterol (LDL-C) in serum by up to ∼15%. The mechanism of action of plant sterols is different from those of 3-hydroxy-3-methylglutaryl coenzyme A inhibitors (statins) and thus their effect is additive. Combining plant sterols with other dietary components known to reduce cholesterol in a portfolio approach has proven to be most effective for reduction of hypercholesterolemia and provide an alternative treatment option for clinicians. Plant sterol-enriched foods provides clinicians with a relatively cheap, safe, and effective way to help patients manage their cardiovascular risk. PMID:17319460

  12. Molecular cloning and biochemical characterization of Candida albicans acyl-CoA:sterol acyltransferase, a potential target of antifungal agents.

    PubMed

    Kim, Ki-Young; Shin, Yu-Kyong; Park, Jong-Chul; Kim, Jung-Ho; Yang, Hongyuan; Han, Dong-Min; Paik, Young-Ki

    2004-07-02

    To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.

  13. Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2.

    PubMed

    Edqvist, Johan; Rönnberg, Elin; Rosenquist, Sara; Blomqvist, Kristina; Viitanen, Lenita; Salminen, Tiina A; Nylund, Matts; Tuuf, Jessica; Mattjus, Peter

    2004-12-17

    This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded