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Sample records for encoding sterol regulatory

  1. Structure of the human gene encoding sterol regulatory element binding protein-1 (SREBF1) and localization of SREBF1 and SREBF2 to chromosomes 17p11.2 and 22q13

    SciTech Connect

    Hua, X.; Wu, J.; Goldstein, J.L.

    1995-02-10

    Sterol regulatory element binding protein-1 (SREBP1) and SREBP2 are structurally related proteins that control cholesterol homeostasis by stimulating transcription of sterol-regulated genes, including those encoding the low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl CoA synthase. SREBP1 and SREBP2 are 47% identical, and they share a novel structure comprising a transcriptionally active NH{sub 2}-terminal basic helix-loop-helix-leucine zipper (bHLH-Zip) domain followed by a membrane attachment domain. Cleavage by a sterol-regulated protease frees the bHLH-Zip domain from the membrane and allows it to enter the nucleus. SREBP1 exists in several forms, possibly as a result of alternative splicing at both the 5{prime} and the 3{prime} ends of the mRNA. The genes for SREBP1 (SREBF1) and SREBP2 (SREBF2) have not been studied. In this paper we describe the cloning and characterization of the human SREBF1 gene. The gene is 26 kb in length and has 22 exons and 20 introns. The 5{prime} and 3{prime} sequences that differ between the two SREBP1 cDNAs are encoded by discrete exons, conforming the hypothesis that they result from alternative splicing. The chromosomal locations of human SREBF1 and SREBF2 were determined by analysis of human-rodent somatic cell hybrids and fluorescence in situ hybridization. The SREBF1 gene mapped to the proximal short arm of chromosome 17 (17p11.2), and the SREBF2 gene was localized to the long arm of chromosome 22 (22q13). 22 refs., 3 figs., 2 tabs.

  2. The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection

    PubMed Central

    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

    2014-01-01

    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. PMID:24516383

  3. Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

    PubMed

    Wen, Gaiping; Eder, Klaus; Ringseis, Robert

    2016-08-01

    The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis. PMID:27321819

  4. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    SciTech Connect

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III ); Billheimer, J.T. )

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  5. Isolation, characterization, and regulation of the Candida albicans ERG27 gene encoding the sterol 3-keto reductase.

    PubMed

    Pierson, C A; Jia, N; Mo, C; Lees, N D; Sturm, A M; Eckstein, J; Barbuct, R; Bard, M

    2004-10-01

    The Candida albicans ERG27 gene which encodes the 3-keto reductase enzyme required for sterol C-4 demethylation was isolated and found to encode a 349 amino acid protein that is 60% identical at the amino acid level to the Saccharomyces cerevisiae Erg27p. A C. albicans erg27 null was created in a strain containing an integrated ERG27 rescue cassette under the control of the pMAL2 inducible promoter. The C. albicans erg27 strain was able to grow only in the presence of maltose indicating that the ERG27 gene is essential. The C. albicans erg27 null showed complete loss of both 3-keto reductase and oxidosqualene cyclase (Erg7p) activities compromising all sterol synthesis. These results suggest that Erg27p inhibitors might be effective antifungals. To explore ERG27 regulation, an erg11 null strain was generated. C. albicans erg6 and erg24 mutants were also employed along with the inhibitors, itraconazole and zaragozic acid A, to characterize ERG27 expression using Northern analysis. Expression was increased two- to fourfold in erg11, erg6 and erg24 backgrounds. However, itraconazole which targets Erg11p (lanosterol demethylase) increased ERG27 expression 10-fold and zaragozic acid A which targets the Erg9p (squalene synthase) increased ERG27 expression fivefold. The azole and erg11 results support other observations that azoles may affect non-sterol targets. PMID:15552648

  6. Sterol regulatory element-binding proteins are regulators of the NIS gene in thyroid cells.

    PubMed

    Ringseis, Robert; Rauer, Christine; Rothe, Susanne; Gessner, Denise K; Schütz, Lisa-Marie; Luci, Sebastian; Wen, Gaiping; Eder, Klaus

    2013-05-01

    The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS. PMID:23542164

  7. 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

  8. Structural Requirements for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast*

    PubMed Central

    Cheung, Rocky; Espenshade, Peter J.

    2013-01-01

    Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis. PMID:23729666

  9. Sterol Synthesis in Diverse Bacteria.

    PubMed

    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

  10. 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

  11. 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

  12. Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus.

    PubMed

    Nakano, Chiaki; Motegi, Akihiro; Sato, Tsutomu; Onodera, Masayuki; Hoshino, Tsutomu

    2007-10-01

    Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed. PMID:17928701

  13. 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

  14. 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.

  15. A clinical isolate of Candida albicans with mutations in ERG11 (encoding sterol 14alpha-demethylase) and ERG5 (encoding C22 desaturase) is cross resistant to azoles and amphotericin B.

    PubMed

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

    2010-09-01

    A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 microg ml(-1), respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 microg ml(-1) for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14alpha-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 microg ml(-1)). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic. PMID:20547793

  16. 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.

  17. 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. PMID:21105651

  18. Sterol Regulatory Element Binding Protein Regulates the Expression and Metabolic Functions of Wild-Type and Oncogenic IDH1.

    PubMed

    Ricoult, Stéphane J H; Dibble, Christian C; Asara, John M; Manning, Brendan D

    2016-09-15

    Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of the enzymes underlying de novo lipid synthesis. However, little is known about the SREBP-mediated control of processes that indirectly support lipogenesis, for instance, by supplying reducing power in the form of NAPDH or directing carbon flux into lipid precursors. Here, we characterize isocitrate dehydrogenase 1 (IDH1) as a transcriptional target of SREBP across a spectrum of cancer cell lines and human cancers. IDH1 promotes the synthesis of lipids specifically from glutamine-derived carbons. Neomorphic mutations in IDH1 occur frequently in certain cancers, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG). We found that SREBP induces the expression of oncogenic IDH1 and influences 2-HG production from glucose. Treatment of cells with 25-hydroxycholesterol or statins, which respectively inhibit or activate SREBP, further supports SREBP-mediated regulation of IDH1 and, in cells with oncogenic IDH1, carbon flux into 2-HG. PMID:27354064

  19. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

    PubMed Central

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

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. PMID:26431033

  20. Noncholesterol sterols.

    PubMed

    Vecka, Marek; Zak, Ales; Tvrzická, Eva

    2008-01-01

    Although most of us are more or less familiar with the term "cholesterol", the world of sterols is far more complicated and interesting. Apart from cholesterol, many non-cholesterol sterols can be found in human plasma and these sterols serve many important functions in human organism. They are either derived from endogenous biosynthesis of cholesterol or they come from dietary sources (phytosterols). The sole cholesterol molecule is used for keeping our cell membranes fit, for signalization purposes as well as a precursor for bile acids and steroid hormones. The compounds prior to cholesterol in its biosynthetic pathway were identified as vitamin D3 precursor, meiosis activating sterols and nowadays it seems that they could play a role in cholesterol homeostasis. The sterols from ingested vegetable sources, the phytosterols, are expelled from enterocytes and thus indirectly help our gut in coping with abundant cholesterol in the lumen. Higher plants synthesize many phytosterols, but in marine organisms, we can find other innumerous sterol molecules. The diversity of sterol molecules produced and resistance of their tetracyclic core to enzymatic activities implies crucial importance of sterols during the ontogenesis of multicellular organisms. First oxygen appeared on the Earth app. 2.7 billion years ago and since that time, every new life form took the advantage of oxygen needed also for build-up of sterol molecules. The last decades changed our view to the sterol molecules on almost at all levels of their appearance in human body. In the gut, the absorption of sterols was proven to be protein dependent and the quest for the transporter was successful. The general concepts of intracellular homeostasis of cholesterol have been described including the covalent interaction unbelievable so far - cholesterol and a protein. The clinical importance of non-cholesterol sterols rises with the effort to discover underlying facts about the causes of atherosclerosis. The

  1. Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.

    PubMed

    Ferdinandusse, S; Kostopoulos, P; Denis, S; Rusch, H; Overmars, H; Dillmann, U; Reith, W; Haas, D; Wanders, R J A; Duran, M; Marziniak, M

    2006-06-01

    In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7). PMID:16685654

  2. 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

  3. Alterations in the predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae.

    PubMed

    Carter, Helen E; Fraaije, Bart A; West, Jonathan S; Kelly, Steven L; Mehl, Andreas; Shaw, Michael W; Cools, Hans J

    2014-06-01

    The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37:erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed. PMID:24298976

  4. Sterol regulatory element binding protein-1 (SREBP-1)c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor α in goat mammary epithelial cells.

    PubMed

    Xu, H F; Luo, J; Wang, H P; Wang, H; Zhang, T Y; Tian, H B; Yao, D W; Loor, J J

    2016-02-01

    Sterol regulatory element binding protein-1 (SREBP-1) is a key transcription factor that regulates lipogenesis in rodent liver. Two isoforms (SREBP-1a and SREBP-1c) of SREBP-1 are transcribed by an alternative promoter on the same gene (SREBF1), and the isoforms differ only in their first exon. Although the regulatory effects of SREBP-1 on lipid and milk fat synthesis have received much attention in ruminants, SREBP-1c promoter and its regulatory mechanisms have not been characterized in the goat. In the present study, we cloned and sequenced a 2,012-bp fragment of the SREBP-1c 5'-flanking region from goat genomic DNA. A luciferase reporter assay revealed that SREBP-1c is transcriptionally activated by the liver X receptor α (LXRα) agonist T0901317, and is decreased by SREBP-1 small interfering (si)RNA. A 5' deletion analysis revealed a core promoter region located -395 to +1 bp upstream of the transcriptional start site (TSS). Site-directed mutagenesis of LXRα binding elements (LXRE1 and LXRE2) and sterol regulatory elements (SRE1 and SRE2) revealed that the full effects of T 4506585 require the presence of both LXRE and SRE. We also characterized a new SRE (SRE1) and demonstrated a direct role of SREBP-1 (auto-loop regulation) in maintaining its basal transcription activity. Results suggest that goat SREBP-1c gene is transcriptionally regulated by mature SREBP-1 (auto-loop circuit regulation) and LXRα in goat mammary epithelial cells. PMID:26709176

  5. A Sterol-Regulatory Element Binding Protein Is Required for Cell Polarity, Hypoxia Adaptation, Azole Drug Resistance, and Virulence in Aspergillus fumigatus

    PubMed Central

    Willger, Sven D.; Puttikamonkul, Srisombat; Kim, Kwang-Hyung; Burritt, James B.; Grahl, Nora; Metzler, Laurel J.; Barbuch, Robert; Bard, Martin; Lawrence, Christopher B.; Cramer, Robert A.

    2008-01-01

    At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds. PMID:18989462

  6. 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. PMID:26822063

  7. The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis.

    PubMed

    Prakash, A; Kasbekar, D P

    2002-01-01

    Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor. We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa. The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N. crassa mutant ( erg-3). The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae. The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH. PMID:11810252

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

    PubMed Central

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

    2014-01-01

    An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10−7). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism. PMID:25201972

  9. Andrographolide prevents high-fat diet-induced obesity in C57BL/6 mice by suppressing the sterol regulatory element-binding protein pathway.

    PubMed

    Ding, Lili; Li, Jinmei; Song, Baoliang; Xiao, Xu; Huang, Wendong; Zhang, Binfeng; Tang, Xiaowen; Qi, Meng; Yang, Qiming; Yang, Qiaoling; Yang, Li; Wang, Zhengtao

    2014-11-01

    Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the expression of genes involved in biosynthesis of cholesterol, fatty acids, and triglycerides. We investigated the effect of the specific SREBP suppressor andrographolide, a natural compound isolated from Andrographis paniculata, on the regulation of SREBP signaling by use of Western blot, reporter gene assay, and quantitative real-time polymerase chain reaction analysis. In addition, the antiobesity effects of andrographolide were evaluated in C57BL/6 mice with high-fat diet (HFD)-induced obesity. Our results showed that andrographolide downregulated the expressions of SREBPs target genes and decreased cellular lipid accumulation in vitro. Further, andrographolide (100 mg/kg per day) attenuated HFD-induced body weight gain and fat accumulation in liver or adipose tissues, and improved serum lipid levels and insulin or glucose sensitivity in HFD-induced obese mice. Andrographolide effectively suppressed the respiratory quotient, energy expenditure, and oxygen consumption, which may have contributed to the decreased body-weight gain of the obese mice fed with a HFD. Consistently, andrographolide regulated SREBP target genes and metabolism-associated genes in liver or brown adipose tissue, which may have directly contributed to the lower lipid levels and enhanced insulin sensitivity. Taken together, our results indicated that andrographolide ameliorated lipid metabolism and improved glucose use in mice with HFD-induced obesity. Andrographolide has potential as a leading compound in the prevention or treatment of obesity and insulin resistance. PMID:25204338

  10. 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. PMID:25659129

  11. Renoprotective effect of myricetin restrains dyslipidemia and renal mesangial cell proliferation by the suppression of sterol regulatory element binding proteins in an experimental model of diabetic nephropathy.

    PubMed

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-11-15

    Myricetin is a natural flavonoid used in various health management systems. In this present study myricetin tested to evaluate the effect on lipids and lipid metabolism enzymes in normal and streptozotocin (STZ) with cadmium (Cd) induced diabetic nephrotoxic rats. Diabetic nephrotoxic rats were significantly (P<0.05) increased the levels of urinary albumin and lipid profiles: total cholesterol (TC), triglycerides (TGs), free fatty acids (FFAs), phospholipids (PLs), low density lipoprotein (LDL), very low-density lipoproteins (VLDL), and decreased in the levels of high-density lipoproteins (HDL). In addition, the activity of lipoprotein lipase (LPL) and lecithin cholesterol acyl transferase (LCAT) were decreased significantly, whereas the 3-hydroxy 3-methylglutaryl coenzyme A (HmgCoA) reductase activity was increased. The upregulation of sterol regulatory element binding protein-1a (SREBP-1a), SREBP-1c, SREBP-2, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF) and downregulation peroxisome proliferator-activated receptor alpha (PPAR-α) proteins expression levels were noticed. An administration of myricetin (1.0 mg/kg body weight (b/w)) for 12 weeks was brought the above parameters towards normal level. Histopathological study of kidney samples showed that extracellular mesangial matrix expansion, glomerulosclerosis and interstitial fibrosis in diabetic nephrotoxic rats was suppressed by myricetin treatment. Further our results indicate that administration of myricetin afforded remarkable protection against STZ-Cd induced alterations in lipid metabolism and thereby reduced the diabetic nephropathy in experimental rats. PMID:25240712

  12. Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

    PubMed Central

    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. PMID:22384276

  13. Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways.

    PubMed

    Hatori, Megumi; Hirota, Tsuyoshi; Iitsuka, Michiko; Kurabayashi, Nobuhiro; Haraguchi, Shogo; Kokame, Koichi; Sato, Ryuichiro; Nakai, Akira; Miyata, Toshiyuki; Tsutsui, Kazuyoshi; Fukada, Yoshitaka

    2011-03-22

    The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland. PMID:21383147

  14. 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

  15. 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

    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

  16. 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

  17. 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. PMID:26327595

  18. FoxO4 interacts with the sterol regulatory factor SREBP2 and the hypoxia inducible factor HIF2α at the CYP51 promoter

    PubMed Central

    Zhu, Jun; Jiang, Xiangning; Chehab, Farid F.

    2014-01-01

    The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression. PMID:24353279

  19. 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.

  20. The relationship of sterol regulatory element-binding protein cleavage-activation protein and apolipoprotein E gene polymorphisms with metabolic changes during weight reduction.

    PubMed

    Nieminen, Tuomo; Matinheikki, Jussi; Nenonen, Arja; Kukkonen-Harjula, Katriina; Lindi, Virpi; Hämelahti, Päivi; Laaksonen, Reijo; Fan, Yue-Mei; Kähönen, Mika; Fogelholm, Mikael; Lehtimäki, Terho

    2007-07-01

    Sterol regulatory element-binding protein cleavage-activating protein (SCAP) and apolipoprotein E (apo E) regulate cellular and plasma lipid metabolism. Therefore, variations in the corresponding genes might influence weight reduction and obesity-associated metabolic changes. We investigated the relationships of SCAP (Ile796Val) and apo E polymorphisms on metabolic changes during weight reduction by using a 12-week very low-energy diet. Body composition, serum lipids, plasma glucose, and insulin were assessed in 78 healthy premenopausal women (initial body mass index, 34 +/- 4 kg/m(2); age, 40 +/- 4 years) before and after the intervention. The SCAP genotype groups did not differ in the responses of any parameters measured during weight reduction. Apo E did not differentiate the weight loss, but the changes in total and low-density lipoprotein cholesterol for the genotype groups apo E epsilon2/3, epsilon3/3, as well as epsilon3/4 and epsilon4/4 combined were -0.94 +/- 0.56 and -0.59 +/- 0.32, -0.71 +/- 0.49 and -0.49 +/- 0.45, and -0.55 +/- 0.47 and -0.37 +/- 0.39 mmol/L, respectively (P < .05 for both). In conclusion, neither the SCAP Ile796Val nor the apo E polymorphism was associated with weight loss in obese premenopausal women. However, the apo E-but not SCAP genotype-seems to be one of the modifying factors for serum cholesterol concentrations during very low-energy diet in obese premenopausal women. PMID:17570245

  1. 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.

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

    PubMed

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

    2016-02-12

    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

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

    PubMed

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

    2016-05-13

    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

  4. 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. PMID:16122768

  5. 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. PMID:22225954

  6. 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

  7. 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

  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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. PMID:26552797

  11. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    PubMed Central

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  12. Recent advances in sterol research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since 1970, the AOCS has been a regular host to the sterol symposia. The 2008 Sterol Symposium, “Recent Advances in Sterol Research,” was held at the AOCS Annual Meeting in Seattle, Washington. This year the symposium held special significance, for it hosted the presentation of the fourth G.J. Schro...

  13. 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

  14. 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

  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. Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.

    PubMed

    Gsaller, Fabio; Hortschansky, Peter; Furukawa, Takanori; Carr, Paul D; Rash, Bharat; Capilla, Javier; Müller, Christoph; Bracher, Franz; Bowyer, Paul; Haas, Hubertus; Brakhage, Axel A; Bromley, Michael J

    2016-07-01

    Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis. PMID:27438727

  17. Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex

    PubMed Central

    Gsaller, Fabio; Furukawa, Takanori; Carr, Paul D.; Rash, Bharat; Capilla, Javier; Müller, Christoph; Bracher, Franz; Bowyer, Paul; Haas, Hubertus; Brakhage, Axel A.; Bromley, Michael J.

    2016-01-01

    Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis. PMID:27438727

  18. Plant Sterols, Stanols, and Sitosterolemia

    PubMed Central

    Ajagbe, Bridget O.; Othman, Rgia A.; Myrie, Semone B.

    2015-01-01

    Phytosterolemia (sitosterolemia) is a rare autosomal recessive sterol storage disease caused by mutations in either of the adenosine triphosphate (ATP) binding cassette transporter genes; (ABC)G5 or ABCG8, leading to impaired elimination of plant sterols and stanols, with their increased accumulation in the blood and tissues. Thus the disease is characterized by substantially elevated serum plant sterols and stanols, with moderate to high plasma cholesterol levels, and increased risk of premature atherosclerosis. Hematologic abnormalities including macrothrombocytopenia, stomatocytosis and hemolysis are frequently observed in sitosterolemia patients. Currently, ezetimibe, a sterol absorption inhibitor, is used as the routine treatment for sitosterolemia, with reported improvement in plant sterol levels and hemolytic parameters. This review summarizes the research related to the health impact of plant sterols and stanols on sitosterolemia. PMID:25941971

  19. Sterol requirement of Mycoplasma capricolum.

    PubMed Central

    Odriozola, J M; Waitzkin, E; Smith, T L; Bloch, K

    1978-01-01

    Mycoplasmas require an external source of sterol for growth. For Mycoplasma capricolum this requirement is met not only by cholesterol but also by the methylcholestane derivatives lanosterol, cycloartenol, 4,4-dimethylcholesterol, and 4beta-methylcholestanol. Cholesteryl methyl ether and 3alpha-methylcholestanol serve equally well as sterol supplements. None of the growth-supporting sterol derivatives tested was metabolically modified. The unusual acceptance of diverse cholestane derivatives by a mycoplasma species contrasts with the structural attributes thought to be necessary for sterol function in eukaryotic membranes. PMID:279900

  20. 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

  1. Bezafibrate at clinically relevant doses decreases serum/liver triglycerides via down-regulation of sterol regulatory element-binding protein-1c in mice: a novel peroxisome proliferator-activated receptor alpha-independent mechanism.

    PubMed

    Nakajima, Takero; Tanaka, Naoki; Kanbe, Hiroki; Hara, Atsushi; Kamijo, Yuji; Zhang, Xiaowei; Gonzalez, Frank J; Aoyama, Toshifumi

    2009-04-01

    The triglyceride-lowering effect of bezafibrate in humans has been attributed to peroxisome proliferator-activated receptor (PPAR) alpha activation based on results from rodent studies. However, the bezafibrate dosages used in conventional rodent experiments are typically higher than those in clinical use (> or =50 versus < or =10 mg/kg/day), and thus it remains unclear whether such data can be translated to humans. Furthermore, because bezafibrate is a pan-PPAR activator, the actual contribution of PPARalpha to its triglyceride-lowering properties remains undetermined. To address these issues, bezafibrate at clinically relevant doses (10 mg/kg/day; low) was administered to wild-type and Ppara-null mice, and its effects were compared with those from conventionally used doses (100 mg/kg/day; high). Pharmacokinetic analyses showed that maximum plasma concentration and area under the concentration-time curve in bezafibrate-treated mice were similar to those in humans at low doses, but not at high doses. Low-dose bezafibrate decreased serum/liver triglycerides in a PPARalpha-independent manner by attenuation of hepatic lipogenesis and triglyceride secretion. It is noteworthy that instead of PPAR activation, down-regulation of sterol regulatory element-binding protein (SREBP)-1c was observed in mice undergoing low-dose treatment. High-dose bezafibrate decreased serum/liver triglycerides by enhancement of hepatic fatty acid uptake and beta-oxidation via PPARalpha activation, as expected. In conclusion, clinically relevant doses of bezafibrate exert a triglyceride-lowering effect by suppression of the SREBP-1c-regulated pathway in mice and not by PPARalpha activation. Our results may provide novel information about the pharmacological mechanism of bezafibrate action and new insights into the treatment of disorders involving SREBP-1c. PMID:19124612

  2. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  3. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  4. 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

    2016-03-01

    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

  5. The Effects of Sterol Structure upon Sterol Esterification

    PubMed Central

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

    2011-01-01

    Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT1 and ACAT2 (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 percent and in tissues by ACAT was 64 percent. 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 percent and decreased the esterification by LCAT by 5.9 percent. Esterification by ACAT and LCAT was reduced respectively by 25 percent and 11 percent 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. PMID:19679306

  6. 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

  7. Characterization of a novel bile-inducible operon encoding a two-component regulatory system in Lactobacillus acidophilus.

    PubMed

    Pfeiler, Erika A; Azcarate-Peril, M Andrea; Klaenhammer, Todd R

    2007-07-01

    Lactobacillus acidophilus NCFM is an industrially important strain used extensively as a probiotic culture. Tolerance of the presence of bile is an attribute important to microbial survival in the intestinal tract. A whole-genome microarray was employed to examine the effects of bile on the global transcriptional profile of this strain, with the intention of elucidating genes contributing to bile tolerance. Genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. A 7-kb eight-gene operon encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins was significantly upregulated in the presence of bile. Deletion mutations were constructed in six genes of the operon. Transcriptional analysis of the 2CRS mutants showed that mutation of the histidine protein kinase (HPK) had no effect on the induction of the operon, whereas the mutated response regulator (RR) showed enhanced induction when the cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance and that the operon it resides in is negatively controlled by the RR. Mutations in the transporter, the HPK, the RR, and a hypothetical protein each resulted in loss of tolerance of bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. This functional analysis showed that the operon encoded proteins involved in both bile tolerance and bile sensitivity. PMID:17449631

  8. Distinct Roles of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factors in Inflammatory Response and Cancer

    PubMed Central

    Baresova, Petra; Pitha, Paula M.

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Similar to other herpesviruses, KSHV has two life cycles, latency and lytic replication. In latency, the KSHV genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed. In this review, we focus on oncogenic, antiapoptotic, and immunomodulating properties of KSHV-encoded homologues of cellular interferon regulatory factors (IRFs)—viral IRF1 (vIRF1) to vIRF4—and their possible role in the KSHV-mediated antiviral response, apoptosis, and oncogenicity. PMID:23785197

  9. Inhibition of mTOR complex 2 induces GSK3/FBXW7-dependent degradation of sterol regulatory element-binding protein 1 (SREBP1) and suppresses lipogenesis in cancer cells

    PubMed Central

    Li, Shaohua; Oh, You-Take; Yue, Ping; Khuri, Fadlo R.; Sun, Shi-Yong

    2015-01-01

    Cancer cells feature increased de novo lipogenesis. Sterol regulatory element-binding protein 1 (SREBP1), when presented in its mature form (mSREBP1), enhances lipogenesis through increasing transcription of several of its target genes. Mammalian target of rapamycin (mTOR) complexes, mTORC1 and mTORC2, are master regulators of cellular survival, growth and metabolism. A role for mTORC1 in the regulation of SREBP1 activity has been suggested; however the connection between mTORC2 and SREBP1 has not been clearly established and hence is the focus of this study. mTOR kinase inhibitors (e.g., INK128), which inhibit both mTORC1 and mTORC2, decreased mSREBP1 levels in various cancer cell lines. Knockdown of rictor, but not raptor, also decreased mSREBP1. Consistently, reduced mSREBP1 levels were detected in cells deficient in rictor or Sin1 compared to parent or rictor-deficient cells with re-expression of ectopic rictor. Hence it is mTORC2 inhibition that causes mSREBP1 reduction. As a result, expression of the mSREBP1 target genes acetyl-CoA carboxylase and fatty acid synthase was suppressed, accompanied with suppressed lipogenesis in cells exposed to INK128. Moreover, mSREBP1 stability was reduced in cells treated with INK128 or rictor knockdown. Inhibition of proteasome, GSK3 or the E3 ubiquitin ligase, FBXW7, prevented mSREBP1 reduction induced by mTORC2 inhibition. Thus mTORC2 inhibition clearly facilitates GSK3-dependent, FBXW7-mediated mSREBP1 degradation, leading to mSREBP1 reduction. Accordingly, we conclude that mTORC2 positively regulates mSREBP1 stability and lipogenesis. Our findings reveal a novel biological function of mTORC2 in the regulation of lipogenesis and warrant further study in this direction. PMID:25893295

  10. Inhibition of mTOR complex 2 induces GSK3/FBXW7-dependent degradation of sterol regulatory element-binding protein 1 (SREBP1) and suppresses lipogenesis in cancer cells.

    PubMed

    Li, S; Oh, Y-T; Yue, P; Khuri, F R; Sun, S-Y

    2016-02-01

    Cancer cells feature increased de novo lipogenesis. Sterol regulatory element-binding protein 1 (SREBP1), when presented in its mature form (mSREBP1), enhances lipogenesis by increasing transcription of several of its target genes. Mammalian target of rapamycin (mTOR) complexes, mTORC1 and mTORC2, are master regulators of cellular survival, growth and metabolism. A role for mTORC1 in the regulation of SREBP1 activity has been suggested; however, the connection between mTORC2 and SREBP1 has not been clearly established and hence is the focus of this study. mTOR kinase inhibitors (for example, INK128), which inhibit both mTORC1 and mTORC2, decreased mSREBP1 levels in various cancer cell lines. Knockdown of rictor, but not raptor, also decreased mSREBP1. Consistently, reduced mSREBP1 levels were detected in cells deficient in rictor or Sin1 compared with parent or rictor-deficient cells with re-expression of ectopic rictor. Hence it is mTORC2 inhibition that causes mSREBP1 reduction. As a result, expression of the mSREBP1 target genes acetyl-CoA carboxylase and fatty-acid synthase was suppressed, along with suppressed lipogenesis in cells exposed to INK128. Moreover, mSREBP1 stability was reduced in cells treated with INK128 or rictor knockdown. Inhibition of proteasome, GSK3 or the E3 ubiquitin ligase, FBXW7, prevented mSREBP1 reduction induced by mTORC2 inhibition. Thus mTORC2 inhibition clearly facilitates GSK3-dependent, FBXW7-mediated mSREBP1 degradation, leading to mSREBP1 reduction. Accordingly, we conclude that mTORC2 positively regulates mSREBP1 stability and lipogenesis. Our findings reveal a novel biological function of mTORC2 in the regulation of lipogenesis and warrant further study in this direction. PMID:25893295

  11. Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties.

    PubMed Central

    Defechereux, P; Debrus, S; Baudoux, L; Rentier, B; Piette, J

    1997-01-01

    Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type 1: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat- or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA. PMID:9261438

  12. [Sterols in Stevia rebaudiana Bertoni].

    PubMed

    D'Agostino, M; De Simone, F; Pizza, C; Aquino, R

    1984-12-30

    The sterol fraction of Stevia rebaudiana Bertoni contains, essentially, the following sterols: stigmasterol (45,8%), beta-sitosterol (39,4%) and campesterol (13,1%). The individual components were separated, after acetylation, by HPLC with absolute methanol as eluant. The identification of the compounds has been carried out through NMR and MS, while the corresponding percentages have been desumed from the GLC data. PMID:6529501

  13. 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

  14. 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

  15. 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.

  16. MarRA, SoxSR, and Rob encode a signal dependent regulatory network in Escherichia coli.

    PubMed

    Jain, Kirti; Saini, Supreet

    2016-05-24

    When exposed to low concentrations of toxic chemicals, bacteria modulate the expression of a number of cellular processes. Typically, these processes include those related to porin production, dismutases, and metabolic fluxes. In Escherichia coli (E. coli), the expression of these systems is largely controlled by three homologous transcriptional regulators: MarA, SoxS, and Rob. Each of the three regulators responds to distinct chemical signals (salicylate for MarA; paraquat for SoxS; and bipyridyl for Rob) and controls the expression of an overlapping set of downstream targets. In addition, the three systems autoregulate their own expression, and cross-regulate each other's expression. Specifically, MarA is known to activate SoxS expression, and Rob is known to activate MarA expression. In addition, a number of conflicting regulatory interactions are known to exist between the three loci. Thus, the three systems encode a complex regulatory topology with multiple feedback loops, the precise nature of whose interactions or their significance in cellular physiology is not well understood currently. In this work, we focus on understanding the details of this crosstalk between the Mar-Sox-Rob systems in E. coli, and the resulting control and dynamics of the expression of cellular processes by studying gene expression at the population level and at single-cell resolution in wild type and mutants. Our results indicate that the regulatory architecture between MarA, SoxS, and Rob is dependent on the signal (inducer) present in the environment. The regulators, in response to an inducer, form a Feed Forward Loop (FFL), which leads to faster and stronger induction of target genes in the cell, consequently resulting in better cellular growth. Through the FFL, the cell is able to integrate qualitatively different signals in the network, and consequently, control cellular physiology. In addition, we present two intriguing dynamic features of the Mar-Sox-Rob regulon. First, in the

  17. 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.

  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. A new sterol glycoside from Securidaca inappendiculata.

    PubMed

    Zhang, Li-Jie; Yang, Xue-Dong; Xu, Li-Zhen; Zou, Zhong-Mei; Yang, Shi-Lin

    2005-08-01

    From the roots of Securidaca inappendiculata, one new sterol glycoside securisteroside (1) has been isolated, along with two known sterols, spinasterol (2) and 3-O-beta-D-glucopyranosyl-spinasterol (3). The new sterol was characterized by chemical and spectrometric methods, including EIMS, FABMS and one- and two-dimensional NMR experiments. PMID:16087640

  20. Sterol dynamics during endocytic trafficking in Arabidopsis.

    PubMed

    Stanislas, Thomas; Grebe, Markus; Boutté, Yohann

    2014-01-01

    Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking. PMID:25117272

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

    PubMed Central

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

    2014-01-01

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

  2. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene (SCP2)

    SciTech Connect

    Ohba, Takashi; Rennert, H.; Pfeifer, S.M.

    1994-11-15

    Sterol carrier protein X (SCPx) is a 58-kDa protein that is localized to peroxisomes. The amino acid sequence of the protein suggests that SCPx may function as a thiolase. The gene encoding SCPx also codes for a 15.3-kDa protein called sterol carrier protein 2 (SCP{sub 2}). Here the authors report the structure of this gene (SCP2), which spans approximately 80 kb and consists of 16 exons and 15 introns. Multiple transcription start sites were identified. The 5{prime} flanking region has characteristics of other peroxisomal protein promoters, which include the absence of a TATA box and G+C-enriched region containing several reverse GC boxes. 24 refs., 3 figs., 1 tab.

  3. FACKEL is a sterol C-14 reductase required for organized cell division and expansion in Arabidopsis embryogenesis

    PubMed Central

    Schrick, Kathrin; Mayer, Ulrike; Horrichs, Andrea; Kuhnt, Christine; Bellini, Catherine; Dangl, Jeff; Schmidt, Jürgen; Jürgens, Gerd

    2000-01-01

    In flowering plants, the developing embryo consists of growing populations of cells whose fates are determined in a position-dependent manner to form the adult organism. Mutations in the FACKEL (FK) gene affect body organization of the Arabidopsis seedling. We report that FK is required for cell division and expansion and is involved in proper organization of the embryo. We isolated FK by positional cloning. Expression analysis in embryos revealed that FK mRNA becomes localized to meristematic zones. FK encodes a predicted integral membrane protein related to the vertebrate lamin B receptor and sterol reductases across species, including yeast sterol C-14 reductase ERG24. We provide functional evidence that FK encodes a sterol C-14 reductase by complementation of erg24. GC/MS analysis confirmed that fk mutations lead to accumulation of intermediates in the biosynthetic pathway preceding the C-14 reductase step. Although fk represents a sterol biosynthetic mutant, the phenotype was not rescued by feeding with brassinosteroids (BRs), the only plant sterol signaling molecules known so far. We propose that synthesis of sterol signals in addition to BRs is important in mediating regulated cell growth and organization during embryonic development. Our results indicate a novel role for sterols in the embryogenesis of plants. PMID:10859166

  4. 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

  5. Sterol phylogenesis and algal evolution

    SciTech Connect

    Nes, W.D.; Norton, R.A.; Crumley, F.G. ); Madigan, S.J.; Katz, E.R. )

    1990-10-01

    The stereochemistry of several sterol precursors and end products synthesized by two fungal-like microorganisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and {sup 1}H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecy-cloartanol, ergosterol, protothecasterol, 4{alpha}-methylergostanol, 4{alpha}-methylclionastanol, clionastanol, 24{beta}-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I (2-{sup 3}H)lanosterol, (2-{sup 3}H)cycloartenol, (24{sup 3}H)lanosterol, and (methyl-{sup 2}H{sub 3})methionine and by feeding to II (methyl-{sup 2}H{sub 3})methionine. The results demonstrate that the 24{beta} configuration is formed by different alkylation routes in I and II. The authors conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors.

  6. The sterols of the echinoderm Asterias rubens

    PubMed Central

    Smith, Andrew G.; Rubinstein, Ian; Goad, L. John

    1973-01-01

    1. Twenty-two sterols were identified in the starfish Asterias rubens (Phylum, Echinodermata; Class, Asteroidea). 2. The major 4-demethyl sterols had a Δ7 bond and the C27 compound 5α-cholest-7-en-3β-ol predominated over other mono- and di-unsaturated sterols belonging to the C26, C27, C28 and C29 series. 3. Small amounts of cholest-5-en-3β-ol and 5α-cholestan-3β-ol were also present. 4. The minor sterols identified all contained either one or two methyl groups at C-4 and are considered to be potential biosynthetic precursors of 5α-cholest-7-en-3β-ol. 5. Three sterols possessing a 9β,19-cyclopropane ring were also isolated and were probably derived by the starfish from a dietary source. PMID:4772271

  7. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. 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. PMID:14645274

  10. 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

  11. A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis.

    PubMed

    Khunajakr, N; Liu, C Q; Charoenchai, P; Dunn, N W

    1999-03-18

    Two regulatory genes (lcoR and lcoS) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (cat). RT-PCR analysis indicates that lcoR and lcoS are organized within an operon, controlling the transcription of cat in a copper-inducible manner. The amino acid sequences deduced from lcoR and lcoS show homology to the response and sensor proteins of known two-component regulatory systems. Deletion within either lcoS or both genes inactivated the copper-dependent activity, suggesting the presence of no trans-acting lcoR and lcoS homologs in the lactococcal host chromosome. The transcription start site involved in copper induction was mapped by primer extension. PMID:10095123

  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. PMID:9148788

  13. Characterization of a Novel Human Herpesvirus 8-Encoded Protein, vIRF-3, That Shows Homology to Viral and Cellular Interferon Regulatory Factors

    PubMed Central

    Lubyova, Barbora; Pitha, Paula M.

    2000-01-01

    The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10.6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity. PMID:10933732

  14. 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

  15. Sterols from the Madagascar sponge Fascaplysinopsis sp.

    PubMed

    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, 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. PMID:21339959

  16. Sterol synthesis in the human arterial intima

    PubMed Central

    Chobanian, Aram V.

    1968-01-01

    Intimal sterol synthesis was examined in isolated human arterial segments obtained at surgery or at postmortem examination. The tissues were incubated with acetate-1-14C and mevalonate-2-14C and the incorporation of these precursors into sterols was determined. Intimal sterols were isolated by multiple chromatographic techniques and purified by bromination and oxidation procedures. The results indicate that the arterial intima can incorporate acetate and mevalonate into cholesterol, cholestanol, and squalene. Cholestanol was the major sterol synthesized by the arterial wall, but cholesterol production was also consistently observed. The findings suggest that local synthesis is a potential source of sterol accumulation within the arterial wall. The conversion of cholesterol to other sterols was also studied in terminally ill patients receiving labeled cholesterol before death. Tissue analyses revealed the presence of labeled cholestanol as well as cholesterol in the tissue 5-104 days after labeled cholesterol administration. The results demonstrate the conversion of cholesterol to cholestanol in man and suggest that the exchange of cholestanol between the blood and tissues is similar to that of cholesterol. PMID:5637146

  17. 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

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

    PubMed

    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. Design of modular "plug-and-play" expression platforms derived from natural riboswitches for engineering novel genetically encodable RNA regulatory devices.

    PubMed

    Trausch, Jeremiah J; Batey, Robert T

    2015-01-01

    Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA leaders and regulating their expression. These regulatory elements are generally composed of two domains: an aptamer that binds a specific effector molecule and an expression platform that informs the transcriptional or translational machinery. While it was long established that riboswitch aptamers are modular and portable, capable of directing different output domains including ribozymes, switches, and fluorophore-binding modules, the same has not been demonstrated until recently for expression platforms. We have engineered and validated a set of expression platforms that regulate transcription through a secondary structural switch that can host a variety of different aptamers, including those derived through in vitro selection methods, to create novel chimeric riboswitches. These synthetic switches are capable of a highly specific regulatory response both in vitro and in vivo. Here we present the methodology for the design and engineering of chimeric switches using biological expression platforms. PMID:25605380

  20. Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis.

    PubMed

    Berkow, Elizabeth L; Manigaba, Kayihura; Parker, Josie E; Barker, Katherine S; Kelly, Stephen L; Rogers, P David

    2015-10-01

    While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis. PMID:26169412

  1. Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis

    PubMed Central

    Berkow, Elizabeth L.; Manigaba, Kayihura; Parker, Josie E.; Barker, Katherine S.; Kelly, Stephen L.

    2015-01-01

    While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis. PMID:26169412

  2. The Drosophila gene taranis encodes a novel trithorax group member potentially linked to the cell cycle regulatory apparatus.

    PubMed Central

    Calgaro, Stéphane; Boube, Muriel; Cribbs, David L; Bourbon, Henri-Marc

    2002-01-01

    Genes of the Drosophila Polycomb and trithorax groups (PcG and trxG, respectively) influence gene expression by modulating chromatin structure. Segmental expression of homeotic loci (HOM) initiated in early embryogenesis is maintained by a balance of antagonistic PcG (repressor) and trxG (activator) activities. Here we identify a novel trxG family member, taranis (tara), on the basis of the following criteria: (i) tara loss-of-function mutations act as genetic antagonists of the PcG genes Polycomb and polyhomeotic and (ii) they enhance the phenotypic effects of mutations in the trxG genes trithorax (trx), brahma (brm), and osa. In addition, reduced tara activity can mimic homeotic loss-of-function phenotypes, as is often the case for trxG genes. tara encodes two closely related 96-kD protein isoforms (TARA-alpha/-beta) derived from broadly expressed alternative promoters. Genetic and phenotypic rescue experiments indicate that the TARA-alpha/-beta proteins are functionally redundant. The TARA proteins share evolutionarily conserved motifs with several recently characterized mammalian nuclear proteins, including the cyclin-dependent kinase regulator TRIP-Br1/p34(SEI-1), the related protein TRIP-Br2/Y127, and RBT1, a partner of replication protein A. These data raise the possibility that TARA-alpha/-beta play a role in integrating chromatin structure with cell cycle regulation. PMID:11861561

  3. Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

    PubMed Central

    Alexandersen, S; Carpenter, S; Christensen, J; Storgaard, T; Viuff, B; Wannemuehler, Y; Belousov, J; Roth, J A

    1993-01-01

    The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown. Images PMID:8380084

  4. 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

  5. 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.

  6. Monitoring sterol uptake, acetylation, and export in yeast.

    PubMed

    Choudhary, Vineet; Schneiter, Roger

    2009-01-01

    Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however, occurs only under anaerobic conditions, where the cells become sterol auxotroph, or in mutant cells that cannot synthesize heme. The reverse sterol transport pathway, however, is more amenable to experimental studies, because arrival of the sterol in the ER membrane can be monitored unambiguously by following the formation of steryl esters. Apart from sterol acylation, we have recently described a reversible sterol acetylation cycle that is operating in the lumen of the ER. Acetylation occurs on both cholesterol and pregnenolone, a steroid precursor, and serves as a signal for export of the acetylated sterols into the culture media. The time-dependent appearance of acetylated sterols in the culture supernatant thus provides a new means to monitor the forward transport of chemically modified sterols out of the ER. PMID:19784602

  7. 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

  8. 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

    2016-01-01

    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

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

    PubMed Central

    Caron, Kathleen M.; Soo, Shiu-Ching; Wetsel, William C.; Stocco, Douglas M.; Clark, Barbara J.; Parker, Keith L.

    1997-01-01

    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. PMID:9326645

  10. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  12. Sterol composition of Bulgarian soya and corn oils.

    PubMed

    Milkova, T; Popov, A; Selva, A; Vettori, U

    1977-01-01

    The free sterols, the sterol esters and the sterol glycosides of the raw soya and corn oils as well as those of the technical lecithin and the deodorizer distillated of the latter oils were isolated by preparative TLC. The composition of each of the isolated sterol derivatives was determined by GLC and MS. Sitosterol, campesterol, stigmasterol and an unknown sterol with a molecular weight of 428 are contained in almost all of the examined fractions of the soya oil and its refinement byproducts. Dehydrocampesterol is present in the free sterols of the raw soya oil and the soya lecithin. Stigmasterol is contained in the soya deodorizer distillate in high amounts. It was established that cholesterol was present in the sterol esters of the raw soya oil high amounts. Delta7-stigmastenol occurs only in the sterol esters of the latter oil. Sitosterol, campesterol and stimgasterol are the main components of all sterol fractions of the corn oil and its refinement products. Dehydrocampesterol and unknown sterols with molecular weights of 428 are present in the free sterols of the raw corn oil. Some sterol glycosides of the soya and corn lecithin are esterified with the same major fatty acid components of the glycerides, palmitic acid being the main one. The fatty acid compositon of sterol esters of the raw soya and corn oil roughly corresponds to the fatty acid composition of oils. PMID:558512

  13. p53 Tumor Suppressor Protein Stability and Transcriptional Activity Are Targeted by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factor 3

    PubMed Central

    Baresova, Petra; Musilova, Jana; Pitha, Paula M.

    2014-01-01

    Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth. PMID:24248600

  14. 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. PMID:12946407

  15. Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

    PubMed Central

    Platero, Ana Isabel; García-Jaramillo, Manuel; Santero, Eduardo

    2012-01-01

    The atzS-atzT-atzU-atzV-atzW gene cluster of the Pseudomonas sp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream from atzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operon atzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzR promoter to form the atzRSTUVW operon. A second, stronger promoter, PatzT, is found within atzS and directs transcription of the four distal genes. PatzT is σN dependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination of in vivo mutational analysis and primer extension allowed us to locate the PatzT promoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along with in vivo expression studies and in vitro binding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzT transcription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process. PMID:23042989

  16. Mutations in GAS8, a Gene Encoding a Nexin-Dynein Regulatory Complex Subunit, Cause Primary Ciliary Dyskinesia with Axonemal Disorganization.

    PubMed

    Jeanson, Ludovic; Thomas, Lucie; Copin, Bruno; Coste, André; Sermet-Gaudelus, Isabelle; Dastot-Le Moal, Florence; Duquesnoy, Philippe; Montantin, Guy; Collot, Nathalie; Tissier, Sylvie; Papon, Jean-François; Clement, Annick; Louis, Bruno; Escudier, Estelle; Amselem, Serge; Legendre, Marie

    2016-08-01

    Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans. PMID:27120127

  17. Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

    PubMed Central

    Puhl, Henry L.; Ikeda, Stephen R.

    2008-01-01

    Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

  18. Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update.

    PubMed

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

    2010-04-01

    PRKAR1A encodes the regulatory subunit type 1-alpha (RIalpha) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (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 (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIalpha haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIalpha haploinsufficiency, but also by the expression of altered RIalpha protein, as proven by analysis of expressed mutations in the gene, consisting of amino acid 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-these are frame-shifts in the 3' end of the coding sequence that shift the stop codon downstream of the normal one. The 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 report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. PMID:20358582

  19. A sterol with an unusual side chain from Anoectochilus koshunensis.

    PubMed

    Ito, A; Yasumoto, K; Kasai, R; Yamasaki, K

    1994-08-01

    A new sterol with a non-conventional side chain has been isolated from the whole plant of Anoectochilus koshunensis, together with four known sterols, a megastigmane glucoside and 2'-deoxyadenosine. The structure of the new sterol was elucidated as 26-methylstigmasta-5,22,25, (27)-trien-3 beta-ol based on chemical and detailed spectroscopic evidence. PMID:7765430

  20. 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. PMID:24211569

  1. 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. PMID:12398993

  2. Nuclear hormone receptors put immunity on sterols.

    PubMed

    Santori, Fabio R

    2015-10-01

    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 nonclassic (all others) NHRs; 17 nonclassic 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 nonsterol intermediates and derivatives, is a source of ligands for many classic and nonclassic 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 nonclassic NHRs and their potential ligands in the immune system. PMID:26222181

  3. Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.

    PubMed Central

    Moldes, M; Boizard, M; Liepvre, X L; Fève, B; Dugail, I; Pairault, J

    1999-01-01

    We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes. PMID:10585876

  4. 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.

  5. 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. PMID:23283302

  6. 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.

  7. 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. PMID:24267242

  8. 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. PMID:26993747

  9. 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

  10. Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements

    PubMed Central

    Mayho, Matthew; Fenn, Katelyn; Craddy, Paul; Crosthwaite, Susan; Matthews, Keith

    2006-01-01

    Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain. Components of this complex represent a form of post-transcriptional operon because trypanosome mitochondrial activity is unusual in being developmentally programmed. By genome analysis we identified the genes for seven components of the COX complex. Each mRNA exhibits bloodstream stage-specific instability, which is not mediated by the RNA silencing pathway but which is alleviated by cycloheximide. Reporter assays have identified regulatory regions within the 3′-untranslated regions of three COX mRNAs operating principally at the translational level, but also via mRNA stability. Interrogation of the mapped regions via oligonucleotide frequency scoring provides evidence for genome-wide conservation of regulatory sequences among a large cohort of procyclic-enriched transcripts. Analysis of the co-regulated subunits of a stage-specific enzyme is therefore a novel approach to uncover cryptic regulatory sequences controlling gene expression at the post-transcriptional level. PMID:17012283

  11. 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

  12. 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). PMID:25942633

  13. 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 ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Fatty acid and sterol composition of three phytomonas species.

    PubMed

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

    1999-01-01

    Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine. PMID:10446013

  16. 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. PMID:26041193

  17. 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. PMID:27060921

  18. 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. PMID:26996016

  19. 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

  20. 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. PMID:25757602

  1. ENCODE data at the ENCODE portal.

    PubMed

    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

  2. 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

  3. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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. PMID:24689228

  6. Analysis of sterol oxidation products in foods.

    PubMed

    Guardiola, Francesc; Bou, Ricard; Boatella, Josep; Codony, Rafael

    2004-01-01

    The main aspects related to the analysis of sterol oxidation products (SOP) in foods are comprehensively reviewed. Special emphasis is placed on the critical and controversial points of this analysis because these points affect crucial analytical parameters such as precision, accuracy, selectivity, and sensitivity. The effect of sample preparation and the conditions of quantification by gas chromatography and liquid chromatography on these parameters are also reviewed. The results show that, in order to choose an adequate method to analyze SOP in a certain food, the analyst must consider its SOP concentration and matrix complexity. The term SOP includes both cholesterol oxidation products (COP) and phytosterol oxidation products (POP). The state of the art of COP and POP analysis is quite different; many more studies have dealt with the analysis of COP than of POP. However, most of the results presented here about COP analysis may be extrapolated to POP analysis because both groups of compounds show similar structures and characteristics. PMID:15164841

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

    PubMed

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

    2015-11-01

    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. PMID:26484739

  8. 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

  9. 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

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

    1991-12-01

    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

  10. 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

  11. 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).

  12. 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.

  13. 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.

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

    PubMed Central

    Marler, Thomas E.; Shaw, Christopher A.

    2010-01-01

    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. PMID:20157628

  15. Encoding Dictionaries.

    ERIC Educational Resources Information Center

    Ide, Nancy

    1995-01-01

    Describes problems in devising a Text Encoding Initiative (TEI) encoding format for dictionaries. Asserts that the high degree of structuring and compression of information are among the most complex text types treated in the TEI. Concludes that the source of some TEI problems lies in the design of Standard Generalized Markup Language (SGML). (CFR)

  16. 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. PMID:25623279

  17. 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...

  18. 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

  19. The biosynthesis of sterols in higher plants

    PubMed Central

    Goad, L. J.; Goodwin, T. W.

    1966-01-01

    1. [2-14C]Mevalonate was incorporated into squalene and the major phytosterols of pea and maize leaves; it was also incorporated into compounds belonging to the 4,4-dimethyl and 4α-methyl steroid groups and which may be possible phytosterol intermediates. 2. l-[Me-14C]Methionine was incorporated into the major sterols and also into the 4,4-dimethyl and 4α-methyl steroid groups. No radioactivity was detected in squalene. 3. Under anaerobic conditions incorporation of [2-14C]-mevalonate into the non-saponifiable lipid of pea leaves was drastically decreased but radioactive squalene was accumulated. 4. Cycloartenol, 24-methylenecycloartanol, 24-methylenelophenol, 24-ethylidenelophenol, fucosterol, β-sitosterol, stigmasterol and campesterol have been identified by gas–liquid chromatography in pea leaves. 5. The significance of these results in connexion with phytosterol biosynthesis and the introduction of the alkyl group at C-24 into phytosterols is discussed. ImagesFig. 1. PMID:5964970

  20. 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. PMID:26370196

  1. Spatial and temporal regulation of sterol biosynthesis in Nicotiana benthamiana.

    PubMed

    Suza, Walter P; Chappell, Joe

    2016-06-01

    Nicotiana benthamiana was used as a model to investigate the spatial and developmental relationship between sterol synthesis rates and sterol content in plants. Stigmasterol levels were approximately twice the level in roots as that found in aerial tissues, while its progenitor sterol sitosterol was the inverse. When incorporation of radiolabeled precursors into sterols was used as measure of in vivo synthesis rates, acetate incorporation was similar across all tissue types, but approximately twofold greater in roots than any other tissue. In contrast, mevalonate incorporation exhibited the greatest differential with the rate of incorporation in roots approximately one-tenth that in apical shoots. Similar to acetate, incorporation of farnesol was higher in roots but remained fairly constant in aerial tissues, suggesting less regulation of the downstream sterol biosynthetic steps. Consistent with the precursor incorporation data, analysis of gene transcript and measurements of putative rate-limiting enzyme activities for 3-hydroxy-3-methylglutaryl-coenzyme A synthase (EC 2.3.3.10) and reductase (EC 1.1.1.34) showed the greatest modulation of levels, while the activity levels for isopentenyl diphosphate isomerase (EC 5.3.3.2) and prenyltransferases (EC 2.5.1.10 and EC 2.5.1.1) also exhibited a strong but moderate correlation with the development age of the aerial tissues of the plants. Overall, the data suggest a multitude of means from transcriptional to posttranslational control affecting sterol biosynthesis and accumulation across an entire plant, and point to some particular control points that might be manipulated using molecular genetic approaches to better probe the role of sterols in plant growth and development. PMID:26671544

  2. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.

    PubMed Central

    Paz-Ares, J; Ghosal, D; Wienand, U; Peterson, P A; Saedler, H

    1987-01-01

    The structure of the wild-type c1 locus of Zea mays was determined by sequence analysis of one genomic and two cDNA clones. The coding region is composed of three exons (150 bp, 129 bp and one, at least 720 bp) and two small introns (88 bp and 145 bp). Transcription of the mRNAs corresponding to the two cDNA clones cLC6 (1.1 kb) and cLC28 (2.1 kb) starts from the same promoter. Both cDNAs are identical except that cLC28 extends further at its 3' end. A putative protein, 273 amino acids in length was deduced from the sequence of both transcripts. It contains two domains, one basic and the other acidic and might function as a transcriptional activator. The basic domain of this c1-encoded protein shows 40% sequence homology to the protein products of animal myb proto-oncogenes. Images Fig. 3. PMID:3428265

  3. The Inactivation of KlNOT4, a Kluyveromyces lactis Gene Encoding a Component of the CCR4-NOT Complex, Reveals New Regulatory Functions

    PubMed Central

    Mazzoni, Cristina; Serafini, Agnese; Falcone, Claudio

    2005-01-01

    We have isolated the KlNOT4 gene of the yeast Kluyveromyces lactis, which encodes a component of the evolutionarily conserved CCR4-NOT complex. We show that inactivation of the gene leads to pleiotropic defects that were differentially suppressed by the NOT4 gene of S. cerevisiae, indicating that these genes have overlapping, but not identical, functions. K. lactis strains lacking Not4p are defective in fermentation and show reduced transcription of glucose transporter and glycolytic genes, which are phenotypes that are not found in the corresponding mutant of S. cerevisiae. We also show that Not4 proteins control the respiratory pathway in both yeasts, although with some differences. They activate transcription of KlACS2 and KlCYC1, but repress KlICL1, ScICL1, ScACS1, and ScCYC1. Altogether, our results indicate that Not4p is a pivotal factor involved in the regulation of carbon metabolism in yeast.

  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. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

    PubMed

    Márkus, Nóra M; Hasel, Philip; Qiu, Jing; Bell, Karen F S; Heron, Samuel; Kind, Peter C; Dando, Owen; Simpson, T Ian; Hardingham, Giles E

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms. PMID:26828201

  6. Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling

    PubMed Central

    Márkus, Nóra M.; Hasel, Philip; Qiu, Jing; Bell, Karen F. S.; Heron, Samuel; Kind, Peter C.; Dando, Owen; Simpson, T. Ian; Hardingham, Giles E.

    2016-01-01

    Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms. PMID:26828201

  7. The Proprotein Convertase Encoded by amontillado (amon) Is Required in Drosophila Corpora Cardiaca Endocrine Cells Producing the Glucose Regulatory Hormone AKH

    PubMed Central

    Rhea, Jeanne M.; Wegener, Christian; Bender, Michael

    2010-01-01

    Peptide hormones are potent signaling molecules that coordinate animal physiology, behavior, and development. A key step in activation of these peptide signals is their proteolytic processing from propeptide precursors by a family of proteases, the subtilisin-like proprotein convertases (PCs). Here, we report the functional dissection of amontillado (amon), which encodes the Drosophila homolog of the mammalian PC2 protein, using cell-type specific inactivation and rescue experiments, and we show that amon is required in the islet-like adipokinetic hormone (AKH)–producing cells that regulate sugar homeostasis. In Drosophila, AKH acts analogously to vertebrate glucagon to increase circulating sugar levels from energy stores, while insulin-like peptides (DILPs) act to decrease sugar levels. amon mutant larvae have significantly reduced hemolymph sugar levels, and thus phenocopy larvae where the AKH–producing cells in the corpora cardiaca have been ablated. Reduction of amon expression in these cells via cell-specific RNA inactivation also results in larvae with reduced sugar levels while expression of amon in AKH cells in an amon mutant background rescues hypoglycemia. Hypoglycemia in larvae resulting from amon RNA inactivation in the AKH cells can be rescued by global expression of the akh gene. Finally, mass spectrometric profiling shows that the production of mature AKH is inhibited in amon mutants. Our data indicate that amon function in the AKH cells is necessary to maintain normal sugar homeostasis, that amon functions upstream of akh, and that loss of mature AKH is correlated with loss of amon activity. These observations indicate that the AKH propeptide is a proteolytic target of the amon proprotein convertase and provide evidence for a conserved role of PC2 in processing metabolic peptide hormones. PMID:20523747

  8. The proprotein convertase encoded by amontillado (amon) is required in Drosophila corpora cardiaca endocrine cells producing the glucose regulatory hormone AKH.

    PubMed

    Rhea, Jeanne M; Wegener, Christian; Bender, Michael

    2010-05-01

    Peptide hormones are potent signaling molecules that coordinate animal physiology, behavior, and development. A key step in activation of these peptide signals is their proteolytic processing from propeptide precursors by a family of proteases, the subtilisin-like proprotein convertases (PCs). Here, we report the functional dissection of amontillado (amon), which encodes the Drosophila homolog of the mammalian PC2 protein, using cell-type specific inactivation and rescue experiments, and we show that amon is required in the islet-like adipokinetic hormone (AKH)-producing cells that regulate sugar homeostasis. In Drosophila, AKH acts analogously to vertebrate glucagon to increase circulating sugar levels from energy stores, while insulin-like peptides (DILPs) act to decrease sugar levels. amon mutant larvae have significantly reduced hemolymph sugar levels, and thus phenocopy larvae where the AKH-producing cells in the corpora cardiaca have been ablated. Reduction of amon expression in these cells via cell-specific RNA inactivation also results in larvae with reduced sugar levels while expression of amon in AKH cells in an amon mutant background rescues hypoglycemia. Hypoglycemia in larvae resulting from amon RNA inactivation in the AKH cells can be rescued by global expression of the akh gene. Finally, mass spectrometric profiling shows that the production of mature AKH is inhibited in amon mutants. Our data indicate that amon function in the AKH cells is necessary to maintain normal sugar homeostasis, that amon functions upstream of akh, and that loss of mature AKH is correlated with loss of amon activity. These observations indicate that the AKH propeptide is a proteolytic target of the amon proprotein convertase and provide evidence for a conserved role of PC2 in processing metabolic peptide hormones. PMID:20523747

  9. 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

  10. Phytoalexin-Deficient Mutants of Arabidopsis Reveal That Pad4 Encodes a Regulatory Factor and That Four Pad Genes Contribute to Downy Mildew Resistance

    PubMed Central

    Glazebrook, J.; Zook, M.; Mert, F.; Kagan, I.; Rogers, E. E.; Crute, I. R.; Holub, E. B.; Hammerschmidt, R.; Ausubel, F. M.

    1997-01-01

    We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates. PMID:9136026

  11. A Potential Regulatory Role for Intronic microRNA-338-3p for Its Host Gene Encoding Apoptosis-Associated Tyrosine Kinase

    PubMed Central

    Kos, Aron; Olde Loohuis, Nikkie F. M.; Wieczorek, Martha L.; Glennon, Jeffrey C.; Martens, Gerard J. M.; Kolk, Sharon M.; Aschrafi, Armaz

    2012-01-01

    MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration. PMID:22363537

  12. Deciphering ENCODE.

    PubMed

    Diehl, Adam G; Boyle, Alan P

    2016-04-01

    The ENCODE project represents a major leap from merely describing and comparing genomic sequences to surveying them for direct indicators of function. The astounding quantity of data produced by the ENCODE consortium can serve as a map to locate specific landmarks, guide hypothesis generation, and lead us to principles and mechanisms underlying genome biology. Despite its broad appeal, the size and complexity of the repository can be intimidating to prospective users. We present here some background about the ENCODE data, survey the resources available for accessing them, and describe a few simple principles to help prospective users choose the data type(s) that best suit their needs, where to get them, and how to use them to their best advantage. PMID:26962025

  13. Characterization of fatty alcohol and sterol fractions in olive tree.

    PubMed

    Orozco-Solano, Mara; Ruiz-Jimenez, José; Luque De Castro, María D

    2010-07-14

    The determination of sterols and fatty alcohols is a part of the study of the metabolomic profile of the unsaponifiable fraction in olive tree. Leaves and drupes from three varieties of olive tree (Arbequina, Picual, and Manzanilla) were used. The content of the target compounds was studied in five ripeness stages and three harvesting periods for olive drupes and leaves, respectively. A method based on ultrasound-assisted extraction and derivatization for the individual identification and quantitation of sterols and fatty alcohols, involving chromatographic separation and mass spectrometry detection by selected ion monitoring, was used. The concentrations of alcohols and sterols in the drupes ranged between 0.1 and 1086.9 mug/g and between 0.1 and 5855.3 mug/g, respectively, which are higher than in leaves. Statistical studies were developed to show the relationship between the concentration of the target analytes and variety, ripeness stage, and harvesting period. PMID:20550122

  14. Transbilayer distribution of sterols in mycoplasma membranes: a review.

    PubMed Central

    Bittman, R.; Clejan, S.; Rottem, S.

    1983-01-01

    The polyene antibiotic, filipin, binds to 3 beta-hydroxysterols. The initial rate of filipin-sterol association, monitored in a stopped-flow spectrophotometer, was first order in each reacting partner. The ratio of rate constants in intact mycoplasma cells relative to isolated, unsealed membranes provides an estimate of sterol distribution in the membrane bilayer. Cholesterol is distributed symmetrically in the bilayer of M. gallisepticum cells from the early exponential phase. However, in the M. capricolum membrane two-thirds of the unesterified cholesterol is localized in the outer leaflet; alkyl-sterols are distributed predominantly in the external monolayer. Cholesterol is translocated rapidly in the bilayer of M. capricolum cells. Exogenous phospholipids incorporated into the membrane had no effect on the cholesterol distribution in M. capricolum. PMID:6382819

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. 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

    2016-03-01

    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

  17. 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

  18. 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

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

    PubMed

    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 C14alpha-demethylase, and (f) azasterols, which inhibit Delta(24(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

  20. Simultaneous Effects of Light Intensity and Phosphorus Supply on the Sterol Content of Phytoplankton

    PubMed Central

    Piepho, Maike; Martin-Creuzburg, Dominik; Wacker, Alexander

    2010-01-01

    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. PMID:21209879

  1. A novel sterol 14alpha-demethylase/ferredoxin fusion protein (MCCYP51FX) from Methylococcus capsulatus represents a new class of the cytochrome P450 superfamily.

    PubMed

    Jackson, Colin J; Lamb, David C; Marczylo, Timothy H; Warrilow, Andrew G S; Manning, Nigel J; Lowe, David J; Kelly, Diane E; Kelly, Steven L

    2002-12-01

    Sterol 14alpha-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. This M. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass approximately 62 kDa on SDS/PAGE and bound lanosterol as a putative substrate. Sterol 14alpha-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general. PMID:12235134

  2. Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication.

    PubMed

    Kuo, Rei-Lin; Li, Zong-Hua; Li, Li-Hsin; Lee, Kuo-Ming; Tam, Ee-Hong; Liu, Helene M; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-05-01

    Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication. PMID:27096427

  3. 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. PMID:26928592

  4. 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.

  5. Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

    PubMed

    Pierce, H D; Pierce, A M; Srinivasan, R; Unrau, A M; Oehlschlager, A C

    1978-06-23

    The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time. PMID:352402

  6. 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. PMID:25416794

  7. 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

  8. Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor.

    PubMed

    Kim, Yeon-Ki; Wang, Yuhuan; Liu, Zhi-Mei; Kolattukudy, Pappachan E

    2002-04-01

    Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP-glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase. PMID:12000454

  9. Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots.

    PubMed

    Campagnac, Estelle; Fontaine, Joël; Sahraoui, Anissa Lounès-Hadj; Laruelle, Frédéric; Durand, Roger; Grandmougin-Ferjani, Anne

    2008-12-01

    Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2mgl(-1)) and fenhexamid (0.02; 0.2; 2; 20mgl(-1)), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Delta(5)-sterols were replaced by unusual compounds such as 9beta,19-cyclopropylsterols (24-methylpollinastanol), Delta(8,14)-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Delta(8)-sterols (Delta(8) sitosterol) and Delta(7)-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected. Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization. PMID:19007946

  10. 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. PMID:15884765

  11. 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.

  12. 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

  13. The sterols of Cucurbita moschata ("calabacita") seed oil.

    PubMed

    Rodriguez, J B; Gros, E G; Bertoni, M H; Cattaneo, P

    1996-11-01

    From the sterol fraction of seed oil from commercial Cucurbita moschata Dutch ("calabacita") delta 5 and delta 7 sterols having saturated and unsaturated side chain were isolated by chromatographic procedures and characterized by spectroscopic (1H and 13C-nuclear magnetic resonance, mass spectrometry) methods. The main components were identified as 24S-ethyl 5 alpha-cholesta-7,22E-dien-3 beta-ol (alpha-spinasterol); 24S-ethyl 5 alpha-cholesta-7,22E,25-trien-3 beta-ol (25-dehydrochondrillasterol); 24S-ethyl 5 alpha-cholesta-7,25-dien-3 beta-ol; 24R-ethyl-cholesta-7-en-3 beta-ol (delta 7-stigmastenol) and 24-ethyl-cholesta-7, 24(28)-dien-3 beta-ol (delta 7,24(28)-stigmastadienol). PMID:8934454

  14. Cytotoxic sterols from the formosan brown alga Turbinaria ornata.

    PubMed

    Sheu, J H; Wang, G H; Sung, P J; Chiu, Y H; Duh, C Y

    1997-12-01

    Two hydroperoxysterols 24-hydroperoxy-24-vinyl-cholesterol (1) and 29-hydroperoxystigmasta-5,24(28)-dien-3beta-ol (2), and fucosterol (3) were isolated from the brown alga Turbinaria ornata (Sargassaceae). Hydroperoxide 2 is a new natural compound and was converted into 29-hydroxystigmasta-5,24 (28)-dien-3beta-ol (4) by reaction with LAH. Sterols 1, 2, and 4 exhibited cytotoxicity against various cancer cell lines. PMID:17252381

  15. 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. PMID:27180904

  16. Distribution of free and glycosylated sterols within Cycas micronesica plants

    PubMed Central

    Marler, Thomas E.; Shaw, Christopher A.

    2010-01-01

    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. PMID:20157629

  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. 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

  19. 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. PMID:22398861

  20. 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. PMID:26729489

  1. 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. PMID:22878342

  2. The PsychENCODE project

    PubMed Central

    Akbarian, Schahram; Liu, Chunyu; Knowles, James A; Vaccarino, Flora M; Farnham, Peggy J; Crawford, Gregory E; Jaffe, Andrew E; Pinto, Dalila; Dracheva, Stella; Geschwind, Daniel H; Mill, Jonathan; Nairn, Angus C; Abyzov, Alexej; Pochareddy, Sirisha; Prabhakar, Shyam; Weissman, Sherman; Sullivan, Patrick F; State, Matthew W; Weng, Zhiping; Peters, Mette A; White, Kevin P; Gerstein, Mark B; Senthil, Geetha; Lehner, Thomas; Sklar, Pamela; Sestan, Nenad

    2015-01-01

    Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE. PMID:26605881

  3. 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. PMID:25250339

  4. 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

  5. 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)

  6. 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. PMID:24422629

  7. Serum response element-like sequences of the human low density lipoprotein receptor promoter: possible regulation sites for sterol-independent transcriptional activation.

    PubMed

    Pak, Y K

    1996-02-01

    Serum factors stimulate low density lipoprotein receptor (LDLR) gene expression in HepG2 cells through sterol-independent pathways. Promoter element other than sterol regulatory element-1 (SRE-1) seems to be necessary. Protein binding activity of the human LDLR promoter fragment (550bp) beyond the SRE-1 was determined by DNase I footprint assay. Five different promoter regions were protected from DNase I digestion; -226 to -258, -291 to -304, -324 to -336, -360 to -373, and -521 to -528. The regions of -324 to -336 and -521 to -528 showed serum response element (SRE)-like consensus sequence of CC(A/T)6GG. Serum incubation affected the protection degree of the SRE-like elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 showed serum response elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 in human LDLR gene may be responsible for the rapid activation of the gene transcription by serum factor in a sterol-independent manner. PMID:8932516

  8. 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

  9. AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism.

    PubMed

    Wang, Penghua; Zhang, Yong; Li, Hongzhe; Chieu, Hai Kee; Munn, Alan L; Yang, Hongyuan

    2005-09-01

    The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport. PMID:16096648

  10. 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%). PMID:25682566

  11. Phylogenomics of Sterol Synthesis: Insights into the Origin, Evolution, and Diversity of a Key Eukaryotic Feature

    PubMed Central

    Desmond, Elie

    2009-01-01

    The availability of complete genomes from a wide sampling of eukaryotic diversity has allowed the application of phylogenomics approaches to study the origin and evolution of unique eukaryotic cellular structures, but these are still poorly applied to study unique eukaryotic metabolic pathways. Sterols are a good example because they are an essential feature of eukaryotic membranes. The sterol pathway has been well dissected in vertebrates, fungi, and land plants. However, although different types of sterols have been identified in other eukaryotic lineages, their pathways have not been fully characterized. We have carried out an extensive analysis of the taxonomic distribution and phylogeny of the enzymes of the sterol pathway in a large sampling of eukaryotic lineages. This allowed us to tentatively indicate features of the sterol pathway in organisms where this has not been characterized and to point out a number of steps for which yet-to-discover enzymes may be at work. We also inferred that the last eukaryotic common ancestor already harbored a large panel of enzymes for sterol synthesis and that subsequent evolution over the eukaryotic tree occurred by tinkering, mainly by gene losses. We highlight a high capacity of sterol synthesis in the myxobacterium Plesiocystis pacifica, and we support the hypothesis that the few bacteria that harbor homologs of the sterol pathway have likely acquired these via horizontal gene transfer from eukaryotes. Finally, we propose a potential candidate for the elusive enzyme performing C-3 ketoreduction (ERG27 equivalent) in land plants and probably in other eukaryotic phyla. PMID:20333205

  12. 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. PMID:26874877

  13. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. A potential biochemical mechanism underlying the influence of sterol deprivation stress on Caenorhabditis elegans longevity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Applying Clustering and Phylogeny Analysis to Study Dinoflagellates based on Sterol Composition.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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

  18. Fungal genomes mining to discover novel sterol esterases and lipases as catalysts

    PubMed Central

    2013-01-01

    Background Sterol esterases and lipases are enzymes able to efficiently catalyze synthesis and hydrolysis reactions of both sterol esters and triglycerides and due to their versatility could be widely used in different industrial applications. Lipases with this ability have been reported in the yeast Candida rugosa that secretes several extracellular enzymes with a high level of sequence identity, although different substrate specificity. This versatility has also been found in the sterol esterases from the ascomycetes Ophiostoma piceae and Melanocarpus albomyces. Results In this work we present an in silico search of new sterol esterase and lipase sequences from the genomes of environmental fungi. The strategy followed included identification and search of conserved domains from these versatile enzymes, phylogenetic studies, sequence analysis and 3D modeling of the selected candidates. Conclusions Six potential putative enzymes were selected and their kinetic properties and substrate selectivity are discussed on the basis of their similarity with previously characterized sterol esterases/lipases with known structures. PMID:24138290

  19. Serum lipid and antioxidant responses in hypercholesterolemic men and women receiving plant sterol esters vary by apolipoprotein E genotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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

  1. Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk.

    PubMed

    Calandra, Sebastiano; Tarugi, Patrizia; Speedy, Helen E; Dean, Andrew F; Bertolini, Stefano; Shoulders, Carol C

    2011-11-01

    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

  2. Lessons from modENCODE.

    PubMed

    Brown, James B; Celniker, Susan E

    2015-01-01

    The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems. PMID:26133010

  3. 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

  4. 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

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

    PubMed Central

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

    2014-01-01

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

  6. Roles of Sterol Derivatives in Regulating the Properties of Phospholipid Bilayer Systems.

    PubMed

    Bui, Tham Thi; Suga, Keishi; Umakoshi, Hiroshi

    2016-06-21

    Liposomes are considered an ideal biomimetic environment and are potential functional carriers for important molecules such as steroids and sterols. With respect to the regulation of self-assembly via sterol insertion, several pathways such as the sterol biosynthesis pathway are affected by the physicochemical properties of the membranes. However, the behavior of steroid or sterol molecules (except cholesterol (Chl)) in the self-assembled membranes has not been thoroughly investigated. In this study, to analyze the fundamental behavior of steroid molecules in fluid membranes, Chl, lanosterol, and ergosterol were used as representative sterols in order to clarify how they regulate the physicochemical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Membrane properties such as surface membrane fluidity, hydrophobicity, surface membrane polarity, inner membrane polarity, and inner membrane fluidity were investigated using fluorescent probes, including 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, 8-anilino-1-naphthalenesulfonic acid, 6-propionyl-2-(dimethylamino) naphthalene, 6-dodecanoyl-2-dimethylaminonaphthalene, and 1,6-diphenyl-1,3,5-hexatriene. The results indicated that each sterol derivative could regulate the membrane properties in different ways. Specifically, Chl successfully increased the packing of the DOPC/Chl membrane proportional to its concentration, and lanosterol and ergosterol showed lower efficiencies in ordering the membrane in hydrophobic regions. Given the different binding positions of the probes in the membranes, the differences in membrane properties reflected the relationship between sterol derivatives and their locations in the membrane. PMID:27158923

  7. The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis.

    PubMed

    Alfaro, Gabriel; Johansen, Jesper; Dighe, Shubha A; Duamel, Giselle; Kozminski, Keith G; Beh, Christopher T

    2011-11-01

    Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein. PMID:21819498

  8. 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. PMID:26626241

  9. Cyclopropyl Sterol and Phospholipid Composition of Membrane Fractions from Maize Roots Treated with Fenpropimorph

    PubMed Central

    Grandmougin, Anne; Bouvier-Navé, Pierrette; Ullmann, Pascaline; Benveniste, Pierre; Hartmann, Marie-Andrée

    1989-01-01

    Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ5-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ5-sterols. PMID:16666813

  10. 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). PMID:12872919

  11. 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.

  12. 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.

  13. 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

    2013-07-01

    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 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. PMID:23823476

  14. Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism.

    PubMed

    Zhou, Xiaoye; He, Wei; Huang, Zhiping; Gotto, Antonio M; Hajjar, David P; Han, Jihong

    2008-01-25

    Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions

  15. 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. PMID:19641030

  16. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

    PubMed

    Li, Nancy C; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  17. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

    PubMed Central

    Li, Nancy C.; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  18. 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

  19. Inhibition of sterol but not fatty acid synthesis by valproate in developing rat brain in vivo.

    PubMed Central

    Bolaños, J P; Medina, J M; Williamson, D H

    1990-01-01

    The effect of administration of valproate on lipogenesis in the developing rat brain in vivo was studied. Valproate inhibited by 21-38% the rate of 3H2O incorporation into brain sterols, without significantly affecting fatty acid synthesis. Similarly, R-[2-14C]mevalonate incorporation into sterols was inhibited by 33-54%; the low rate of fatty acid synthesis under these conditions was not affected by valproate. Plasma ketone bodies decreased after treatment with valproate. Valproate inhibited (about 50%) both sterol and fatty acid synthesis in livers of weanling rats. It is concluded that valproate can specifically inhibit sterol synthesis in the brain during development, in part at a stage after mevalonate formation, and also by decreased exogenous precursor supply. PMID:2264830

  20. Excretion of sterols from the skin of normal and hypercholesterolemic humans

    PubMed Central

    Bhattacharyya, Ashim K.; Connor, William E.; Spector, Arthur A.

    1972-01-01

    The 24 hr sterol excretion from the entire skin surface was determined in six normal and five hypercholesterolemic (Type II) patients fed a controlled, eucaloric diet containing 400 mg of plant sterols. All subjects received radiolabeled cholesterol intravenously in order to measure cholesterol turnover and exchange. The 24 hr skin surface lipids were collected subsequently at intervals of 7-10 days. Sterols were quantified and identified by a combination of thin-layer and gas-liquid chromatographic methods. The mean 24 hr excretion of cholesterol in milligrams was 82.6 in the normal subjects and 82.7 in the hypercholesterolemic patients. Cholesterol constituted 89% of the total sterol excretion through the skin surface in both groups. The specific radioactivity of cholesterol in the skin surface lipids increased gradually after the intravenous administration of the isotope. Within 4-5 wk the specific activity equaled and then remained higher than that of the plasma up to 10 wk. These specific activity curves suggested that, for at least some of skin surface cholesterol, there was a precursor-product relationship between the plasma cholesterol and the skin cholesterol. The presence of plant sterols, β-sitosterol, campesterol, and stigmasterol in the skin surface lipids of man has not been reported previously. We identified these sterols in the skin surface lipids of all of our subjects. They constituted about 7% of the total skin surface sterols. The occurrence of plant sterols in the skin surface lipids suggested that plasma sterols were transferred from the plasma into the skin. 1-2% of the skin surface sterols were tentatively identified as lathosterol and lanosterol. The present study documented that a significant amount of cholesterol was excreted from the skin surface and that probably there was a net transfer of plasma cholesterol into the skin surface lipids. Both normal subjects and hypercholesterolemic patients excreted similar amounts of cholesterol per

  1. Major Sterol Fluxes in Sinking Particles and Surface Sediments in the Cariaco Basin.

    NASA Astrophysics Data System (ADS)

    Woodworth, M. P.; Goni, M. A.; Thunell, R.; Tappa, E.; Astor, Y.

    2004-12-01

    Sterols in sediments are used to trace past ecosystem dynamics in the upper ocean. It is therefore important to know what factors control the creation of sterol fluxes, degradation of sterols in the water column and eventual burial in the sediments. To this end we examined the major sterols fluxes in sediment traps during 1996-1997 (at depths of 275, 455 and 975m) and surface sediments in the Cariaco Basin. Sterol flux data in the sediment traps were compared with hydrographic data collected as part of the CARIACO Project. Diatom sterols 24-methylcholesta-5,22-dien-3b-ol (brassicasterol), 24-methylcholesta-5,24(28)-dien-3b-ol (24methlyene-cholesterol) fluxes were greatest during upwelling. 24methlyene-cholesterol was well correlated with biogenic opal flux (r2 = 0.88) suggesting that 24methlyene-cholesterol is an excellent biomarker for diatom production. 4a,23,24-trimethyl-5a(H)-cholest-22-en-3b-ol (dinosterol) exhibited a post upwelling maximum indicating that fluxes of dinoflagellate-derived materials were dominant during stratified conditions. Sterols were degraded with depth but the relative composition of the major sterols remained fairly constant. There is a sharp decrease in the magnitude of total sterol fluxes between the sediment traps (955m; 143 ug m-2 d-1) and the surface sediments (core depth 460m; 11.7 ug m-2 -1) indicating that a large portion of the flux is lost at the sediment water interface. It is at this transition in which the relative compositions of the sterols are also altered. Dinosterol, which is a minor component of the sediment trap fluxes, is 3 to 4 times greater than that of cholesterol in the sediment. While the ratio of dinosterol to cholesterol changed significantly, the ratio between the two diatom sterol fluxes, brassicasterol and 24methlyene-cholesterol, and cholesterol remained within the range of values observed in the sediment traps.

  2. Expression and purification of two recombinant sterol-carrier proteins: SCPX and SCP2.

    PubMed

    Manfra, D J; Baum, C L; Reschley, E; Lundell, D; Zavodny, P; Dalie, B

    1995-04-01

    We report the cloning, expression, and purification of the rat sterol carrier proteins SCPX and SCP2. The cDNA's encoding rat SCPX and SCP2 were isolated from a lambda gt11 rat liver cDNA library. To maximize expression and to facilitate the purification of the recombinant proteins, the SCPX and SCP2 proteins were expressed as carboxy-terminal fusion proteins to the glutathione S-transferase (GST). The GST-SCPX and GST-SCP2 fusion proteins contained a thrombin recognition site between the GST and SCPX or SCP2 polypeptides. The expression of the fusion proteins was controlled by the inducible tac promoter. Under optimal conditions, the approximately 85-kDa GST-SCPX and the approximately 41-kDa GST-SCP2 proteins represented approximately 1-2% of the total cell lysate. Both fusion proteins were easily purified under nondenaturing conditions from the soluble fraction of total cell lysate by glutathione-Sepharose 4B affinity chromatography. Thrombin cleavage resulted in the release of the SCPX and SCP2 proteins from the GST-SCPX and GST-SCP2 fusions, respectively. Amino terminal protein sequencing confirmed the authenticity of the recombinant proteins. Furthermore, functional assay revealed that recombinant SCP2 is highly active in facilitating the conversion of 7-dehydrocholesterol to cholesterol. Recombinant SCPX is also active in this assay but only 50% as active as SCP2. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of these proteins. PMID:7606169

  3. Identification of ergosterol and inhibition of sterol synthesis by. Delta. sup 5 -sterols in GL7, an auxotrophic mutant of yeast

    SciTech Connect

    Dhanuka, I.C.

    1988-01-01

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

  4. Influence of salinity and natural organic matter on the solid phase extraction of sterols and stanols: application to the determination of the human sterol fingerprint in aqueous matrices.

    PubMed

    Jeanneau, L; Jardé, E; Gruau, G

    2011-05-01

    Faecal sterols have been proposed as direct chemical markers for the determination of faecal contamination in inland and coastal waters. In this study, we assess the impact of (a) the concentration of dissolved organic carbon (DOC), (b) the nature of DOC, (c) the salinity and (d) the concentration of sterols and stanols on their solid phase extraction. When natural organic matter (NOM) is modelled by humic acid, increasing DOC concentration from 2.7 to 15.4 mg/L has no significant impact on the recovery of sterols and stanols. The modelling of NOM by a mixture of humic acid and succinoglycan induces a significant (24%) decrease in the recovery of sterols and stanols. For all concentrations of target compounds, no significant increase in recovery is associated with increasing the salinity. Moreover, an increase in the recovery of target compounds is induced by an increase in their concentration. The nine target compounds and the recovery standard (RS) exhibit the same behaviour during the extraction step. Thus, we propose that (a) the concentration of target compounds can be corrected by the RS to calculate more realistic concentrations without modifying their profile and (b) the sterol fingerprint can be investigated in the colloidal fraction of aqueous samples without altering the information it could provide about the source. The application of this analytical method to waste water treatment plant influent and effluents yields results in agreement with previous studies concerning the use of those compounds to differentiate between sources of faecal contamination. We conclude that this analytical method is fully applicable to the determination of sterol fingerprints in the dissolved phase (<0.7 μm) of natural aqueous samples. PMID:21420686

  5. Foliar Sterols in Soybeans Exposed to Chronic Levels of Ozone 1

    PubMed Central

    Grunwald, Claus; Endress, Anton G.

    1985-01-01

    Soybeans (Glycine max) exposed to chronic levels of ozone showed a linear decrease in biomass with increasing concentration. The foliar free sterols increased while the steryl ester, and the steryl glycosides, a minor component, decreased with increasing pollutant concentration. Of the free sterols, stigmasterol showed the largest increase, followed by sitosterol; campesterol, however, decreased. All steryl esters decreased; sitosterol showed the largest decrease and campesterol the least. PMID:16664020

  6. 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

  7. The role of regulatory genes nifA, vnfA, anfA, nfrX, ntrC, and rpoN in expression of genes encoding the three nitrogenases of Azotobacter vinelandii.

    PubMed

    Walmsley, J; Toukdarian, A; Kennedy, C

    1994-01-01

    Several regulatory gene mutants of Azotobacter vinelandii were tested for ability to synthesize functional nitrogenase-1 (Nif phenotype), nitrogenase-2 (Vnf), or nitrogenase-3 (Anf). While nifA mutants were Nif-, Vnf+, and Anf+/-, and ntrC mutants were Nif+, Vnf+, and Anf+, nifA ntrC double mutants were Nif-, Vnf-, and Anf-. A vnfA mutant was Nif+, Vnf+/-, and Anf+/-, and an anfA strain was Nif+, Vnf+, and Anf-. lacZ fusions in the nifH, vnfH, vnfD, anfH, and nifM genes of Azotobacter vinelandii were constructed and introduced into wild-type and regulatory mutants of A. vinelandii. Expression of these operons correlated with the growth phenotype of the regulatory mutants. Apparently either NifA or NtrC can activate expression of nifM. Also, expression of the anf operon required the NifA transcriptional activator, although there are no NifA binding sites at appropriate locations upstream of anfH (or anfA). The results confirm previous reports that VnfA and AnfA are required for expression of vnf and anf genes, respectively, and that VnfA is involved in repression of the nifHDK operon in the absence of molybdenum and of the anfHDGK operon in the presence of vanadium. PMID:7872838

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

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

  11. Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport.

    PubMed

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

    2015-01-01

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

  12. Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular Cholesterol Transport

    PubMed Central

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

    2015-01-01

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

  13. Quantitation of fatty acids, sterols, and tocopherols in turpentine (Pistacia terebinthus Chia) growing wild in Turkey.

    PubMed

    Matthäus, Bertrand; Ozcan, Mehmet Musa

    2006-10-01

    The chemical composition (fatty acids, tocopherols, and sterols) of the oil from 14 samples of turpentine (Pistacia terebinthus L.) fruits is presented in this study. The oil content of the samples varied in a relatively small range between 38.4 g/100 g and 45.1 g/100 g. The dominating fatty acid of the oil is oleic acid, which accounted for 43.0 to 51.3% of the total fatty acids. The total content of vitamin E active compounds in the oils ranged between 396.8 and 517.7 mg/kg. The predominant isomers were alpha- and gamma-tocopherol, with approximate equal amounts between about 110 and 150 mg/kg. The seed oil of P. terebinthus also contained different tocotrienols, with gamma-tocotrienol as the dominate compound of this group, which amounted to between 79 and 114 mg/kg. The total content of sterols of the oils was determined to be between 1341.3 and 1802.5 mg/kg, with beta-sitosterol as the predominent sterol that accounted for more than 80% of the total amount of sterols. Other sterols in noteworthy amounts were campesterol, Delta5-avenasterol, and stigmasterol, which came to about 3-5% of the total sterols. PMID:17002437

  14. Postprandial plasma oxyphytosterol concentrations after consumption of plant sterol or stanol enriched mixed meals in healthy subjects.

    PubMed

    Baumgartner, Sabine; Mensink, Ronald P; Konings, Maurice; Schött, Hans-F; Friedrichs, Silvia; Husche, Constanze; Lütjohann, Dieter; Plat, Jogchum

    2015-07-01

    Epidemiological studies have reported inconsistent results on the relationship between increased plant sterol concentrations with cardiovascular risk, which might be related to the formation of oxyphytosterols (plant sterol oxidation products) from plant sterols. However, determinants of oxyphytosterol formation and metabolism are largely unknown. It is known, however, that serum plant sterol concentrations increase after daily consumption of plant sterol enriched products, while concentrations decrease after plant stanol consumption. Still, we have earlier reported that fasting oxyphytosterol concentrations did not increase after consuming a plant sterol- or a plant stanol enriched margarine (3.0g/d of plant sterols or stanols) for 4weeks. Since humans are in a non-fasting state for most part of the day, we have now investigated effects on oxyphytosterol concentrations during the postprandial state. For this, subjects consumed a shake (50g of fat, 12g of protein, 67g of carbohydrates), containing no, or 3.0g of plant sterols or plant stanols. Blood samples were taken up to 8h and after 4h subjects received a second shake (without plant sterols or plant stanols). Serum oxyphytosterol concentrations were determined in BHT-enriched EDTA plasma via GC-MS/MS. 7β-OH-campesterol and 7β-OH-sitosterol concentrations were significantly higher after consumption of a mixed meal enriched with plant sterol esters compared to the control and plant stanol ester meal. These increases were seen only after consumption of the second shake, illustrative for a second meal effect. Non-oxidized campesterol and sitosterol concentrations also increased after plant sterol consumption, in parallel with 7β-OH concentrations and again only after the second meal. Apparently, plant sterols and oxyphytosterols follow the same second meal effect as described for dietary cholesterol. However, the question remains whether the increase in oxyphytosterols in the postprandial phase is due to

  15. Evaluation of potential sewage contamination by fecal sterol biomarkers adsorbed in natural biofilms.

    PubMed

    Froehner, Sandro; Sánez, Juan

    2013-10-01

    The use of biofilms for adsorption of sterols was investigated for the first time to evaluate sewage contamination in the Barigüi River, Curitiba (Brazil). The characteristics of a biofilm that favor its use in monitoring include the relatively rapid development of biofilms and their capacity to sorb hydrophobic compounds. Some fecal sterols considered to be biomarkers for human and animal feces have relatively high octanol-water partitioning coefficients (log KOW); thus, sterols were expected to be readily sorbed in the biofilms. The biofilms were developed on glass plates (0.48 m(2)) previously coated with a fine layer of stearic acid and supported by a PVC tube that was submersed in the river 20 cm above the river bottom. After a certain period of incubation time, the biofilm growth was scraped from the plates and analyzed for the following fecal steroids: coprostanol (5β-cholestan-3β-ol), epicoprostanol (5β-cholestan-3α-ol), cholesterol (5,6-cholesten-3β-ol), cholestanol (5α-cholestan-3β-ol), stigmastanol (24β-ethyl-5α-cholestan-3β-ol) and coprostanone (5β-cholestan-3-one). Six samples were collected between March 2012 and June 2012. All analyzed compounds were detected, and in general, cholesterol was present in high amounts (23 160-41.9 ng g(-1) dry biofilm). Variation among campaigns was observed in the distribution of sterols, with cholestanol showing the least variation among the samples. Sterol ratios that are commonly used for evaluating sewage contamination were calculated; these ratios indicated some periods of potential sewage influence. However, these sterol ratios are intended to be applied primarily for sediments and not for biological compartments; thus, the results must be carefully interpreted. Biofilms developed under natural conditions can be a tool for monitoring some important sterols that are used as biomarkers of fecal pollution. PMID:24064988

  16. Distribution of fecal sterols in surface sediment of Sungai Tebrau, Johor

    NASA Astrophysics Data System (ADS)

    Nordin, N.; Ali, M. M.

    2013-11-01

    Decreasing quality of aquatic environments may harm human health in general. Sewage pollution from human and animal excretions is a major cause of environmental quality depletion. This study investigates the distribution of sewage contamination level in twenty surface sediment samples taken from Sungai Tebrau, Johor. Four principal fecal sterols have been identified and were found in all sediment samples, which are coprostanol, cholesterol, epicoprostanol and also cholestanol. Cholesterol as the major sterol and most abundant compound derived from a variety of sources ranged from 32.92 to 1,100.55 ngg-1 dry weights. Meanwhile, major fecal sterol, coprostanol has the lowest quantity of total sterol in all samples, constituting only 13% of total sterol. It ranged from 12.63 to 565.42 ngg-1 dry weights, but only two stations (ST12 and ST14) are sewage contaminated. Squatters and residential areas are a major contributor of poorly treated sewage into the aquatic environment. Coprostanol concentration alone is not reliable to indicate sewage contamination; diagnostic indices enhance reliability of sterols as a marker for sewage contamination. Indices applied in this study are coprostanol/cholesterol, coprostanol/(coprostanol+cholestanol) and also epicoprostanol/coprostanol. Resultsof coprostanol/cholesterol, coprostanol/(coprostanol+cholestanol) indices supported the findings that both ST12 and ST14 samples are contaminated with sewage. All samples consist of relativelyhigh concentration of epicoprostanol and high ratio value of epicoprostanol/coprostanol. Generally, it can be concluded that these sampling sites are not contaminated with sewage even though fecal sterols were detected in all samples as they were found to be at low concentration.

  17. In vivo studies of sterol and squalene secretion by human skin.

    PubMed

    Nikkari, T; Schreibman, P H; Ahrens, E H

    1974-11-01

    This work was aimed at studying the quantity and composition of sterols and squalene secreted by the human skin. Lipids secreted by the entire skin were recovered by Soxhlet extraction of the clothing worn by a patient for 24 hr with a chloroform-methanol azeotrope and by extracting the water of a shower taken by the patient at the end of the 24-hr period. Squalene and sterols were quantified by gas-liquid chromatography. Plant sterols were separated from total sterols by thin-layer chromatography. Free and esterified cholesterol were separated by digitonin precipitation. In eight adults, seven of them with hyperlipoproteinemia, the total skin secretion of cholesterol ranged from 59 to 108 mg/day, with a mean of 88 +/- 17 (SD) mg/day. There was no difference in cholesterol secretion between the normocholesterolemic individual and the hypercholesterolemic ones, nor were there any differences according to type of hyperlipoproteinemia. Free cholesterol amounted to 54 +/- 5% of the total cholesterol. The secretion of squalene ranged from 125 to 475 mg/day in five patients. The secretion of both squalene and cholesterol was quite constant for any individual on a given diet. Cholesterol constituted 95.6 +/- 0.5% of the digitonin-precipitable total body surface sterols of eight patients, and lathosterol, the next largest fraction, 3.4 +/- 0.4%. Total plant sterols formed only 0.65 +/- 0.38% and beta-sitosterol 0.35 +/- 0.23% of the skin surface sterols in six patients whose dietary beta-sitosterol intake ranged from 230 to 3400 mg/day. PMID:4430879

  18. Triterpene alcohols and sterols from rice bran lower postprandial glucose-dependent insulinotropic polypeptide release and prevent diet-induced obesity in mice.

    PubMed

    Fukuoka, Daisuke; Okahara, Fumiaki; Hashizume, Kohjiro; Yanagawa, Kiyotaka; Osaki, Noriko; Shimotoyodome, Akira

    2014-12-01

    Obesity is now a worldwide health problem. Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone that is secreted following the ingestion of food and modulates energy metabolism. Previous studies reported that lowering diet-induced GIP secretion improved energy homeostasis in animals and humans, and attenuated diet-induced obesity in mice. Therefore, food-derived GIP regulators may be used in the development of foods that prevent obesity. Rice bran oil and its components are known to have beneficial effects on health. Therefore, the aim of the present study was to clarify the effects of the oil-soluble components of rice bran on postprandial GIP secretion and obesity in mice. Triterpene alcohols [cycloartenol (CA) and 24-methylene cycloartanol (24Me)], β-sitosterol, and campesterol decreased the diet-induced secretion of GIP in C57BL/6J mice. Mice fed a high-fat diet supplemented with a triterpene alcohol and sterol preparation (TASP) from rice bran for 23 wk gained less weight than control mice. Indirect calorimetry revealed that fat utilization was higher in TASP-fed mice than in control mice. Fatty acid oxidation-related gene expression in the muscles of mice fed a TASP-supplemented diet was enhanced, whereas fatty acid synthesis-related gene expression in the liver was suppressed. The treatment of HepG2 cells with CA and 24Me decreased the gene expression of sterol regulatory element-binding protein (SREBP)-1c. In conclusion, we clarified for the first time that triterpene alcohols and sterols from rice bran prevented diet-induced obesity by increasing fatty acid oxidation in muscles and decreasing fatty acid synthesis in the liver through GIP-dependent and GIP-independent mechanisms. PMID:25257874

  19. Developmental changes in the sterol composition and the glycerol content of cuticular and internal lipids of three species of flies.

    PubMed

    Gołębiowski, Marek; Cerkowniak, Magdalena; Boguś, Mieczysława I; Włóka, Emilia; Przybysz, Elżbieta; Stepnowski, Piotr

    2013-08-01

    The glycerol concentration and the composition of cuticular and internal sterols in three medically and forensically important fly species, viz., Musca domestica, Sarcophaga carnaria, and Calliphora vicina, were analyzed. The cuticular and internal lipid extracts were separated by HPLC-LLSD, after which the sterol fraction was characterized by GC/MS in total ion current (TIC) mode. The cuticular lipids of M. domestica larvae contained seven sterols, while in pupae and females, six sterols were identified. Five sterols were found in the cuticular lipids of M. domestica males. The internal lipids of M. domestica larvae and pupae contained six and seven sterols, respectively, while those of male and female flies contained only five sterols. Sitosterol, cholesterol, and campesterol were the dominant sterols in M. domestica, while campestanol, stigmasterol, sitostanol, and fucosterol were identified in low concentrations or in traces. In contrast, cuticular and internal lipids of S. carnaria and C. vicina contained only cholesterol. Glycerol was identified in all stages of M. domestica, S. carnaria, and C. vicina. For all the three examined fly species, the present study clearly showed species-specific developmental changes in the composition of cuticular and internal sterols as well as in the glycerol concentration. PMID:23939800

  20. Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia.

    PubMed

    Muñoz-Bertomeu, Jesús; Sales, Ester; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2007-11-01

    Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, beta-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T1 plants derived from self-pollinated seeds of T0 lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species. PMID:17714440

  1. Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

    PubMed Central

    Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.

    2015-01-01

    The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313

  2. Sterol composition of shellfish species commonly consumed in the United States

    PubMed Central

    Phillips, Katherine M.; Ruggio, David M.; Exler, Jacob; Patterson, Kristine Y.

    2012-01-01

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

  3. Effect of plant sterols on the lipid profile of patients with hypercholesterolaemia. Randomised, experimental study

    PubMed Central

    2011-01-01

    Background Studies have been conducted on supplementing the daily diet with plant sterol ester-enriched milk derivatives in order to reduce LDL-cholesterol levels and, consequently, cardiovascular risk. However, clinical practice guidelines on hypercholesterolaemia state that there is not sufficient evidence to recommend their use in subjects with hypercholesterolaemia. The main objective of this study is to determine the efficacy of the intake of 2 g of plant sterol esters a day in lowering LDL-cholesterol levels in patients diagnosed with hypercholesterolaemia. The specific objectives are: 1) to quantify the efficacy of the daily intake of plant sterol esters in lowering LDL-cholesterol, total cholesterol and cardiovascular risk in patients with hypercholesterolaemia; 2) to evaluate the occurrence of adverse effects of the daily intake of plant sterol esters; 3) to identify the factors that determine a greater reduction in lipid levels in subjects receiving plant sterol ester supplements. Methods/Design Randomised, double-blind, placebo controlled experimental trial carried out at family doctors' surgeries at three health centres in the Health Area of Albacete (Spain). The study subjects will be adults diagnosed with "limit" or "defined" hypercholesterolaemia and who have LDL cholesterol levels of 130 mg/dl or over. A dairy product in the form of liquid yoghurt containing 2 g of plant sterol ester per container will be administered daily after the main meal, for a period of 24 months. The control group will receive a daily unit of yogurt not supplemented with plant sterol esters that has a similar appearance to the enriched yoghurt. The primary variable is the change in lipid profile at 1, 3, 6, 12, 18 and 24 months. The secondary variables are: change in cardiovascular risk, adherence to the dairy product, adverse effects, adherence to dietary recommendations, frequency of food consumption, basic physical examination data, health problems, lipid

  4. Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

    PubMed Central

    2012-01-01

    Background The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Results Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. Conclusions The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress

  5. Plant sterols: factors affecting their efficacy and safety as functional food ingredients

    PubMed Central

    Berger, Alvin; Jones, Peter JH; Abumweis, Suhad S

    2004-01-01

    Plant sterols are naturally occurring molecules that humanity has evolved with. Herein, we have critically evaluated recent literature pertaining to the myriad of factors affecting efficacy and safety of plant sterols in free and esterified forms. We conclude that properly solubilized 4-desmetyl plant sterols, in ester or free form, in reasonable doses (0.8–1.0 g of equivalents per day) and in various vehicles including natural sources, and as part of a healthy diet and lifestyle, are important dietary components for lowering low density lipoprotein (LDL) cholesterol and maintaining good heart health. In addition to their cholesterol lowering properties, plant sterols possess anti-cancer, anti-inflammatory, anti-atherogenicity, and anti-oxidation activities, and should thus be of clinical importance, even for those individuals without elevated LDL cholesterol. The carotenoid lowering effect of plant sterols should be corrected by increasing intake of food that is rich in carotenoids. In pregnant and lactating women and children, further study is needed to verify the dose required to decrease blood cholesterol without affecting fat-soluble vitamins and carotenoid status. PMID:15070410

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

    PubMed

    Hammann, Simon; Vetter, Walter

    2016-05-01

    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. PMID:27064103

  7. Following intracellular cholesterol transport by linear and non-linear optical microscopy of intrinsically fluorescent sterols.

    PubMed

    Wüstner, Daniel

    2012-02-01

    Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow the cellular movement of this essential lipid molecule. In this article, a survey of the various methods being used for analysis of sterol trafficking is given. Various classical biochemical methods are presented and their suitability for analysis of sterol trafficking is assessed. Special emphasis is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented. PMID:21470123

  8. Study of thermodynamic parameters for solubilization of plant sterol and stanol in bile salt micelles.

    PubMed

    Matsuoka, Keisuke; Nakazawa, Tomomi; Nakamura, Ai; Honda, Chikako; Endo, Kazutoyo; Tsukada, Masamichi

    2008-08-01

    We investigated the difference between the molecular structures of plant sterols and stanols that affect the solubilization of cholesterol in bile salt micelles (in vitro study). First, the aqueous solubility of beta-sitosterol, beta-sitostanol, and campesterol was determined by considering the specific radioactivity by using a fairly small quantity of each radiolabeled compound. The order of their aqueous solubilities was as follows: cholesterol > campesterol > beta-sitostanol > beta-sitosterol. The maximum solubility of cholesterol and the above mentioned sterol/stanol in sodium taurodeoxycholate and sodium taurocholate solutions (single solubilizate system) was measured. Moreover, the preferential solubilization of cholesterol in bile salt solutions was systematically studied by using different types of plant sterols/stanols. The solubilization results showed that the cholesterol-lowering effect was similar for sterols and stanol. Thermodynamic analysis was applied to these experimental results. The Gibbs energy change (Delta G degrees ) for the solubilization of plant sterols/stanols showed a negative value larger than that for cholesterol. PMID:18544343

  9. Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi.

    PubMed Central

    Kerwin, J L; Duddles, N D

    1989-01-01

    Several genera of oomycete fungi which are incapable of de novo sterol synthesis do not require these compounds for vegetative growth. The requirement for an exogenous source of sterols for sexual reproduction by several members of the Pythiaceae has been questioned by reports of apparent induction and maturation of oospores on defined media supplemented with phospholipids in the absence of sterols. A more detailed examination of this phenomenon suggested that trace levels of sterols in the inoculum of some pythiaceous fungi act synergistically with phospholipid medium supplements containing unsaturated fatty acid moieties to induce oosporogenesis. Phospholipid analysis of one species, Pythium ultimum, suggested that only the fatty acid portion of the exogenous phospholipid is taken up by the fungus. Enrichment of the phospholipid fraction of total cell lipid of P. ultimum with unsaturated fatty acids promoted oospore induction, and enhanced levels of unsaturated fatty acids in the neutral lipid fraction increased oospore viability. For some pythiaceous fungi, the levels of sterols required for the maturation of oospores with appropriate phospholipid medium supplementation suggest that these compounds are necessary only for the sparking and critical domain roles previously described in other fungi. PMID:2738023

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

    PubMed

    Stong, Rachel A; Kolodny, Eli; Kelsey, Rick G; González-Hernández, M P; Vivanco, Jorge M; Manter, Daniel K

    2013-06-01

    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 white oak) in order to evaluate the effect of differing sterol/tannin contents on Phytophthora ramorum growth. For all three species, high levels of foliage inhibited P. ramorum growth and sporulation, with a steeper concentration dependence for the two oak samples. Phytophthora ramorum growth and sporulation were inhibited by either phytosterols or tannin-enriched extracts. High levels of sterols diminished elicitin gene expression in P. ramorum; whereas the tannin-enriched extract decreased the amount of 'functional' or ELISA-detectable elicitin, but not gene expression. Across all treatment combinations, P. ramorum growth and sporulation correlated strongly with the amount of ELISA-detectable elicitin (R (2) = 0.791 and 0.961, respectively). PMID:23689874

  11. Analysis of Vascular Development in the hydra Sterol Biosynthetic Mutants of Arabidopsis

    PubMed Central

    Pullen, Margaret; Clark, Nick; Zarinkamar, Fatemeh; Topping, Jennifer; Lindsey, Keith

    2010-01-01

    Background The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning. Methodology/Principal Findings Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development. Conclusions The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development. PMID:20808926

  12. Towards New Insights in the Sterol/Amphotericin Nanochannels Formation: A Molecular Dynamic Simulation Study.

    PubMed

    Boukari, Khaoula; Balme, Sébastien; Janot, Jean-Marc; Picaud, Fabien

    2016-06-01

    Amphotericin B (AmB) is a well-known polyene which self-organizes into membrane cell in order to cause the cell death. Its specific action towards fungal cell is not fully understood but was proved to become from sterol composition. The mechanism was shown experimentally to require the formation of stable sterol/polyene couples which could then organize in a nanochannel. This would allow the leakage of ions responsible for the death of fungal cells, only. In this present study, we investigate the arrangement of AmB/sterols in biological membrane using molecular dynamic simulations in order to understand the role of the sterol structure on the antifungal action of the polyene. We show in particular that the nanochannels tend to close up when cell was composed with cholesterol (animal cell) due to strong interaction between amphotericin and sterol. On the other side, with ergosterol (fungal cell) the largest interactions between amphotericin and lipid membrane lead to the appearance of large hole that could favor the important leakage of ions and thus, the fungal cell death. This work appears as a good complement in the extensive studies linked to the understanding of the antifungal molecules in membrane cells. PMID:26700625

  13. Shading Influence on the Sterol Balance of Nicotiana tabacum L. 1

    PubMed Central

    Grunwald, Claus

    1978-01-01

    Tobacco plants (Nicotiana tabacum L.) were grown in the field and the apex was removed at the 42-day stage. Shading screens were set up which produced 0, 26, 67, and 90% shade. Plants were grown an additional 25 days before leaves from top, middle, and bottom stalk positions were harvested. Each leaf group was analyzed for free sterol, steryl ester, steryl glycoside, and acylsteryl glycoside. The free sterol content was lowest in top leaves and highest in bottom leaves; however, the top leaves had more steryl ester than the bottom leaves. Leaf position had no effect on steryl glycosides and acylsteryl glycosides. Shading did not influence the level of any sterol class; but in general, shading increased stigmasterol and decreased sitosterol. This trend was observed for all sterol classes, and the free sterols showed the largest and most consistent change. The younger top leaves showed a greater response than the older bottom leaves, but bottom leaves always had more stigmasterol than sitosterol even without shade. PMID:16660242

  14. Farnesol biosynthesis in Candida albicans: cellular response to sterol inhibition by zaragozic acid B.

    PubMed

    Hornby, Jacob M; Kebaara, Bessie W; Nickerson, Kenneth W

    2003-07-01

    The dimorphic fungus Candida albicans produces farnesol as a quorum-sensing molecule that regulates cellular morphology. The biosynthetic origin of farnesol has been resolved by treating these cells with zaragozic acid B, a potent inhibitor of squalene synthase in the sterol biosynthetic pathway. Treatment with zaragozic acid B leads to an eightfold increase in the amount of farnesol produced by C. albicans. Furthermore, C. albicans cell extracts contain enzymatic activity to convert [(3)H]farnesyl pyrophosphate to [(3)H]farnesol. Many common antifungal antibiotics (e.g., zaragozic acids, azoles, and allylamines) target steps in sterol biosynthesis. We suggest that the fungicidal activity of zaragozic acid derives in large part from the accumulation of farnesol that accompanies the inhibition of sterol biosynthesis. PMID:12821501

  15. Sewage contamination of sediments from two Portuguese Atlantic coastal systems, revealed by fecal sterols.

    PubMed

    Rada, Jesica P A; Duarte, Armando C; Pato, Pedro; Cachada, Anabela; Carreira, Renato S

    2016-02-15

    Fecal sterols in sediments were used to assess the degree of sewage contamination in Ria de Aveiro lagoon and Mondego River estuary for the first time. Coprostanol, the major fecal sterol, averaged 1.82 ± 4.12 μg g(-1), with maxima of 16.6 μg g(-1). The northwestern sector of the Ria and a marina at Mondego estuary showed the highest level of sewage contamination. This scenario was confirmed by several diagnostic ratios based on fecal sterols and other phytosterols. Our data revealed that in spite of the improvements achieved in the last decades, there is still a need for control the organic inputs into the aquatic environment in the studied regions. PMID:26778497

  16. A rapid method to determine sterol, erythrodiol, and uvaol concentrations in olive oil.

    PubMed

    Mathison, Brian; Holstege, Dirk

    2013-05-15

    A rapid, accurate, and efficient method for determining the sterol, uvaol, and erythrodiol concentrations was developed to meet International Olive Council (IOC) certification criteria for extra virgin olive oil (EVOO). The unsaponifiable fraction of the sample (0.2 g) was separated with a diatomaceous earth column, and the sterol and triterpenic dialcohols were isolated with a novel base-activated silica solid-phase extraction (SPE) cartridge cleanup protocol. The improved method and the IOC method provided identical pass/fail results (n = 34) for each of the six sterol and erythrodiol/uvaol IOC criteria used to assess olive oil. This method was validated, and recoveries of stigmasterol (88%) and β-sitosterol (84%) were greater than previously published values obtained using the IOC method. This method requires approximately one-third the time required to complete the IOC method and has great utility for the rapid screening of EVOO to detect adulteration, false labeling, and an inferior product. PMID:23587059

  17. Visualization of Sterol-Rich Membrane Domains with Fluorescently-Labeled Theonellamides

    PubMed Central

    Nishimura, Shinichi; Ishii, Kumiko; Iwamoto, Kunihiko; Arita, Yuko; Matsunaga, Shigeki; Ohno-Iwashita, Yoshiko; Sato, Satoshi B.; Kakeya, Hideaki; Kobayashi, Toshihide; Yoshida, Minoru

    2013-01-01

    Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions. PMID:24386262

  18. Pentacyclic hemiacetal sterol with antifouling and cytotoxic activities from the soft coral Nephthea sp.

    PubMed

    Zhang, Jun; Li, Liang-Chun; Wang, Kai-Ling; Liao, Xiao-Jian; Deng, Zhou; Xu, Shi-Hai

    2013-02-15

    A novel unusual pentacyclic hemiacetal sterol nephthoacetal (1), was isolated from soft coral Nephthea sp. The structure of this sterol was inferred from its two acetyl derivatives (2) and (3), by means of spectroscopic methods, and quantum chemical calculations. Anti-fouling activity of compounds 1-3 against Bugula neritina larvae was evaluated, sterol (1) exhibited significant inhibitory effect with EC(50) value of 2.5 μg/mL, while having low toxicity with LC(50)>25.0 μg/mL. The in vitro cytotoxic activity of compounds 1-3 against HeLa cells was also evaluated, all of them exhibited moderate cytotoxicity with IC(50) values of 12.3 (1), 10.1 (2), and 19.6 μg/mL (3), respectively. PMID:23294699

  19. Lanosterol biosynthesis in the prokaryote Methylococcus capsulatus: insight into the evolution of sterol biosynthesis.

    PubMed

    Lamb, David C; Jackson, Colin J; Warrilow, Andrew G S; Manning, Nigel J; Kelly, Diane E; Kelly, Steven L

    2007-08-01

    A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general. PMID:17567593

  20. Lipid and stress dependence of amphotericin B ion selective channels in sterol-free membranes.

    PubMed

    Ruckwardt, T; Scott, A; Scott, J; Mikulecky, P; Hartsel, S C

    1998-07-17

    The idea that amphotericin B (AmB) may not require sterols to form ion selective channels has recently been criticized on the grounds that egg phospholipids commonly used in experiments may contain small amounts of sterol which associate with AmB to form AmB/sterol pore channel structures. It was recently shown in this laboratory that modest osmotic stress can enhance the formation of AmB channels in sterol-free egg phosphatidylcholine (eggPC) membranes. We have tested AmB's ability to form ion channels/defects in synthetic palmitoyl oleoyl (POPC), dieicosenyl (DEPC) and natural eggPC osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. These sterol-free POPC LUV exhibit greatly increased sensitivity to cation selective AmB channel formation when osmotically stressed; even more than eggPC. Under these stressed conditions, AmB activity was observed at [AmB]/POPC ratios as low as 3.5x10(-4), corresponding to about 34 AmB molecules/vesicle. DEPC vesicles were almost completely unresponsive, demonstrating a strong bilayer thickness dependence. These results prove conclusively that AmB can form sterol-free channels and do so within therapeutic concentration ranges (>0.5-10x10(-6) M) in a stress-dependent manner. This phenomenon may allow us to use osmotic stress changes in simple model systems to spectroscopically isolate and characterize the thus-far elusive AmB channel forming aggregate. In addition, this stress dependence may be responsible for the potentiation of renal toxicity of AmB in the ascending branch of the loop of Henle which is under greatest osmotic stress. PMID:9675313

  1. Activation of Sterol-response Element-binding Proteins (SREBP) in Alveolar Type II Cells Enhances Lipogenesis Causing Pulmonary Lipotoxicity*

    PubMed Central

    Plantier, Laurent; Besnard, Valérie; Xu, Yan; Ikegami, Machiko; Wert, Susan E.; Hunt, Alan N.; Postle, Anthony D.; Whitsett, Jeffrey A.

    2012-01-01

    Pulmonary inflammation is associated with altered lipid synthesis and clearance related to diabetes, obesity, and various inherited metabolic disorders. In many tissues, lipogenesis is regulated at the transcriptional level by the activity of sterol-response element-binding proteins (SREBP). The role of SREBP activation in the regulation of lipid metabolism in the lung was assessed in mice in which both Insig1 and Insig2 genes, encoding proteins that bind and inhibit SREBPs in the endoplasmic reticulum, were deleted in alveolar type 2 cells. Although deletion of either Insig1 or Insig2 did not alter SREBP activity or lipid homeostasis, deletion of both genes (Insig1/2Δ/Δ mice) activated SREBP1, causing marked accumulation of lipids that consisted primarily of cholesterol esters and triglycerides in type 2 epithelial cells and alveolar macrophages. Neutral lipids accumulated in type 2 cells in association with the increase in mRNAs regulating fatty acid, cholesterol synthesis, and inflammation. Although bronchoalveolar lavage fluid phosphatidylcholine was modestly decreased, lung phospholipid content and lung function were maintained. Insig1/2Δ/Δ mice developed lung inflammation and airspace abnormalities associated with the accumulation of lipids in alveolar type 2 cells, alveolar macrophages, and within alveolar spaces. Deletion of Insig1/2 activated SREBP-enhancing lipogenesis in respiratory epithelial cells resulting in lipotoxicity-related lung inflammation and tissue remodeling. PMID:22267724

  2. Fungal sterol C22-desaturase is not an antimycotic target as shown by selective inhibitors and testing on clinical isolates.

    PubMed

    Müller, Christoph; Binder, Ulrike; Maurer, Elisabeth; Grimm, Christian; Giera, Martin; Bracher, Franz

    2015-09-01

    Inhibition of concise enzymes in ergosterol biosynthesis is one of the most prominent strategies for antifungal chemotherapy. Nevertheless, the enzymes sterol C5-desaturase and sterol C22-desaturase, which introduce double bonds into the sterol core and side chain, have not been fully investigated yet for their potential as antifungal drug targets. Lathosterol side chain amides bearing N-alkyl groups of proper length are known as potent inhibitors of the enzymes sterol C5-desaturase and sterol Δ(24)-reductase in mammalian cholesterol biosynthesis. Here we present the results of our evaluation of these amides for their ability to inhibit enzymes in fungal ergosterol biosynthesis. In the presence of inhibitor(s) an accumulation of sterols lacking a double bond at C22/23 (mainly ergosta-5,7-dien-3β-ol) was observed in Candida glabrata, Saccharomyces cerevisiae, and Yarrowia lipolytica. Hence, the lathosterol side chain amides were identified as selective inhibitors of the fungal sterol C22-desaturase, which was discussed as a specific target for novel antifungals. One representative inhibitor, (3S,20S)-20-N-butylcarbamoylpregn-7-en-3β-ol was subjected to antifungal susceptibility testing on patient isolates according to modified EUCAST guidelines. But, the test organisms showed no significant reduction of cell growth and/or viability up to an inhibitor concentration of 100μg/mL. This leads to the conclusion that sterol C22-desaturase is not an attractive target for the development of antifungals. PMID:26022150

  3. Seasonal and geographical variations of sterol composition in snow crab hepatopancreas and pelagic fish viscera from Eastern Quebec.

    PubMed

    Souchet, Nathalie; Laplante, Serge

    2007-07-01

    Sterol composition was determined in snow crab hepatopancreas and mackerel and herring viscera for various locations and collection periods. A simple and valuable method, using direct saponification and extraction with water-cyclohexane has been optimized to recover total sterol. They were identified and quantified as trimethylsilyl ether derivatives by GC-MS analysis. Method validation indicated excellent sensitivity (limit of quantification: 1.25 mg/100 g wet basis for cholesterol and desmosterol; 0.03-0.05 mg/100 g for other sterols), good reproducibility (CV%: 1.5-6.8) and accuracy (recovery%: 94-107). In crab hepatopancreas, cholesterol was the main sterol (67-76%), followed by desmosterol (19-24%). Phytosterols and molluscan sterols were also present in low quantity. A lower total sterol content with different composition was found in crabs from Magdalen Islands compared to those from Gaspé Peninsula or North Shore of the St-Lawrence Gulf. No seasonal variation was observed between collection periods, which were probably too close. Mackerel and herring viscera contained the same sterols as crab except for campesterol and sitosterol, but the cholesterol proportion was higher (93-98%). The higher abundance of sterols in herring caught in September vs. May would be related to an increase of the body lipid content during the summer. PMID:17374564

  4. Concentrations of surfactants and sterols in the surface microlayer of the estuarine areas of Selangor River, Malaysia

    NASA Astrophysics Data System (ADS)

    Alsalahi, Murad Ali; Talib Latif, Mohd; Mohd Ali, Masni; Dominick, Doreena; Firoz Khan, Md; Bahiyah Abd Wahid, Nurul; Ili Hamizah Mustaffa, Nur

    2016-04-01

    This study determined 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 in SML 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 ionisation 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. 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%).

  5. Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts 1

    PubMed Central

    Moeller, Curt H.; Mudd, J. Brian

    1982-01-01

    Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16662716

  6. Biosynthesis of Sterols and Triterpenoids in Tissue Cultures of Cucurbita maxima.

    PubMed

    Caputo, O; Delprino, L; Viola, F; Caramiello, R; Balliano, G

    1983-11-01

    The biosynthesis of sterols and triterpenoids in CUCURBITA MAXIMA was studied by analysis of unsaponifiable fraction of tissues from different development stages of the plant (seeds, seedlings, adult plant and tissue culture) and by feeding germinating seeds and tissue cultures with [2- (14)C]-acetate. Synthesis of cucurbitacins does not occur in callus tissues of CUCURBITA MAXIMA, whereas a wide variety of 4,4-dimethylsterols present in these tissues testifies of a high level of squaleneoxide cyclase activity in growing callus. The peculiarity of Cucurbitaceae among the higher plants is also discussed comparing the side chain biosynthesis of sterols in CUCURBITA MAXIMA to that operating in other higher plants. PMID:17405044

  7. Fecal free and conjugated bile acids and neutral sterols in vegetarians, omnivores, and patients with colorectal cancer.

    PubMed

    Korpela, J T; Adlercreutz, H; Turunen, M J

    1988-04-01

    Increased excretion and intestinal bacterial metabolism of bile acids and neutral sterols have been suggested to be associated with an increased risk of colorectal cancer. We determined fecal neutral sterol and bile acid profiles by new capillary column gas-liquid chromatographic methods in 18 patients with colorectal cancer, 10 omnivores, and 10 vegetarians. The methods also determine concentrations of esterified neutral sterols and saponifiable bile acids formed by intestinal bacterial action. Patients with colorectal cancer had the highest concentrations of neutral animal sterols, the lowest degree of esterification of neutral sterols, the lowest relative amount of saponifiable bile acids, and the highest concentrations of unconjugated primary bile acids. These differences were statistically significant (p less than 0.05) and more profound when the patients were compared with vegetarians than with omnivores. Since epidemiologic studies suggest that vegetarians have a lower risk of colorectal cancer than omnivores, these differences are discussed as possible risk factors for colorectal cancer. PMID:3387891

  8. Genomewide location analysis of Candida albicans Upc2p, a regulator of sterol metabolism and azole drug resistance.

    PubMed

    Znaidi, Sadri; Weber, Sandra; Al-Abdin, Osman Zin; Bomme, Perrine; Saidane, Saloua; Drouin, Simon; Lemieux, Sébastien; De Deken, Xavier; Robert, François; Raymond, Martine

    2008-05-01

    Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5'-VNCGBDTR that includes the previously characterized Upc2p binding site 5'-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2Delta/upc2Delta strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes. PMID:18390649

  9. Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

    PubMed

    Leonardsen, L; Strömstedt, M; Jacobsen, D; Kristensen, K S; Baltsen, M; Andersen, C Y; Byskov, A G

    2000-01-01

    Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in

  10. Comparative health effects of margarines fortified with plant sterols and stanols on a rat model for hemorrhagic stroke.

    PubMed

    Ratnayake, W M N; Plouffe, L; L'Abbé, M R; Trick, K; Mueller, R; Hayward, S

    2003-12-01

    There is increased acceptance of fortifying habitual foods with plant sterols and their saturated derivatives, stanols, at levels that are considered safe. These sterols and stanols are recognized as potentially effective dietary components for lowering plasma total and LDL cholesterol. Our previous studies have shown that daily consumption of plant sterols promotes strokes and shortens the life span of stroke-prone spontaneously hypertensive (SHRSP) rats. These studies question the safety of plant sterol additives. The present study was performed to determine whether a large intake of plant stanols would cause nutritional effects similar to those seen with plant sterols in SHRSP rats. Young SHRSP rats (aged 26-29 d) were fed semipurified diets containing commercial margarines fortified with either plant stanols (1.1 g/100 g diet) or plant sterols (1.4 g/100 g diet). A reference group of SHRSP rats was fed a soybean oil diet (0.02 g plant sterols/100 g diet and no plant stanols). Compared to soybean oil, both plant stanol and plant sterol margarines significantly (P < 0.05) reduced the life span of SHRSP rats. At the initial stages of feeding, there was no difference in the survival rates between the two margarine groups, but after approximately 50 d of feeding, the plant stanol group had a slightly, but significantly (P < 0.05), lower survival rate. Blood and tissue (plasma, red blood cells, liver, and kidney) concentrations of plant sterols in the plant sterol margarine group were three to four times higher than the corresponding tissue concentrations of plant stanols in the plant stanol group. The deformability of red blood cells and the platelet count of SHRSP rats fed the plant sterol margarine were significantly (P < 0.05) lower than those of the plant stanol margarine and soybean oil groups at the end of the study. These parameters did not differ between the soybean oil and plant stanol margarine groups. These results suggest that, at the levels tested in

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Evaluation of anthropogenic contamination using sterol markers in a tropical estuarine system of northeast Brazil.

    PubMed

    Frena, Morgana; Souza, Michel R R; Damasceno, Flaviana C; Madureira, Luiz A S; Alexandre, Marcelo R

    2016-08-15

    The São Francisco River estuarine system, located in the Northeast coast of Brazil, has great economic, tourist and social importance. Its waters are used for activities such as agriculture, aquaculture, navigation and fishery, which supplies the surrounding communities. In this study, sterols markers were determined in twenty-eight sediment samples from São Francisco River estuary by gas chromatography - mass spectrometry (GC-MS). Sterol analysis was useful to distinguish between anthropogenic and biogenic organic matter (OM) sources in the studied area. Six sterols were quantified, suggesting different sources. Concentrations of fecal sterol (coprostanol) were lower than 500ngg(-1), suggesting no indicative of severe sewage contamination.However, two stations showed concentrations around 100ngg(-1) and the values for the coprostanol/(coprostanol+cholestanol) and coprostanol/cholesterol ratios indicates sewage contamination. The results in this study may be considered as baseline concentrations to be used as future reference for monitoring programs to prevent anthropogenic impacts. PMID:27207024

  13. The biological activity of a-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening. Alpha-mangostin was tested for its larvicidal activity against 3rd instar larvae of six mosquito species and the LC50 values range from 0.84 to 2.90 ppm....

  14. Sterol Biosynthesis Pathway as an Alternative for the Anti-Protozoan Parasite Chemotherapy.

    PubMed

    de Macedo-Silva, Sara Teixeira; de Souza, Wanderley; Rodrigues, Juliany C Fernandes

    2015-01-01

    Sterols play an essential role in the physiology of eukaryotic cells; they play a pivotal role in the normal structure and function of cell membranes and also act as precursors for the synthesis of several different molecules like steroid hormones. Trypanosomatids and fungi have an essential requirement of ergosterol and other 24-alkyl sterols, which are absent in mammalian cells, for their survival and growth. At least 20 metabolic steps are necessary to synthesize sterols as cholesterol and ergosterol with the involvement of different specific enzymes. Some enzymes have been studied in detail in order to find new inhibitors that are able to abolish the parasite growth in vitro; besides, they also promote the curative efficacy in murine models of infection, thus opening new possibilities to introduce new drugs for the treatment of leishmaniasis and Chagas' disease. Sterols biosynthesis inhibitors (SBIs) can potentially be used as a chemotherapeutic agent against trypanosomatids. Actually, there are several drugs that interfere with the SB pathway, and some of them are already in clinical trials, such as posaconazole, and a new pro-drug, the ravuconazole. Furthermore, new approaches are being used, such as the combination of drugs, to reduce the resistance and minimize toxic effects. In this review, we discuss the main steps of the SB pathway, showing each enzyme involved in the steps, as well as the antiproliferative, physiological, biochemical, and ultrastructural effects of the several known inhibitors. PMID:25787966

  15. Absence of sterols constrains carbon transfer between cyanobacteria and a freshwater herbivore (Daphnia galeata).

    PubMed Central

    von Elert, Eric; Martin-Creuzburg, Dominik; Le Coz, Jean R

    2003-01-01

    A key process in freshwater plankton food webs is the regulation of the efficiency of energy and material transfer. Cyanobacterial carbon (C) in particular is transferred very inefficiently to herbivorous zooplankton, which leads to a decoupling of primary and secondary production and the accumulation of cyanobacterial biomass, which is associated with reduced recreational quality of water bodies and hazards to human health. A recent correlative field study suggested that the low transfer efficiency of cyanobacterial C is the result of the absence of long-chain polyunsaturated fatty acids (PUFA) in the diet of the zooplankton. By supplementation of single-lipid compounds in controlled growth experiments, we show here that the low C transfer efficiency of coccal and filamentous cyanobacteria to the keystone herbivore Daphnia is caused by the low sterol content in cyanobacteria, which constrains cholesterol synthesis and thereby growth and reproduction of the herbivore. Estimations of sterol requirement in Daphnia suggest that, when cyanobacteria comprise more than 80% of the grazed phytoplankton, growth of the herbivore may be limited by sterols and Daphnia may subsequently fail to control phytoplankton biomass. Dietary sterols therefore may play a key role in freshwater food webs and in the control of water quality in lakes dominated by cyanobacteria. PMID:12816661

  16. Plant sterol consumption frequency affects plasma lipid levels and cholesterol kinetics in humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Objectives: To compare the efficacy of single versus multiple doses of plant sterols on circulating lipid level and cholesterol trafficking. Subjects/Methods: A randomized, placebo-controlled, three-phase (6 days/phase) crossover, supervised feeding trial was conducted in 19 subjects. Sub...

  17. Effect of frequency of dosing of plant sterols on plasma cholesterol levels and synthesis rate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to compare the effects of plant sterols (PS) consumed as a single dose (single) at breakfast or as three doses consumed with breakfast, lunch and dinner (divided) on plasma lipoprotien levels and cholesterol endogenous fractional synthesis rate (FSR). A randomized, placebo-controll...

  18. Effects of Temperature and Nutrients on Sterol Concentration in Marine Diatoms and Implications for Productivity Reconstructions

    NASA Astrophysics Data System (ADS)

    Ding, Y.; Bi, R.; Zhao, M.; Zhang, L. H.; Li, L.

    2015-12-01

    Sterols as phytoplankton productivity and community structure proxies have been widely applied for paleo-reconstructions, while quantitative reconstructions using sterols remain understudied. In this study, we aimed to determine the quantitative relationship between sterols and biomass in three species of marine diatoms under different temperature (15℃, 20℃ and25℃) and different nutrient supply (N:P=10:1, 24:1 and 63:1). Brassicasterol is the major sterol in Phaeodactylum tricornutum Bohli, an important species in marginal seas. The effects of temperature on the cellular concentration of brassicasterol is minimum, with values of 1.01×10 -4 ng cell-1 at 15℃, 1.07×10 -4 ng cell-1 at 20℃ and 1.17×10 -4 ng cell-1 at 25℃. Work is underway to evaluate the effects of nutrients on the cellular concentration of brassicasterol. Our preliminary results suggest that brassicasterol could be used to quantitatively reconstruct diatom productivity, and we will report the results of its application in several sediment cores.

  19. Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption.

    PubMed

    Wood, W G; Schroeder, F; Hogy, L; Rao, A M; Nemecz, G

    1990-06-27

    Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids. PMID:2364080

  20. Unsaturated lipid matrices protect plant sterols from degradation during heating treatment.

    PubMed

    Barriuso, Blanca; Astiasarán, Iciar; Ansorena, Diana

    2016-04-01

    The interest in plant sterols enriched foods has recently enhanced due to their healthy properties. The influence of the unsaturation degree of different fatty acids methyl esters (FAME: stearate, oleate, linoletate and linolenate) on a mixture of three plant sterols (PS: campesterol, stigmasterol and β-sitosterol) was evaluated at 180 °C for up to 180 min. Sterols degraded slower in the presence of unsaturated FAME. Both PS and FAME degradation fit a first order kinetic model (R(2)>0.9). Maximum oxysterols concentrations were achieved at 20 min in neat PS and 120 min in lipid mixtures and this maximum amount decreased with increasing their unsaturation degree. In conclusion, the presence of FAME delayed PS degradation and postponed oxysterols formation. This protective effect was further promoted by increasing the unsaturation degree of FAME. This evidence could help industries to optimize the formulation of sterol-enriched products, so that they could maintain their healthy properties during cooking or processing. PMID:26593514

  1. Building Developmental Gene Regulatory Networks

    PubMed Central

    Li, Enhu; Davidson, Eric H.

    2009-01-01

    Animal development is an elaborate process programmed by genomic regulatory instructions. Regulatory genes encode transcription factors and signal molecules, and their expression is under the control of cis-regulatory modules that define the logic of transcriptional responses to the inputs of other regulatory genes. The functional linkages amongst regulatory genes constitute the gene regulatory networks (GRNs) that govern cell specification and patterning in development. Constructing such networks requires identification of the regulatory genes involved and characterization of their temporal and spatial expression patterns. Interactions (activation/repression) among transcription factors or signals can be investigated by large-scale perturbation analysis, in which the function of each gene is specifically blocked. Resultant expression changes are then integrated to identify direct linkages, and to reveal the structure of the GRN. Predicted GRN linkages can be tested and verified by cis-regulatory analysis. The explanatory power of the GRN was shown in the lineage specification of sea urchin endomesoderm. Acquiring such networks is essential for a systematic and mechanistic understanding of the developmental process. PMID:19530131

  2. An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

    PubMed Central

    Keller, Petra; Müller, Christoph; Engelhardt, Isabel; Hiller, Ekkehard; Lemuth, Karin; Eickhoff, Holger; Wiesmüller, Karl-Heinz; Burger-Kentischer, Anke; Bracher, Franz

    2015-01-01

    Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway. PMID:26248360

  3. Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases.

    PubMed

    Yu, Shan; Li, Sijia; Henke, Adam; Muse, Evan D; Cheng, Bo; Welzel, Gustav; Chatterjee, Arnab K; Wang, Danling; Roland, Jason; Glass, Christopher K; Tremblay, Matthew

    2016-07-01

    Liver X receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR-mediated transcription by synthetic agonists, such as T0901317 and GW3965, attenuates progression of inflammatory disease in animal models. However, the adverse effects of these conventional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. Here, we explore the ability of a series of sterol-based LXR agonists to alleviate inflammatory conditions in mice without hepatotoxicity. We show that oral treatment with sterol-based LXR agonists in mice significantly reduces dextran sulfate sodium colitis-induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. The anti-inflammatory property of these agonists is recapitulated in vitro in mouse lamina propria mononuclear cells, human colonic epithelial cells, and human peripheral blood mononuclear cells. In addition, treatment with LXR agonists dramatically suppresses inflammatory cytokine expression in a model of traumatic brain injury. Importantly, in both disease models, the sterol-based agonists do not affect the liver, and the conventional agonist T0901317 results in significant liver lipid accumulation and injury. Overall, these results provide evidence for the development of sterol-based LXR agonists as novel therapeutics for chronic inflammatory diseases.-Yu, S., Li, S., Henke, A., Muse, E. D., Cheng, B., Welzel, G., Chatterjee, A. K., Wang, D., Roland, J., Glass, C. K., Tremblay, M. Dissociated sterol-based liver X receptor agonists as therapeutics for chronic inflammatory diseases. PMID:27025962

  4. An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols.

    PubMed

    Keller, Petra; Müller, Christoph; Engelhardt, Isabel; Hiller, Ekkehard; Lemuth, Karin; Eickhoff, Holger; Wiesmüller, Karl-Heinz; Burger-Kentischer, Anke; Bracher, Franz; Rupp, Steffen

    2015-10-01

    Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway. PMID:26248360

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

    PubMed

    Kessler, Rafael Luis; Soares, Maurilio José; Probst, Christian Macagnan; Krieger, Marco Aurélio

    2013-01-01

    The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC(50)/72 h) or killing all cells within 24 hours (EC(100)/24 h). Incubation with inhibitors at the EC(50)/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC(50)/72 h. By contrast, treatment with SBIs at the EC(100)/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the "point of no return" in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite. PMID:23383204

  6. Sterols in red and green algae: quantification, phylogeny, and relevance for the interpretation of geologic steranes.

    PubMed

    Kodner, R B; Pearson, A; Summons, R E; Knoll, A H

    2008-08-01

    Steroids, a class of triterpenoid lipids with high preservation potential, are widely distributed in sedimentary rocks. All eukaryotes have a physiological requirement for these molecules, making steroids important biomarkers for aiding our understanding of eukaryote molecular evolution and geologic history. C(26)-C(30) sterols are the molecules most commonly incorporated or synthesized by eukaryotes, and correspond to C(26)-C(30) steranes ubiquitously and abundantly preserved in petroleums and sedimentary bitumens. Because these sterols occur in evolutionarily diverse taxa, it can be difficult to associate any particular compound with a single group of organisms. Nevertheless, geochemists have still been able to draw parallels between the empirical patterns in geologic sterane abundances and the age of petroleum source rocks. Paleobiologists have also used sterane data, in particular the patterns in C(29) and C(28) steranes, to support fossil evidence of an early radiation of green algae in latest Proterozoic and Paleozoic and the succession of the major modern phytoplankton groups in the Mesozoic. Although C(29) sterols are found in many eukaryotes, organisms that produce them in proportional abundances comparable to those preserved in Proterozoic and Paleozoic rocks are limited. Based on a large, phylogenetically based survey of sterol profiles from the kingdom Plantae, we conclude that modern ulvophyte and early diverging prasinophyte green algae produce high abundances of C(29) relative to C(27) and C(28) sterols most consistent with the sterane profiles observed in Paleozoic rocks. Our analysis also suggests that ancestral stem groups among the Plantae, including the glaucocystophytes and early divergent red algae are also plausible candidates. PMID:18624688

  7. History and development of plant sterol and stanol esters for cholesterol-lowering purposes.

    PubMed

    Thompson, Gilbert R; Grundy, Scott M

    2005-07-01

    Plant stanol esters provide a novel approach to lowering plasma low-density lipoprotein (LDL) cholesterol by dietary means. Their development was preceded by a long period of research into the cholesterol-lowering properties of plant sterols and, recently, plant stanols. Both classes of compound competitively inhibit the absorption of cholesterol and thus lower its level in plasma. Initial impressions were that stanols were more effective and safer than sterols, but the negative outcome of a study led to the recognition that the lipid solubility of free stanols was very limited. This was overcome by esterifying them with fatty acids, with the resultant stanol esters being freely soluble in fat spreads. This led to the launch of Benecol (margarine; Raisio Group, Raisio, Finland) in 1995. The coincident publication of the year-long North Karelia study conclusively demonstrated the long-term LDL-lowering efficacy of plant stanol esters. Variables that might influence the efficacy of stanol esters include dose, frequency of administration, food vehicle in which the stanol ester is incorporated, and background diet. The effective dose is 1 to 3 g/day, expressed as free stanol, which, in placebo-controlled studies, decreased LDL cholesterol by 6% to 15%. This effect is maintained, appears to be similar with once-daily or divided dosage, and is independent of the fat content of the food vehicle. Short-term studies suggest that equivalent amounts of plant sterol and stanol esters are similarly effective in lowering LDL, the main difference being that plasma plant sterol levels increase on plant sterols and decrease on plant stanols. The clinical significance of these changes remains to be determined. PMID:15992509

  8. Phosphatidyl alcohols: effect of head group size on domain forming properties and interactions with sterols.

    PubMed

    Jaikishan, Shishir; Björkbom, Anders; Slotte, J Peter

    2010-08-01

    In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature ( approximately 49 degrees C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC ( approximately 40-41 degrees C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 degrees C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical

  9. Sterol Modulation of the Plasma Membrane H+-ATPase Activity from Corn Roots Reconstituted into Soybean Lipids.

    PubMed Central

    Grandmougin-Ferjani, A.; Schuler-Muller, I.; Hartmann, M. A.

    1997-01-01

    A partially purified H+-ATPase from the plasma membrane (PM) of corn (Zea mays L.) roots was inserted into vesicles prepared with soybean (Glycine max L.) phospholipids and various concentrations of individual sterols using either a freeze-thaw sonication or an octylglucoside dilution procedure. Both methods yielded a functional enzyme that retained its native characteristics. We have investigated the effects of typical plant sterols (i.e. sitosterol, stigmasterol, and 24-methylcholesterol) on both ATP hydrolysis and H+ pumping by the reconstituted corn root PM ATPase. We have also checked the influence of cholesterol and of two unusual sterols, 24-methylpollinastanol and 14[alpha],24-dimethylcholest-8-en-3[beta]-ol. Here we present evidence for a sterol modulation of the plant PM H+-ATPase activity. In particular, cholesterol and stigmasterol were found to stimulate the pump, especially when present at 5 mol%, whereas all of the other sterols tested behaved as inhibitors at any concentration in proteoliposomes. In all situations H+ pumping was shown to be more sensitive to a sterol environment than was ATP hydrolysis. Our results suggest the occurrence of binding sites for sterols on the plant PM H+-ATPase. PMID:12223599

  10. Effect of unialgal diets on the composition of fatty acids and sterols in juvenile ark shell Tegillarca granosa Linnaeus.

    PubMed

    Xu, Jilin; Zhou, Haibo; Yan, Xiaojun; Zhou, Chengxu; Zhu, Peng; Ma, Bin

    2012-04-18

    This study has investigated the effects of six different unialgal diets ( Chaetoceros calcitrans , Platymonas helgolandica , Chlorella sp., Isochrysis galbana , Nannochloropsis oculata , and Pavlova viridis ) on the composition of fatty acids and sterols in juvenile ark shell Tegillarca granosa Linnaeus. The best feeding effects on the growth of shellfish were found in C. calcitrans, followed by I. galbana and P. viridis, whereas Chlorella sp. and N. oculata exhibited relatively poor effects. The fatty acid and sterol compositions in the six microalgae and the juvenile ark shell after feeding were analyzed, and 39 fatty acids and 18 sterols were identified. Although the results demonstrate a close correlation between the sterol compositions in algal species and juvenile ark shell, a similar correlation was not observed between fatty acids. In the juvenile ark shell fed microalgae, the ratio of total saturated fatty acids (SFA) rapidly decreases, whereas the proportion of total polyunsaturated fatty acids (PUFAs) increases considerably. The abundances of AA, EPA, and DHA increase most significantly in shellfish with better growth (fed C. calcitrans, I. galbana, and P. viridis). The number of sterol species is reduced, but the total sterol content in groups fed corresponding microalgae increases, and abundant plant sterols, instead of cholesterol, are accumulated in juvenile ark shell fed appropriate microalgae I. galbana and P. viridis. Therefore, to be more conducive to human health, I. galbana and P. viridis, of the six experimental microalgae, are recommended for artificial ark shell culture. PMID:22443233

  11. Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A 13C MAS NMR Study

    PubMed Central

    Ciesielski, Filip; Griffin, David C.; Loraine, Jessica; Rittig, Michael; Delves-Broughton, Joss; Bonev, Boyan B.

    2016-01-01

    The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state 13C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol 13C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B. PMID:27379235

  12. Sterol glycosyltransferases-identification of members of gene family and their role in stress in Withania somnifera.

    PubMed

    Chaturvedi, Pankaj; Mishra, Manoj; Akhtar, Nehal; Gupta, Parul; Mishra, Pratibha; Tuli, Rakesh

    2012-10-01

    Sterol glycosyltransferases (SGTs) catalyze the transfer of sugar molecules to diverse sterol molecules, leading to a change in their participation in cellular metabolism. Withania somnifera is a medicinal plant rich in sterols, sterol glycosides and steroidal lactones. Sterols and their modified counterparts are medicinally important and play a role in adaptation of the plant to stress conditions. We have identified 3 members of SGT gene family through RACE (Rapid Amplification of cDNA Ends) in addition to sgtl1 reported earlier. The amino acid sequence deduced from the ORF's showed homology (45-67 %) to the reported plant SGTs. The expression of the genes was differentially modulated in different organs in W. somnifera and in response to external stimuli. Salicylic acid and methyl jasmonate treatments showed up to 10 fold increase in the expression of sgt genes suggesting their role in defense. The level of expression increased in heat and cold stress indicating the role of sterol modifications in abiotic stress. One of the members, was expressed in E. coli and the enzyme assay showed that the crude enzyme glycosylated stigmasterol. W. somnifera expresses a family of sgt genes and there is a functional recruitment of these genes under stress conditions. The genes which are involved in sterol modification are important in view of medicinal value and understanding stress. PMID:22744427

  13. Sterol-dependent nuclear import of ORP1S promotes LXR regulated trans-activation of apoE

    SciTech Connect

    Lee, Sungsoo; Wang, Ping-Yuan; Jeong, Yangsik; Mangelsdorf, David J.; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 ; Anderson, Richard G.W.; Michaely, Peter

    2012-10-01

    Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements. -- Highlights: Black-Right-Pointing-Pointer ORP1S translocates to the nucleus in response to sterol binding. Black-Right-Pointing-Pointer The sterols that best promote nuclear import of ORP1S are LXR agonists. Black-Right-Pointing-Pointer ORP1S binds to LXRs, enhances binding of LXRs to LXREs and promotes LXR-dependent transcription of apoE.

  14. The Origin of Sterol Biosynthesis: A Time-Point for the Evolution of Eukaryotes and the Presence of O2

    NASA Astrophysics Data System (ADS)

    Pearson, A.; Budin, M.; Brocks, J. J.

    2003-12-01

    The evolution of sterol biosynthesis is of critical interest to geoscientists as well as to evolutionary biologists. The first enzyme in the pathway, squalene monooxygenase (Sqmo), requires molecular oxygen (O2), suggesting that this process post-dates the evolution of Cyanobacteria. Additionally, the presence of steranes in ancient rocks marks the suggested time-point of eukaryogenesis(1). Sterol biosynthesis is viewed primarily as a eukaryotic process, and the frequency of its occurrence in bacteria long has been a subject of controversy. In this work, 19 protein gene sequences for Sqmo from eukaryotes were compared to all available complete and partial prokaryotic genomes. Twelve protein gene sequences representing oxidosqualene cyclase (Osc), the second enzyme of the sterol biosynthetic pathway, also were examined. The only unequivocal matches among the bacteria were the alpha-proteobacterium, Methylococcus capsulatus, in which sterol biosynthesis already is known, and the planctomycete, Gemmata obscuriglobus. The latter species contains the most abbreviated sterol pathway yet identified in any organism. Experiments show that the major sterols in Gemmata are lanosterol and its uncommon isomer, parkeol. In bacteria, the sterol biosynthesis genes occupy a contiguous coding region and may represent a single operon. Phylogenetic trees show that the sterol pathway in bacteria and eukaryotes has a common ancestry. Gemmata may retain the most ancient remnants of the pathway's origin, and it is likely that sterol biosynthesis in eukaryotes was acquired through gene transfer from bacteria. However, this work indicates that no known prokaryotes could produce the 24-ethyl steranes found in Archaean rocks(1). Therefore these compounds remain indicative of the presence of both eukaryotes and O2 at 2.7 Ga. 1. J. J. Brocks, G. A. Logan, R. Buick, R. E. Summons, (1999) Science 285, 1033-1036.

  15. Effect of Sterol Structure on the Physical Properties of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Membranes Determined Using (2)H Nuclear Magnetic Resonance.

    PubMed

    Shaghaghi, Mehran; Chen, Mei-Ting; Hsueh, Ya-Wei; Zuckermann, Martin J; Thewalt, Jenifer L

    2016-08-01

    The effect of a series of phytosterols on lipid chain ordering in 1-palmitoyl((2)H31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) multibilayer vesicles was examined by (2)H NMR spectroscopy at 25 °C. These results, along with existing data for other sterols, indicate that the ordering power of sterols in POPC-d31 depends on subtle aspects of sterol structure. Cholesterol, 7-dehydrocholesterol (7-DHC), campesterol, β-sitosterol, ergosterol, brassicasterol, and stigmasterol all increase the lipid chain order as sterol concentration is increased. However, saturation of the ordering occurs at different sterol concentrations for ergosterol (as previously reported), brassicasterol, β-sitosterol, and stigmasterol. Here our interest lies in finding which part of the sterol structure is responsible for the observed saturation of the palmitoyl chain order as a function of sterol concentration. In particular, we propose that the saturation of the ordering of POPC-d31/brassicasterol and POPC-d31/stigmasterol membranes at quite low sterol concentrations is due to the presence of a double bond at C22. We also discuss how the structural differences between the sterols affect their ability to intercalate between the POPC acyl chains. Furthermore, the effective solubility of sterols in POPC is discussed in relation to the dependence of maximum POPC-d31 chain order vs sterol concentration. PMID:27341069

  16. Alignment system for encoders

    NASA Technical Reports Server (NTRS)

    Villani, Daniel D. (Inventor)

    1988-01-01

    An improved encoder alignment system is disclosed which provides an indication of the extent of misalignment and a measure of the rate at which the misalignment may be changing. The invention is adapted for use with a conventional encoder which provides a digital coarse word having at least significant bit and a digital fine word having a least significant bit and a most significant bit. The invention generates the exclusive or of the least significant bit of the coarse digital signal and the least significant bit of the fine digital signal to provide a first signal. The invention then generates the exclusive or of the first signal and the complement of the most significant bit of the fine digital signal to provide an output signal which represents the misalignment of the encoder.

  17. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared. PMID:24690806

  18. The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

    PubMed Central

    Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

    2013-01-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

  19. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  20. Video Time Encoding Machines

    PubMed Central

    Lazar, Aurel A.; Pnevmatikakis, Eftychios A.

    2013-01-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value. PMID:21296708

  1. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  2. Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Troung, T. K.; Reed, I. S.; Deutsch, L. J.; Hsu, I. S.; Wang, K.; Yeh, C. S.

    1985-01-01

    Report presents mathematical principles of Berlekamp bit serial multiplier algorithm and its application to design of very-large-scale integrated (VLSI) encoders for Reed-Solomon error-correcting codes. Structure made readily on single chip of negatively doped channel metal oxide semiconductor.

  3. Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

    PubMed Central

    Warrilow, Andrew G. S.; Hull, Claire M.; Rolley, Nicola J.; Parker, Josie E.; Nes, W. David; Smith, Stephen N.

    2014-01-01

    A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml−1). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities. PMID:25085484

  4. Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14α-demethylase (CYP51).

    PubMed

    Warrilow, Andrew G S; Hull, Claire M; Rolley, Nicola J; Parker, Josie E; Nes, W David; Smith, Stephen N; Kelly, Diane E; Kelly, Steven L

    2014-10-01

    A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities. PMID:25085484

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

    PubMed Central

    Zhao, Donghai; Zheng, Lianwen; Qi, Ling; Wang, Shuran; Guan, Liping; Xia, Yanan; Cai, Jianhui

    2016-01-01

    The purified total sterols and β-sitosterol extracted from Sargassum horneri were evaluated for their antidepressant-like activity using the forced swim test (FST) and tail suspension test (TST) in mice. Total sterols and β-sitosterol significantly reduced the immobility time in the FST and TST. Total sterols were administered orally for 7 days at doses of 50, 100, and 200 mg/kg, and β-sitosterol was administered intraperitoneally at doses of 10, 20, and 30 mg/kg. β-sitosterol had no effect on locomotor activity in the open field test. In addition, total sterols and β-sitosterol significantly increased NE, 5-HT, and the metabolite 5-HIAA in the mouse brain, suggesting that the antidepressant-like activity may be mediated through these neurotransmitters. PMID:27367705

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

    SciTech Connect

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

    1982-05-01

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

  7. The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.

    1992-01-01

    Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the Delta9, Delta10, and Delta11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 C cells and the lowest in 50 C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

  8. The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.

    1992-01-01

    Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

  9. Cloning the sterol carrier protein 2 genes of Japanese toad (Bufo japonicus formosus) and Chinese toad (Bufo gargarizans) and its tissue expression analysis

    PubMed Central

    JI, Yu-Cheng; ZHUGE, Hui; ZHANG, Shan-Shan; ZHANG, Shu-Fang; YANG, Xian-Yu

    2014-01-01

    In this study, to clarify the bioactive polypeptides included in the skins and secretions of Bufo, we screened the Japanese toad (Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction (PCR), and obtained a transcript of 1 075 bp consisting of 1 37 bp 5′ untranslated region (UTR), 515 bp 3′ UTR and a 423 bp open reading frame (ORF) encoding a polypeptide of 140 amino acid residues (GenBank accession number: KF359945). Homolog analysis showed a 70%-96% homology with sterol carrier protein-2 (SCP-2) present in other animals, which is implicated in lipid metabolism of other organisms. The gene SCP-2 of Chinese toad (B. gargarizans) was cloned from a first strand cDNA of Bufo skin (GenBank accession number: KF381341) via PCR, whose encoding polypeptide has only one amino acid difference from that of Japanese toad. Tissue distribution analysis showed that SCP-2 expressed in all organs tested, though in the liver and spleen it manifested lower expression than in other organs. These findings might indicate SCP-2 being one of the active ingredients in toad skin. These findings may in turn have implications for further drug development from traditional Chinese medicine sources. PMID:25297079

  10. Cloning the sterol carrier protein 2 genes of Japanese toad (Bufo japonicus formosus) and Chinese toad (Bufo gargarizans) and its tissue expression analysis.

    PubMed

    Ji, Yu-Cheng; Zhuge, Hui; Zhang, Shan-Shan; Zhang, Shu-Fang; Yang, Xian-Yu

    2014-09-01

    In this study, to clarify the bioactive polypeptides included in the skins and secretions of Bufo, we screened the Japanese toad (Bufo japonicus formosus) skin cDNA liary by colony polymerase chain reaction (PCR), and obtained a transcript of 1 075 bp consisting of 1 37 bp 5' untranslated region (UTR), 515 bp 3' UTR and a 423 bp open reading frame (ORF) encoding a polypeptide of 140 amino acid residues (GenBank accession number: KF359945). Homolog analysis showed a 70%-96% homology with sterol carrier protein-2 (SCP-2) present in other animals, which is implicated in lipid metabolism of other organisms. The gene SCP-2 of Chinese toad (B. gargarizans) was cloned from a first strand cDNA of Bufo skin (GenBank accession number: KF381341) via PCR, whose encoding polypeptide has only one amino acid difference from that of Japanese toad. Tissue distribution analysis showed that SCP-2 expressed in all organs tested, though in the liver and spleen it manifested lower expression than in other organs. These findings might indicate SCP-2 being one of the active ingredients in toad skin. These findings may in turn have implications for further drug development from traditional Chinese medicine sources. PMID:25297079

  11. Synthesis of oxysterols and nitrogenous sterols with antileishmanial and trypanocidal activities.

    PubMed

    Bazin, Marc-Antoine; Loiseau, Philippe M; Bories, Christian; Letourneux, Yves; Rault, Sylvain; El Kihel, Laïla

    2006-10-01

    Two sterol families have been synthesized: the first one is nitrogenous sterols containing amino, N-hydroxyimino or cyano group and the second one is oxysterols such as ketosterol and hydroxysterols. These compounds were then evaluated in vitro against Leishmania donovani promastigotes and Trypanosoma brucei brucei trypomastigotes. The most active compounds against L. donovani promastigotes were 7beta-aminomethylcholesterol and 7alpha,beta-aminocholesterol (IC50 in a range from 1 to 3 microM, pentamidine: 2.8 microM). These compounds were active on intramacrophage amastigotes with IC50 of 1.3 microM. Such an activity justifies further in vivo antileishmanial evaluation. Against T. b. brucei, (24R,S)-24-hydroxy-24-methylcholesterol (MEC, 12.5 microM) was the most active compound from these series. PMID:16949702

  12. A Sterol and Spiroditerpenoids from a Penicillium sp. Isolated from a Deep Sea Sediment Sample

    PubMed Central

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

    2012-01-01

    A new polyoxygenated sterol, sterolic acid (1), three new breviane spiroditerpenoids, breviones I–K (2–4), and the known breviones (5–8), were isolated from the crude extract of a Penicillium sp. obtained from a deep sea sediment sample that was collected at a depth of 5115 m. The structures of 1–4 were elucidated primarily by NMR experiments, and 1 was further confirmed by X-ray crystallography. The absolute configurations of 2 and 3 were deduced by comparison of their CD spectra with those of the model compounds. Compounds 2 and 5 showed significant cytotoxicity against MCF-7 cells, which is comparable to the positive control cisplatin. PMID:22412815

  13. Antioxidative succinobucol-sterol conjugates: Crystal structures and pseudosymmetry in the crystals

    NASA Astrophysics Data System (ADS)

    Ikonen, Satu; Jurček, Ondřej; Wimmer, Zdeněk; Drašar, Pavel; Kolehmainen, Erkki

    2012-03-01

    An extensive study to attach succinobucol to sterols has provided conjugates which comprise two pharmaceutically important compounds into one entity where the components are expected to have a synergistic effect. The motivation to design these novel conjugates was the need to broaden the armamentarium of current agents used in the treatment of atherosclerotic diseases and type 2 diabetes. In desire for detailed information of these compounds in solid state, which also have an influence to their physiological activity, systematic crystallization experiments were performed and as a result, X-ray quality single crystals were obtained from four succinobucol-sterol conjugates. All of these compounds crystallized in space group P1 with two or four molecules in an asymmetric unit and the crystallographically independent molecules were found to be related by pseudosymmetry (i.e. by pseudoinversion in 1-3 and by pseudoinversion plus pseudotranslation in 4).

  14. Impact of Lipid Components and Emulsifiers on Plant Sterols Bioaccessibility from Milk-Based Fruit Beverages.

    PubMed

    Alvarez-Sala, Andrea; Garcia-Llatas, Guadalupe; Cilla, Antonio; Barberá, Reyes; Sánchez-Siles, Luis Manuel; Lagarda, María Jesús

    2016-07-20

    Sterol bioaccessibility (BA) of three plant sterol (PS)-enriched milk-based fruit beverages (MFb) with different fat contents (1.1-2.4%), lipid sources (animal or vegetable), and without or with emulsifiers (whey proteins enriched with milk fat globule membrane (MFGM) or soy lecithin) was evaluated after simulated gastrointestinal digestion. The BA of total PS followed the order 31.4% (MFbM containing milk fat and whey proteins enriched with MFGM) = 28.2% (MFbO containing extra virgin olive oil and soy lecithin) > 8.7% (MFb without fat addition). Total and individual PS content in the bioaccessible fractions followed the order MFbM > MFbO > MFb. Consequently, formulation with MFGM is proposed in beverages of this kind to ensure optimum bioavailability of PS. Our results suggest that the BA of PS is influenced by the type and quantity of fat and the emulsifier type involved. PMID:27329567

  15. Microbial water quality and sedimentary faecal sterols as markers of sewage contamination in Kuwait.

    PubMed

    Lyons, B P; Devlin, M J; Abdul Hamid, S A; Al-Otiabi, A F; Al-Enezi, M; Massoud, M S; Al-Zaidan, A S; Smith, A J; Morris, S; Bersuder, P; Barber, J L; Papachlimitzou, A; Al-Sarawi, H A

    2015-11-30

    Microbial water quality and concentrations of faecal sterols in sediment have been used to assess the degree of sewage contamination in Kuwait's marine environment. A review of microbial (faecal coliform, faecal streptococci and Escherichia coli) water quality data identified temporal and spatial sources of pollution around the coastline. Results indicated that bacterial counts regularly breach regional water quality guidelines. Sediments collected from a total of 29 sites contained detectable levels of coprostanol with values ranging from 29 to 2420 ng g(-1) (dry weight). Hot spots based on faecal sterol sediment contamination were identified in Doha Bay and Sulaibikhat Bay, which are both smaller embayments of Kuwait Bay. The ratio of epicoprostanol/coprostanol indicates that a proportion of the contamination was from raw or partially treated sewage. Sewage pollution in these areas are thought to result from illegal connections and discharges from storm drains, such as that sited at Al-Ghazali. PMID:26228071

  16. Potential of Ophiostoma piceae sterol esterase for biotechnologically relevant hydrolysis reactions

    PubMed Central

    Barba Cedillo, Víctor; Prieto, Alicia; Martínez, María Jesús

    2013-01-01

    The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity toward p-nitrophenol, glycerol, and sterol esters. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris under the AOX1 methanol-inducible promoter (PAOX1) using sorbitol as co-susbtrate, and the hydrolytic activity of the recombinant protein (OPE*) turned out to be improved from a kinetic point of view. In this study, we analyze the effects of sorbitol during the expression of OPE*, at first added as an additional carbon source, and methanol as inducer. The O. piceae enzyme was successfully used for PVAc hydrolysis, suggesting its potential applicability in recycled paper production to decrease stickies problems. PMID:23138020

  17. Sterol fatty acid esters from the mushroom Hericium erinaceum and their PPAR transactivational effects.

    PubMed

    Li, Wei; Zhou, Wei; Song, Seok Bean; Shim, Sang Hee; Kim, Young Ho

    2014-12-26

    Six new (erinarols A-F, 1-6) and five known (7-11) ergostane-type sterol fatty acid esters were isolated from the methanol extract of the dried fruiting bodies of Hericium erinaceum. Their chemical structures were elucidated using chemical and physical methods as well as through comparison of NMR and mass spectral data with those reported previously. This is the first comprehensive investigation on ergostane-type sterol fatty acid esters from H. erinaceum. The isolated compounds were evaluated for their PPAR transactivational effects using a luciferase reporter system. Compounds 1 and 2 significantly activated the transcriptional activity of PPARs in a dose-dependent manner, with EC50 values of 8.2 and 6.4 μM, respectively. Moreover, compounds 1 and 2 also activated PPARα and PPARγ transcriptional activity, with stimulation from 1.3- to 3.9-fold at 20 μM concentrations. PMID:25437304

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

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Involvement of membrane sterols in hypergravity-induced modifications of growth and cell wall metabolism in plant stems

    NASA Astrophysics Data System (ADS)

    Koizumi, T.; Soga, K.; Wakabayashi, K.; Suzuki, M.; Muranaka, T.; Hoson, T.

    Organisms living on land resist the gravitational force by constructing a tough body Plants have developed gravity resistance responses after having first went ashore more than 500 million years ago The mechanisms of gravity resistance responses have been studied under hypergravity conditions which are easily produced on earth by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which is involved in synthesis of terpenoids such as membrane sterols In the present study we examined the role of membrane sterols in gravity resistance in plants by analyzing sterol levels of stem organs grown under hypergravity conditions and by analyzing responses to hypergravity of the organs whose sterol level was modulated Hypergravity inhibited elongation growth but stimulated lateral expansion of Arabidopsis hypocotyls and azuki bean epicotyls Under hypergravity conditions sterol levels were kept high as compared with 1 g controls during incubation Lovastatin an inhibitor HMGR prevented lateral expansion as the gravity resistance response in azuki bean epicotyls Similar results were obtained in analyses with loss of function mutants of HMGR in Arabidopsis It has been shown that sterols play a role in cellulose biosynthesis probably as the primer In wild type Arabidopsis hypocotyls hypergravity increased the cellulose content but it did not influence the content in HMGR mutants These results suggest that hypergravity increases

  1. Impact of sterol tilt on membrane bending rigidity in cholesterol and 7DHC-containing DMPC membranes

    PubMed Central

    Khelashvili, George; Rappolt, Michael; Chiu, See-Wing; Pabst, Georg; Harries, Daniel

    2012-01-01

    Cholesterol is so essential to the proper function of mammalian cell membranes that even strikingly small inborn errors in cholesterol synthesis can be devastating. Here we combine molecular dynamics simulations with small angle x-ray diffraction experiments to compare mixed sterol/DMPC membranes over a wide range of sterol compositions for two types of sterols: cholesterol and its immediate metabolic precursor 7DHC, that differs from cholesterol by one double bond. We find that while most membrane properties are only slightly affected by the replacement of one sterol by the other, the tilt degree of freedom, as gauged by the tilt modulus, is significantly larger for cholesterol than for 7DHC over a large range of concentrations. In silico mutations of one sterol into the other further support these findings. Moreover, bending rigidities calculated from simulations and estimated in experiments show that cholesterol stiffens membranes to a larger extent than 7DHC. We discuss the possible mechanistic link between sterol tilt and the way it impacts the membrane mechanical properties, and comment on how this link may shed light on the way replacement of cholesterol by 7DHC leads to disease. PMID:23173009

  2. Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols.

    PubMed

    Nagao, Toshihiro; Hirota, Yoshinori; Watanabe, Yomi; Kobayashi, Takashi; Kishimoto, Noriaki; Fujita, Tokio; Kitano, Motohiro; Shimada, Yuji

    2004-08-01

    Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1:2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40 degrees C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7-36.5 degrees C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5 degrees C; JAOCS 80, 341-346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%. PMID:15638248

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

    PubMed

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

    2016-05-01

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

  4. An efficient diethyl ether-based soxhlet protocol to quantify faecal sterols from catchment waters.

    PubMed

    Shah, Vikas Kumar G; Dunstan, Hugh; Taylor, Warren

    2006-03-01

    A study was conducted to evaluate the efficiency and reproducibility of a diethyl ether-based soxhlet extraction procedure for faecal sterols occurring from catchment waters. Water samples spiked with a mixture of faecal sterols were filtered and analytes were extracted using the diethyl ether-based soxhlet method and the Bligh and Dyer chloroform extraction process. For diethyl ether-based soxhlet extraction procedure, solvent extracts were saponified with 100 microL of 10% KOH in methanol (100 degrees C/120 min) and then acidified with 60 microL of 6M HCl. Lipid contents were extracted by ethanol (0.5 mL) from the saponification products. The lipid extracts were then reacted with 100 microL of bis(trimethyl)trifluoroacetamide (BSTFA) containing 1% trimethyl chlorosilane (100 degrees C/60 min) to form the trimethylsilyl (TMS) derivatives. The derivatised extracts were then analyzed by gas chromatography-mass spectrometry. For sterol concentrations ranging from 35 to 175 microg mL(-1), the soxhlet-based extraction process yielded the following recovery efficiencies for coprostanol (101%), epicoprostanol (97%), cholesterol (97%), dihydrocholesterol (97%) and 5alpha-cholestane (111%), whereas the Bligh and Dyer process yielded recoveries of 32, 41, 0, 36 and 51%, respectively. The results suggested that the diethyl ether-based soxhlet extraction method was more efficient and reproducible than the Bligh and Dyer chloroform extraction process for the analyses of trace levels of faecal sterols from water samples. Moreover, it was revealed that the diethyl ether-based soxhlet extraction method used less solvent and was logistically easier. PMID:16430911

  5. Isolation and structure elucidation of azoricasterol, a new sterol of the deepwater sponge Macandrewia azorica

    NASA Astrophysics Data System (ADS)

    Gross, Harald; Reitner, Joachim; König, Gabriele M.

    2004-09-01

    Chemical investigation of the deepwater sponge Macandrewia azorica, collected from the flanks of the Gettysburg and Ormonde Sea Mount, North Atlantic, from a depth of 600 m, has led to the isolation of a new sterol with an unusual side chain (1), along with S-methylergothioneine (2). The structures of compounds 1 and 2 were established by employing spectroscopic techniques (NMR, MS, UV, IR and polarimetry). This is the first report of metabolites of a sponge belonging to the genus Macandrewia.

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

    PubMed Central

    Jahnke, L L; Nichols, P D

    1986-01-01

    Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids. PMID:3087955

  7. Effects of chronic ethanol consumption on sterol transfer proteins in mouse brain.

    PubMed

    Myers-Payne, S C; Fontaine, R N; Loeffler, A; Pu, L; Rao, A M; Kier, A B; Wood, W G; Schroeder, F

    1996-01-01

    Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross-reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein-2 (SCP-2) and the liver form of fatty acid-binding protein (L-FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP-2 and L-FABP were used to identify the lipid transfer proteins in the brains of alcohol-treated and control mice. L-FABP was not detectable in brain of either control or chronic ethanol-treated mice. In contrast, SCP-2 not only was present, but its level was significantly (p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP-2 reflected true levels of SCP-2 in brain, the cDNA sequence for brain SCP-2 was isolated from a brain cDNA library. The mouse brain SCP-2 sequence was 99.99% identical to the mouse liver SCP-2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP-2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl-CoA binding protein, a lipid-binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP-2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP-2 may contribute to membrane tolerance to ethanol. PMID:8522969

  8. MECHANISMS UNDERLYING THE MICRON-SCALE SEGREGATION OF STEROLS AND GM1 IN LIVE MAMMALIAN SPERM

    PubMed Central

    Selvaraj, Vimal; Asano, Atsushi; Buttke, Danielle E.; Sengupta, Prabuddha; Weiss, Robert S.; Travis, Alexander J.

    2009-01-01

    We demonstrate for the first time that a stable, micron-scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, GM1, localizes to this same domain in live sperm. Interestingly, the GM1 undergoes an unexplained redistribution upon cell death. We now demonstrate that GM1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing GM1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB-bound endogenous GM1 and exogenous BODIPY-GM1 in live murine sperm demonstrate that the sub-acrosomal ring functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol- and GM1-enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of sub-types of membrane raft microdomains enriched in sterols and/or GM1 that can be separated biochemically. PMID:19012288

  9. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications. PMID:25108239

  10. Short-Term Water Deficit Changes Cuticular Sterol Profile in the Eggplant (Solanum melongena).

    PubMed

    Haliński, Łukasz P; Stepnowski, Piotr

    2016-06-01

    Crop irrigation uses a majority of a total world water supply, at the same time displaying low efficiency. As the expected, future water requirements are higher than the current ones; there is a risk of a growing deficit of water for the agricultural use. Hence, there is an arising need for better understanding the effects of water deprivation on the crop plants. Eggplant (Solanum melongena L.) is a vegetable crop cultivated in arid and semi-arid parts of the world. Because of its high water demands, the eggplant is a convenient model organism for studies concerning the effects of water deficit on the plant growth. The objective of the study was to determine the impact of short-term water deficit on eggplant leaf cuticular waxes and total sterols. Water deprivation did not affect the amount and composition of aliphatic components of cuticular waxes. Significant decrease in the total cuticular sterols and the increase in cuticular cholesterol were observed as an effect of water deficit. In contrast, some of the free internal sterols were more abundant in water-deprived plants. The possible importance of these observations, including increased biosynthesis of defensive compounds and the need to maintain the cell membrane stability, was discussed. PMID:27127890

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

    PubMed

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

    2016-07-01

    Phytosterols are important bioactive compounds which have several health benefits including reduction of serum cholesterol and preventing cardiovascular diseases. The most widely used method in the synthesis of its ester analogous form is the use of catalysts and solvents. These methods have been found to present some safety and health concern. In this paper, an alternative method of synthesizing phytosterol ester from soybean sterol and acetic anhydride was investigated. Process parameters such as mole ratio, temperature and time were optimized. The structure and physicochemical properties of phytosterol acetic ester were analyzed. By the use of gas chromatography, the mole ratio of soybean sterol and acetic anhydride needed for optimum esterification rate of 99.4% was 1:1 at 135 °C for 1.5 h. FTIR spectra confirmed the formation of phytosterol ester with strong absorption peaks at 1732 and 1250 cm(-1) , which corresponds to the stretching vibration of C=O and C-O-C, respectively. These peaks could be attributed to the formation of ester links which resulted from the reaction between the hydroxyl group of soybean sterol and the carbonyl group of acetic anhydride. This paper provides a better alternative to the synthesis of phytosterol ester without catalyst and solvent residues, which may have potential application in the food, health-care food, and pharmaceutical industries. PMID:27240315

  12. Formulation and antifungal performance of natamycin-loaded liposomal suspensions: the benefits of sterol-enrichment.

    PubMed

    Bouaoud, Clotilde; Lebouille, Jérôme G J L; Mendes, Eduardo; De Braal, Henriette E A; Meesters, Gabriel M H

    2016-06-01

    The aim of this study is to develop and evaluate food-grade liposomal delivery systems for the antifungal compound natamycin. Liposomes made of various soybean lecithins are prepared by solvent injection, leading to small unilamellar vesicles (<130 nm) with controlled polydispersity, able to encapsulate natamycin without significant modification of their size characteristics. Presence of charged phospholipids and reduced content of phosphatidylcholine in the lecithin mixture are found to be beneficial for natamycin encapsulation, indicating electrostatic interactions of the preservative with the polar head of the phospholipids. The chemical instability of natamycin upon storage in these formulations is however significant and proves that uncontrolled leakage out of the liposomes occurs. Efficient prevention of natamycin degradation is obtained by incorporation of sterols (cholesterol, ergosterol) in the lipid mixture and is linked to higher entrapment levels and reduced permeability of the phospholipid membrane provided by the ordering effect of sterols. Comparable action of ergosterol is observed at concentrations 2.5-fold lower than cholesterol and attributed to a preferential interaction of natamycin-ergosterol as well as a higher control of membrane permeability. Fine-tuning of sterol concentration allows preparation of liposomal suspensions presenting modulated in vitro release kinetics rates and enhanced antifungal activity against the model yeast Saccharomyces cerevisiae. PMID:26009272

  13. The Text Encoding Initiative: Flexible and Extensible Document Encoding.

    ERIC Educational Resources Information Center

    Barnard, David T.; Ide, Nancy M.

    1997-01-01

    The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

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

    NASA Astrophysics Data System (ADS)

    Lu, Y.; Philp, P. R.

    2014-12-01

    Organic wastes, in particular fecal material, are qualified as one of the major causes of water quality deterioration. Their accumulation in water bodies may increase algal proliferation and eutrophication and the number of pathogenic organisms, which are responsible for many intestinal diseases especially when the water is used for recreational activities and/or as a supply for drinking water. In order to estimate the risk level associated with primary body contact in recreational water bodies, enumeration of some specific micro-organisms, such as Enterococci and Escherichia coli, are commonly used. Sterol distributions can provide some relevant information on the origin of fecal material in water system, since they are ubiquitous organic compounds and their distributions in many warm-blooded animal feces can be used as evidence for their source. In this study, we monitored fecal material contamination in three Oklahoma watersheds based on sterol fingerprints over a one-year period (2012 ~ 2013). The sterols from sediments and water samples (sterols associated to suspended particles as well as free sterols in water) were recovered using sonication and solid phase extraction (SPE), respectively, using different organic solvents. They were then identified and quantified by gas chromatography - mass spectrometry (GC-MS) using an internal standard. The GC-MS was previously calibrated with a sterol mixture injected at different concentrations. Our primary results show that the concentration of total sterols generally increases from the Upper Canadian < Neosho Grand < Cimarron - Upper Arkansas Basins in Oklahoma. The fecal sterols commonly represent a small proportion (<15%) within the total sterols quantified in these three basins. Their distributions show a significant contribution from herbivore feces. By means of this monitoring, we are able to determine the presence of fecal contamination and provide a better understanding on the ability of using sterol

  15. VLSI Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1983-01-01

    Modular Reed-solomon encoder uses identical custom VLSI chips called "symbol slices." By cascading and properly interconnecting group of these chips, encoder is made for any desired error-correcting capability and interleaving level. VLSI encoder requires only one-tenth the number of chips required by conventional Reed-Solomon Circuit implemented with discrete IC's.

  16. Effect of plant sterol-enriched diets on plasma and egg yolk cholesterol concentrations and cholesterol metabolism in laying hens.

    PubMed

    Liu, X; Zhao, H L; Thiessen, S; House, J D; Jones, P J H

    2010-02-01

    Egg exists as a major dietary source of cholesterol in Western diets. In North America, laying hen diets are usually devoid of cholesterol when diets are formulated to exclude animal-based products. Hence, laying hens meet their physiological cholesterol requirement through de novo synthesis. Plant sterols exert a cholesterol-lowering effect in humans by interfering with intestinal sterol absorption. However, it is unknown whether plant sterol supplementation could be effective in reducing intestinal reabsorption of biliary cholesterol in laying hens, thus modulating whole body cholesterol in favor of lower plasma and yolk cholesterol content. The current study was designed to investigate the effect of diets enriched with 0, 0.5, 1, and 2% plant sterols on cholesterol absorption, synthesis, as well as plasma, liver, and egg yolk cholesterol concentrations in laying hens. After 8 wk of plant sterol intervention (first 2 wk were acclimatization), feed intake, BW, egg weight, egg yolk weight, egg production, Haugh units, liver mass, plasma, and hepatic cholesterol concentrations did not differ as a function of plant sterol supplementation. Egg cholesterol concentrations (mg/g) fluctuated during the 6-wk experimental period. At wk 6, a minor reduction in egg yolk cholesterol concentration (mg per g of yolk, P<0.05, vs. control) was observed in hens fed 1 and 2% cholesterol-enriched diets, respectively. However, such result failed to affect total egg cholesterol content. No statistical difference was observed across treatments over 6 wk. Neither cholesterol absorption rates nor synthesis differed as a function of treatment. Results suggested that overall cholesterol content in egg yolk was not affected by feeding hens plant sterol-enriched diets over 6 wk. PMID:20075279

  17. A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

    PubMed Central

    2011-01-01

    The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

  18. A user's guide to the encyclopedia of DNA elements (ENCODE).

    PubMed

    2011-04-01

    The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

  19. Virus-encoded microRNAs

    PubMed Central

    Grundhoff, Adam; Sullivan, Christopher S.

    2011-01-01

    microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

  20. Sterol Regulatory Transcription Factor-1: Key Regulator of Fasting Response in the Adipose Tissue inGPigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic mechanisms controlling appetite and feeding behaviors are not well understood. In this study, transcriptional profiling was used to identify porcine genes and pathways that respond to a fasting treatment or to a missense mutation (D298N) in the melanocortin-4 receptor (MC4R) gene, which...

  1. Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.

    PubMed

    Corey, E J; Matsuda, S P; Bartel, B

    1993-12-15

    Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis. PMID:7505443

  2. Time encoded radiation imaging

    DOEpatents

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  3. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  4. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  5. Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells.

    PubMed

    Zhao, Guoqun; Hu, Tao; Zhao, Lihua

    2014-03-01

    Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified. PMID:24297326

  6. Kinetic studies on recombinant UDP-glucose: sterol 3-O-β-glycosyltransferase from Micromonospora rhodorangea and its bioconversion potential.

    PubMed

    Hoang, Nguyen Huu; Huong, Nguyen Lan; Kim, Byul; Park, Je Won

    2016-12-01

    Kinetics of a recombinant uridine diphosphate-glucose: sterol glycosyltransferase from Micromonospora rhodorangea ATCC 27932 (MrSGT) were studied using a number of sterols (including phytosterols) as glycosyl acceptors. The lowest K m value and the highest catalytical efficiency (k cat/K m) were found when β-sitosterol was the glycosyl acceptor in the enzymatic reaction. In contrast to the enzyme's flexibility toward the glycosyl acceptor substrate, this recombinant enzyme was highly specific to uridine diphosphate (UDP)-glucose as the donor substrate. Besides, the UDP-glucose-dependent MrSGT was able to attach one glucose moiety specifically onto the C-3 hydroxyl group of other phytosterols such as fucosterol and gramisterol, yielding stereo-specific fucosterol-3-O-β-D-glucoside and gramisterol-3-O-β-D-glucoside, respectively. Based on kinetic data obtained from the enzyme's reactions using five different sterol substrates, the significance of the alkene (or ethylidene) side chains on the C-24 position in the sterol scaffolds was described and the possible relationship between the substrate structure and enzyme activity was discussed. This is the first report on the enzymatic bioconversion of the above two phytosteryl 3-O-β-glucosides, as well as on the discovery of a stereospecific bacterial SGT which can attach a glucose moiety in β-conformation at the C-3 hydroxyl group of diverse sterols, thus highlighting the catalytic potential of this promiscuous glycosyltransferase to expand the structural diversity of steryl glucosides. PMID:27485517

  7. A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids.

    PubMed

    John, Clara; Werner, Philipp; Worthmann, Anna; Wegner, Katrin; Tödter, Klaus; Scheja, Ludger; Rohn, Sascha; Heeren, Joerg; Fischer, Markus

    2014-12-01

    Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79-92% in liver, 76-98% in adipose tissue, 93-104% in feces and 62-79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation. PMID:25456597

  8. Regulatory network rewiring for secondary metabolism in Arabidopsis thaliana under various conditions

    PubMed Central

    2014-01-01

    Background Plant secondary metabolites are critical to various biological processes. However, the regulations of these metabolites are complex because of regulatory rewiring or crosstalk. To unveil how regulatory behaviors on secondary metabolism reshape biological processes, we constructed and analyzed a dynamic regulatory network of secondary metabolic pathways in Arabidopsis. Results The dynamic regulatory network was constructed through integrating co-expressed gene pairs and regulatory interactions. Regulatory interactions were either predicted by conserved transcription factor binding sites (TFBSs) or proved by experiments. We found that integrating two data (co-expression and predicted regulatory interactions) enhanced the number of highly confident regulatory interactions by over 10% compared with using single data. The dynamic changes of regulatory network systematically manifested regulatory rewiring to explain the mechanism of regulation, such as in terpenoids metabolism, the regulatory crosstalk of RAV1 (AT1G13260) and ATHB1 (AT3G01470) on HMG1 (hydroxymethylglutaryl-CoA reductase, AT1G76490); and regulation of RAV1 on epoxysqualene biosynthesis and sterol biosynthesis. Besides, we investigated regulatory rewiring with expression, network topology and upstream signaling pathways. Regulatory rewiring was revealed by the variability of genes’ expression: pathway genes and transcription factors (TFs) were significantly differentially expressed under different conditions (such as terpenoids biosynthetic genes in tissue experiments and E2F/DP family members in genotype experiments). Both network topology and signaling pathways supported regulatory rewiring. For example, we discovered correlation among the numbers of pathway genes, TFs and network topology: one-gene pathways (such as δ-carotene biosynthesis) were regulated by a fewer TFs, and were not critical to metabolic network because of their low degrees in topology. Upstream signaling pathways of 50

  9. Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii

    PubMed Central

    Pan, Li; Lezama-Davila, Claudio M.; Isaac-Marquez, Angelica P.; Calomeni, Edward P.; Fuchs, James R.; Satoskar, Abhay R.; Kinghorn, A. Douglas

    2012-01-01

    A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (3–16), three coumarins (17–19), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3β-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC50 value of 0.03 μM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds. PMID:22840389

  10. Role of a Disordered Steroid Metabolome in the Elucidation of Sterol and Steroid Biosynthesis

    PubMed Central

    2013-01-01

    In 1937 Butler and Marrian found large amounts of the steroid pregnanetriol in urine from a patient with the adrenogenital syndrome, a virilizing condition known to be caused by compromised adrenal secretion even in this pre-cortisol era. This introduced the concept of the study of altered excretion of metabolites as an in vivo tool for understanding sterol and steroid biosynthesis. This approach is still viable and has experienced renewed significance as the field of metabolomics. From the first cyclized sterol lanosterol to the most downstream product estradiol, there are probably greater than 30 steps. Based on a distinctive metabolome clinical disorders have now been attributed to about seven post-squalene cholesterol (C) biosynthetic steps and around 15 en-route to steroid hormones or needed for further metabolism of such hormones. Forty years ago it was widely perceived that the principal steroid biosynthetic defects were known but interest rekindled as novel metabolomes were documented. In his career this investigator has been involved in the study of many steroid disorders, the two most recent being P450 oxidoreductase deficiency and apparent cortisone reductase deficiency. These are of interest as they are due not to mutations in the primary catalytic enzymes of steroidogenesis but in ancillary enzymes needed for co-factor oxido-reduction A third focus of this researcher is Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder caused by 7-dehydrocholesterol reductase mutations. The late George Schroepfer, in whose honor this article has been written, contributed greatly to defining the sterol metabolome of this condition. Defining the cause of clinically severe disorders can lead to improved treatment options. We are now involved in murine gene therapy studies for SLOS which, if successful could in the future offer an alternative therapy for this severe condition. PMID:21874273

  11. Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures.

    PubMed

    Burlini, Nedda; Iriti, Marcello; Daghetti, Anna; Faoro, Franco; Ruggiero, Antonietta; Bernasconi, Silvana

    2011-11-01

    Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D(3) metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3'-diaminobenzidine (DAB) for hydrogen peroxide (H(2)O(2)) detection and phloroglucinol for lignin staining showed that BTH causes H(2)O(2) accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D(3), and the hydroxylated metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D(3), though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D(3) and 1α,25(OH)(2)D(3) metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol. PMID:21779826

  12. Distinct biochemical activities and heat shock responses of two UDP-glucose sterol glucosyltransferases in cotton.

    PubMed

    Li, Xianliang; Xia, Tao; Huang, Jiangfeng; Guo, Kai; Liu, Xu; Chen, Tingting; Xu, Wen; Wang, Xuezhe; Feng, Shengqiu; Peng, Liangcai

    2014-04-01

    UDP-glucose sterol glucosyltransferase (SGT) are enzymes typically involved in the production of sterol glycosides (SG) in various organisms. However, the biological functions of SGTs in plants remain largely unknown. In the present study, we identified two full-length GhSGT genes in cotton and examined their distinct biochemical properties. Using UDP-[U-(14)C]-glucose and β-sitosterol or total crude membrane sterols as substrates, GhSGT1 and GhSGT2 recombinant proteins were detected with different enzymatic activities for SG production. The addition of Triton (X-100) strongly inhibited the activity of GhSGT1 but caused an eightfold increase in the activity of GhSGT2. The two GhSGTs showed distinct enzyme activities after the addition of NaCl, MgCl2, and ZnCl2, indicating that the two GhSGTs exhibited distinct biochemical properties under various conditions. Furthermore, after heat shock treatment, GhSGT1 showed rapidly enhanced gene expression in vivo and low enzyme activity in vitro, whereas GhSGT2 maintained extremely low gene expression levels and relatively high enzyme activity. Notably, the GhSGT2 gene was highly expressed in cotton fibers, and the biochemical properties of GhSGT2 were similar to those of GhCESA in favor for MgCl2 and non-reduction reaction condition. It suggested that GhSGT2 may have important functions in cellulose biosynthesis in cotton fibers, which must be tested in the transgenic plants in the future. Hence, the obtained data provided insights into the biological functions of two different GhSGTs in cotton and in other plants. PMID:24576758

  13. Structural and Functional Analyses of a Sterol Carrier Protein in Spodoptera litura

    PubMed Central

    Xu, Rui; Zheng, Sichun; He, Hongwu; Wan, Jian; Feng, Qili

    2014-01-01

    Backgrounds In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport. Results In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae. Conclusions Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae. PMID:24454688

  14. THAP and ATF-2 Regulated Sterol Carrier Protein-2 Promoter Activities in the Larval Midgut of the Yellow Fever Mosquito, Aedes aegypti

    PubMed Central

    Peng, Rong; Fu, Qiang; Hong, Huazhu; Schwaegler, Tyler; Lan, Que

    2012-01-01

    Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream −1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the −1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the −1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled −1.6/−1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between −1.6 to −1.3 kb 5′ upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression. PMID:23056538

  15. THAP and ATF-2 regulated sterol carrier protein-2 promoter activities in the larval midgut of the yellow fever mosquito, Aedes aegypti.

    PubMed

    Peng, Rong; Fu, Qiang; Hong, Huazhu; Schwaegler, Tyler; Lan, Que

    2012-01-01

    Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream -1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the -1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the -1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled -1.6/-1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between -1.6 to -1.3 kb 5' upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression. PMID:23056538

  16. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR

    PubMed Central

    2013-01-01

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism. PMID:22414897

  17. Identification of hopanoid, sterol, and tetrahymanol production in the aerobic methanotroph Methylomicrobium alcaliphilum 20Z

    NASA Astrophysics Data System (ADS)

    Welander, P. V.; Summons, R. E.

    2013-12-01

    Correlating the occurrence of molecular biosignatures preserved in the rock record with specific microbial taxa is a compelling strategy for studying microbial life in the context of the Earth's distant past. Polycyclic triterpenoids, including the hopanes and steranes, comprise classes of biomarkers that are readily detected in a variety of ancient sediments and are clearly recognized as the diagenetic products of modern day bacterial hopanoids and eukaryotic sterols. Thus, based on the distribution of these lipids in extant microbes, the occurrence of their diagenetic products in the rock record is often utilized as evidence for the existence of specific bacterial and eukaryotic taxa in ancient ecosystems. However, questions have arisen about our understanding of the taxonomic distribution of many of these molecular biomarkers in extant microbes. This is prompting reassessments of the use of polycyclic triterpenoids as geological proxies for microbial taxa, especially in the light of the poorly defined issue of microbial diversity. Recently, significant effort has been put forth to better understand the biosynthesis, function, and regulation of these lipid molecules in a variety of modern organisms so that a more informed interpretation of their occurrence in the rock record can be reached. Here we report the unprecedented production of three different classes of polycyclic triterpenoid biomarker lipids in one bacterium. Methylomicrobium alcaliphilum 20Z, a member of the Gammaproteobacteria, is a halotolerant alkaliphilic aerobic methanotroph previously isolated from a moderately saline soda lake in Tuva (Central Asia). In this study, M. alcaliphilum is shown to produce C-3 methylated and unmethylated aminohopanoids commonly associated with other mesophilic aerobic methanotrophs. In addition, this organism is also able to produce 4,4-dimethyl sterols and surprisingly, the gammacerane triterpenoid tetrahymanol. Previously, tetrahymanol production has only been

  18. Regulatory gene networks and the properties of the developmental process

    NASA Technical Reports Server (NTRS)

    Davidson, Eric H.; McClay, David R.; Hood, Leroy

    2003-01-01

    Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.

  19. Final report of the amended safety assessment of PEG-5, -10, -16, -25, -30, and -40 soy sterol.

    PubMed

    2004-01-01

    PEGs Soy Sterol are polyethylene glycol (PEG) derivatives of soybean oil sterols used in a variety of cosmetic formulations as surfactants and emulsifying agents, skin-conditioning agents, and cleansing and solubilizing agents. When the safety of these ingredients were first reviewed, the available data were insufficient to support safety. New data have since been received and the safety of these ingredients in cosmetics has been substantiated. Current concentration of use ranges from a low of 0.05% in makeup preparations to 2% in moisturizers and several other products. PEGs Soy Sterol are produced by the reaction of the soy sterol hydroxyl with ethylene oxide. In general, ethoxylated fatty acids can contain 1,4-dioxane as a byproduct of ethoxylation. The soy sterols include campesterol, stigmasterol, and beta-sitosterol. The distribution of sterols found in oils derived from common plants is similar, with beta-sitosterol comprising a major component. Impurities include sterol hydrocarbons and cholesterol (4% to 6%) and triterpine alcohols, keto-steroids, and other steroid-like substances (4% to 6%). No pesticide residues were detected. PEGS: Because PEGs are an underlying structure in PEGs Soy Sterols, the previous assessment of PEGs was considered. It is generally recognized that the PEG monomer, ethylene glycol, and certain of its monoalkyl ethers are reproductive and developmental toxins. Given the methods of manufacture of PEGs Soy Sterol, there is no likelihood of ethylene glycol or its alkyl ethers being present. Also, the soybean oil sterol ethers in this ingredient are chemically different from the ethylene glycol alkyl ethers of concern. PEGs are not carcinogenic, although sensitization and nephrotoxicity were observed in burn patients treated with a PEG-based cream. No evidence of systemic toxicity or sensitization was found in studies with intact skin. Plant Phytosterols: Intestinal absorption of ingested plant phytosterols is on the order of 5%, with

  20. Regulatory link between steryl ester formation and hydrolysis in the yeast Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Korber, Martina; Schmidt, Claudia; Koch, Barbara; Leitner, Erich; Daum, Günther

    2015-07-01

    Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells. PMID:25720564

  1. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  2. Cytoplasmic localization of sterol transcription factors Upc2p and Ecm22p in S.cerevisiae

    PubMed Central

    Marie, Chelsea; Leyde, Sarah; White, Theodore C

    2008-01-01

    Ergosterol homeostasis is a critical process for fungal cells. Paralogous zinc cluster transcription factors Upc2p and Ecm22p are major regulators of ergosterol biosynthesis in Saccharomyces cerevisiae. Upc2p and Ecm22p sense and respond to sterol depletion but their mechanism of activation has not been defined. Subcellular localization and functional expression of Upc2p–GFP and Ecm22p-GFP was monitored by fluorescence microscopy and flow cytometry in live yeast cells. Both fusion proteins localized to intracellular membranes and to perinuclear foci. Perinuclear localization of Upc2p-GFP and Ecm22p-GFP was increased when ergosterol biosynthesis was blocked by azole drug treatment. Nuclear localization in response to sterol depletion is consistent with the hypothesis that Upc2p and Ecm22p are trafficked from a membrane to the nucleus as a post-translational mechanism of sterol sensing. PMID:18675371

  3. Sterols and triterpenoids as potential anti-inflammatories: Molecular docking studies for binding to some enzymes involved in inflammatory pathways.

    PubMed

    Loza-Mejía, Marco A; Salazar, Juan Rodrigo

    2015-11-01

    Triterpenes and sterols are good candidates for the development of anti-inflammatory drugs and use in chemoprevention or chemotherapy of cancer via the interaction with therapeutic targets related to inflammation, such as COX-1 and -2; LOX-5; MPO, PLA2 and i-NOS. In this study, we use molecular docking to evaluate the potential binding of a database of selected sterol and triterpenoid compounds with several skeletons against enzymes related to inflammation to propose structural requirements beneficial for anti-inflammatory activity that can be used for the design of more potent and selective anti-inflammatory and antitumor drugs. Our results suggest that the substitution pattern is important and that there is an important relationship between the class of sterol or triterpenoid skeleton and enzyme binding. PMID:26342572

  4. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  5. Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

    PubMed Central

    Mishra, Manoj K; Singh, Gaurav; Tiwari, Shalini; Singh, Ruchi; Kumari, Nishi; Misra, Pratibha

    2015-01-01

    Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. PMID:26382564

  6. Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress.

    PubMed

    Mishra, Manoj K; Singh, Gaurav; Tiwari, Shalini; Singh, Ruchi; Kumari, Nishi; Misra, Pratibha

    2015-01-01

    Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. PMID:26382564

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

    SciTech Connect

    Hargrove, Tatiana Y.; Wawrzak, Zdzislaw; Liu, Jialin; Nes, W. David; Waterman, Michael R.; Lepesheva, Galina I.

    2012-05-14

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

  8. Potential of the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) as an Unconventional Source of Dietary and Therapeutic Sterols

    PubMed Central

    Cheseto, Xavier; Kuate, Serge Philibert; Tchouassi, David P.; Ndung’u, Mary; Teal, Peter E. A.; Torto, Baldwyn

    2015-01-01

    Insects are increasingly being recognized not only as a source of food to feed the ever growing world population but also as potential sources of new products and therapeutic agents, among which are sterols. In this study, we sought to profile sterols and their derivatives present in the desert locust, Schistocerca gregaria, focusing on those with potential importance as dietary and therapeutic components for humans. Using coupled gas chromatography-mass spectrometry (GC-MS), we analyzed and compared the quantities of sterols in the different sections of the gut and tissues of the locust. In the gut, we identified 34 sterols which showed a patchy distribution, but with the highest composition in the foregut (55%) followed by midgut (31%) and hindgut (14%). Fed ad libitum on wheat seedlings, five sterols unique to the insect were detected. These sterols were identified as 7-dehydrocholesterol, desmosterol, fucosterol, (3β, 5α) cholesta-8, 14, 24-trien-3-ol, 4, 4-dimethyl, and (3β, 20R) cholesta-5, 24-dien-3, 20-diol with the first three having known health benefits in humans. Incubation of the fore-, mid- and hindgut with cholesterol-[4-13C] yielded eight derivatives, three of these were detected in the gut of the desert locust after it had consumed the vegetative diet but were not detected in the diet. Our study shows that the desert locust ingests phytosterols from a vegetative diet and, amplifies and metabolizes them into derivatives with potential salutary benefits and we discuss our findings in this context. PMID:25970517

  9. GC-MS method for determining faecal sterols as biomarkers of human and pastoral animal presence in freshwater sediments.

    PubMed

    Battistel, Dario; Piazza, Rossano; Argiriadis, Elena; Marchiori, Enrico; Radaelli, Marta; Barbante, Carlo

    2015-11-01

    In order to determine sterols and stanols in freshwater sediments to reconstruct the past presence of humans and pastoral animals, we developed an analytical method based on pressurised liquid extraction (PLE), clean-up performed using solid phase extraction (SPE) and sterol determination using gas chromatography-mass spectrometry (GC-MS) analysis. PLE extraction conditions were optimised using dichloromethane (DCM) and DCM/methanol mixtures. Clean-up was performed with 2 g silica SPE cartridges, and the concentrated extracts were eluted with 70 mL DCM. Extraction yield was evaluated using an in-house reference material spiked with (13)C-labelled cholesterol and aged for 10 days. In comparison with pre-extraction, where the sediment is extracted and then spiked with a known analyte concentration, this approach preserves the original composition of the sediment. DCM and DCM/methanol mixtures resulted in high extraction yields ranging from 86 to 92 % with good reproducibility (relative standard deviation (RSD) 5-8 %). PLE extraction yields obtained with DCM as the extracting solvent were about 1.5 times higher than extractions using an ultrasonic bath. The solvent extraction mixture and matrix composition strongly affected the solvent extraction composition where higher overall recoveries (70-80 %) for each compound were obtained with DCM. The extraction mixture and matrix composition also affected the analyte concentrations, resulting in a method precision ranging from 1 to 18 %. Diatomaceous earth spiked with 10 to 100 ng of sterols, and environmental samples fortified with suitable amounts of sterols provided apparent recovery values ranging from 90 to 110 %. We applied the method to environmental samples both close to and upstream from sewage discharge zones, resulting in substantially higher faecal sterol (FeSt) concentrations near the sewage. In addition, we also applied the method to a 37-cm freshwater sediment core in order to evaluate its applicability for

  10. Intake of a Single Morning Dose of Standard and Novel Plant Sterol Preparations for 4 Weeks Does Not Dramatically Affect Plasma Lipid Concentrations in Humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recommendations for decreasing the risk of developing cardiovascular disease include increasing the intake of plant sterols and fish oil. The cholesterol-lowering action of plant sterols, when provided in a fish-oil fatty acids vehicle, remains to be investigated in humans. A randomized, crossover-f...

  11. Fully automated determination of the sterol composition and total content in edible oils and fats by online liquid chromatography-gas chromatography-flame ionization detection.

    PubMed

    Nestola, Marco; Schmidt, Torsten C

    2016-09-01

    Sterol analysis of edible oils and fats is important in authenticity control. The gas chromatographic determination of the sterol distribution and total content is described by ISO norm 12228. Extraction, purification, and detection of the sterols are time-consuming and error-prone. Collaborative trials prove this regularly. Purification by thin-layer chromatography (TLC) and robust GC determination of all mentioned sterols is not straightforward. Therefore, a fully automated LC-GC-FID method was developed to facilitate the determination of sterols. The only manual step left was to weigh the sample into an autosampler vial. Saponification and extraction were performed by an autosampler while purification, separation, and detection were accomplished by online coupled normal-phase LC-GC-FID. Interlacing of sample preparation and analysis allowed an average sample throughput of one sample per hour. The obtained quantitative results were fully comparable with the ISO method with one apparent exception. In the case of sunflower oils, an additional unknown sterol was detected generally missed by ISO 12228. The reason was found in the omission of sterol silylation before subjection to GC-FID. The derivatization reaction changed the retention time and hid this compound behind a major sterol. The compound could be identified as 14-methyl fecosterol. Its structure was elucidated by GC-MS and ensured by HPLC and GC retention times. Finally, validation of the designed method confirmed its suitability for routine environments. PMID:27522150

  12. Effect of Transition Metal Ions on the B Ring Oxidation of Sterols and their Kinetics in Oil-in-Water Emulsions

    PubMed Central

    Lu, Baiyi; Hu, Yinzhou; Huang, Weisu; Wang, Mengmeng; Jiang, Yuan; Lou, Tiantian

    2016-01-01

    This study investigated the effect of metal ions on the oxidation of sterols and their kinetics in oil-in-water emulsions. Sterol substrates were added with different metal ions (Cu2+, Fe2+, Mn2+, Zn2+, Na+, and Mg2+) of five concentrations and investigated after 2 h of heating at 90 °C. The substrates added with Fe2+ and Cu2+ were heated continuously to evaluate the kinetics of four sterols and their corresponding sterol oxidation products (SOPs). Sterol oxidation increased as the metal ion concentration increased and the heating time was prolonged. The capability of the metal ions oxidizing sterols ranked as followed: Fe2+ > Cu2+ > Mn2+ > Zn2+ > Mg2+ ≈ Na+. 7-Ketosterol, 7β/7α-Hydroxysterol, 5β,6β/5α,6α-Epoxysterol, and Triols were the main oxides on the B ring, whereas 6β-Hydroxysterol was not or only slightly influenced. The acceleration of sterol degradation induced by Fe2+ and Cu2+, as well as the formation of oxidation products, followed first-order formation/elimination kinetics. The acceleration effect may be partly ascribed to the increase in elimination rate constant and formation rate constant. Transition metal ions can significantly induce sterol oxidation, which reduces food nutritional quality and triggers the formation of undesirable compounds, such as SOPs. PMID:27328709

  13. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  16. Complete genome sequence of 'Mycobacterium neoaurum' NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols.

    PubMed

    Rodríguez-García, Antonio; Fernández-Alegre, Estela; Morales, Alejandro; Sola-Landa, Alberto; Lorraine, Jess; Macdonald, Sandy; Dovbnya, Dmitry; Smith, Margaret C M; Donova, Marina; Barreiro, Carlos

    2016-04-20

    Microbial bioconversion of sterols into high value steroid precursors, such as 4-androstene-3,17-dione (AD), is an industrial challenge. Genes and enzymes involved in sterol degradation have been proposed, although the complete pathway is not yet known. The genome sequencing of the AD producer strain 'Mycobacterium neoaurum' NRRL B-3805 (formerly Mycobacterium sp. NRRL B-3805) will serve to elucidate the critical steps for industrial processes and will provide the basis for further genetic engineering. The genome comprises a circular chromosome (5 421 338bp), is devoid of plasmids and contains 4844 protein-coding genes. PMID:26988397

  17. Recoverable Pd/C catalyst mediated dehydrogenation of sterols and an improved synthesis of 1α-hydroxydehydroepiandrosterone.

    PubMed

    Yin, Yi-Zhen; Liu, Chao; Tang, Long-Qian; Liu, Zhao-Peng

    2012-11-01

    A novel recyclable Pd/C catalyst mediated dehydrogenation of sterols is developed. The conversion of sterols to 1,4,6-trien-3-ones is best achieved with Pd/C as a catalyst (10%) in the presence of six equivalents of allyl diethyl phosphate (ADP) and excess amount of sodium carbonate in DMF under vigorous reflux conditions. This transformation gives 17,17-ethylenedioxyandrost-1,4,6-trien-3-one in better yield than that of DDQ oxidation and thus provides an improved synthesis of 1α-hydroxydehydroepiandrosterone from DHEA. PMID:23000152

  18. Regulatory RNAs

    PubMed Central

    Vazquez-Anderson, Jorge; Contreras, Lydia M

    2013-01-01

    RNAs have many important functional properties, including that they are independently controllable and highly tunable. As a result of these advantageous properties, their use in a myriad of sophisticated devices has been widely explored. Yet, the exploitation of RNAs for synthetic applications is highly dependent on the ability to characterize the many new molecules that continue to be discovered by large-scale sequencing and high-throughput screening techniques. In this review, we present an exhaustive survey of the most recent synthetic bacterial riboswitches and small RNAs while emphasizing their virtues in gene expression management. We also explore the use of these RNA components as building blocks in the RNA synthetic biology toolbox and discuss examples of synthetic RNA components used to rewire bacterial regulatory circuitry. We anticipate that this field will expand its catalog of smart devices by mimicking and manipulating natural RNA mechanisms and functions. PMID:24356572

  19. Regulatory Physiology

    NASA Technical Reports Server (NTRS)

    Lane, Helen W.; Whitson, Peggy A.; Putcha, Lakshmi; Baker, Ellen; Smith, Scott M.; Stewart, Karen; Gretebeck, Randall; Nimmagudda, R. R.; Schoeller, Dale A.; Davis-Street, Janis

    1999-01-01

    As noted elsewhere in this report, a central goal of the Extended Duration Orbiter Medical Project (EDOMP) was to ensure that cardiovascular and muscle function were adequate to perform an emergency egress after 16 days of spaceflight. The goals of the Regulatory Physiology component of the EDOMP were to identify and subsequently ameliorate those biochemical and nutritional factors that deplete physiological reserves or increase risk for disease, and to facilitate the development of effective muscle, exercise, and cardiovascular countermeasures. The component investigations designed to meet these goals focused on biochemical and physiological aspects of nutrition and metabolism, the risk of renal (kidney) stone formation, gastrointestinal function, and sleep in space. Investigations involved both ground-based protocols to validate proposed methods and flight studies to test those methods. Two hardware tests were also completed.

  20. Regulatory Anatomy

    PubMed Central

    2015-01-01

    This article proposes the term “safety logics” to understand attempts within the European Union (EU) to harmonize member state legislation to ensure a safe and stable supply of human biological material for transplants and transfusions. With safety logics, I refer to assemblages of discourses, legal documents, technological devices, organizational structures, and work practices aimed at minimizing risk. I use this term to reorient the analytical attention with respect to safety regulation. Instead of evaluating whether safety is achieved, the point is to explore the types of “safety” produced through these logics as well as to consider the sometimes unintended consequences of such safety work. In fact, the EU rules have been giving rise to complaints from practitioners finding the directives problematic and inadequate. In this article, I explore the problems practitioners face and why they arise. In short, I expose the regulatory anatomy of the policy landscape. PMID:26139952

  1. Determination of triacyl glycerol and sterol components of fat to authenticate ghee based sweets.

    PubMed

    Kala, A L Amrutha; Sabeena, K; Havanur, Priya Pramod

    2016-04-01

    Method comparison of triacyl glycerol (TAG) and sterol components of fats of ghee based sweets was carried out on dairy ghee, laboratory prepared control sample and market samples. The fat was extracted from control and market samples. Determination of TAG and sterol composition of the fats was carried out using low resolution Gas Chromatography. The quantification of cholesterol and β-sitosterol and TAG classes of dairy ghee, control and market samples fat was also determined using single short column. Adulteration at 5 % level in milk fats showed varied TAG compositions of C50, C52 and C54 as compared to control and pure ghee sample. The cholesterol content of ghee and control sample was 2.30 ± 0.8, 2.00 ± 0.24 g/kg respectively and β-sitosterol content of control was 0.20 ± 0.11 g/kg. The adulterated samples showed varied cholesterol and β-sitosterol contents as compared to control sample fat. PMID:27413245

  2. Sterols and Stanols Preserved in Pond Sediments Track Seabird Biovectors in a High Arctic Environment.

    PubMed

    Cheng, Wenhan; Sun, Liguang; Kimpe, Linda E; Mallory, Mark L; Smol, John P; Gallant, Lauren R; Li, Jinping; Blais, Jules M

    2016-09-01

    Seabirds are major vertebrates in the coastal ecosystems of the Canadian High Arctic, where they transport substantial amounts of marine-derived nutrients and pollutants from oceans to land by depositing guano and stomach oils to their nesting area, which often includes nearby freshwater ponds. Here we present novel indicators for evaluating the impact of seabirds on freshwater ecosystems. The ratio of cholesterol/(cholesterol + sitosterol) in pond sediments showed significant enrichment near a nesting colony of northern fulmars (Fulmarus glacialis) and was significantly correlated with ornithogenic enrichment of sediment as determined by sedimentary δ(15)N. The sterol ratio was also correlated with several bioaccumulative persistent organic pollutants (POPs), suggesting its usefulness in tracking biovector enrichment of contaminants. Human-derived epicoprostanol was also analyzed in the sediments, and its relationship with an abandoned, prehistoric camp was recorded, suggesting its potential as a tracer of prehistoric human activities in the Arctic. Sterols and stanols preserved in sediments appear to be useful geochemical tools that will inform our understanding of migratory species and the presence of prehistoric human populations in the Arctic, and possibly other animal populations. PMID:27409713

  3. Sterols and triterpene diols in olive oil as indicators of variety and degree of ripening.

    PubMed

    Lukić, Marina; Lukić, Igor; Krapac, Marin; Sladonja, Barbara; Piližota, Vlasta

    2013-01-01

    Sterols and triterpene diols in olive oil as indicators of variety and degree of ripening derived from three olive varieties and produced at three different harvesting periods were studied. In order to test the stability of the proposed indicators, oils obtained were stored for 12 months at three different temperatures. Thirty-six samples in total were subjected to GC analysis and results were processed by multivariate chemometric methods (MANOVA, PCA, and SLDA). Campesterol, β-sitosterol, Δ(7)-campesterol/Δ(5,24)-stigmastadienol, clerosterol, uvaol, and campestanol/Δ(7)-avenasterol were established as the indicators of variety of fresh oils, while when stored oils were included in the model, the final three compounds were substituted by 24-methylene-cholesterol/stigmasterol. The most important variables for differentiating fresh oils according to degree of ripening were Δ(7)-campesterol/β-sitosterol, uvaol/stigmasterol, clerosterol/Δ(5)-avenasterol and sitostanol/uvaol, while stored oils were differentiated by campestanol/stigmasterol, erythrodiol, stigmasterol/Δ(7)-campesterol, Δ(5)-avenasterol, 24-methylene-cholesterol/β-sitosterol and 24-methylene-cholesterol. Results demonstrated that sterols and triterpene diols can be used as indicators of variety and degree of ripening among virgin olive oils. PMID:23017420

  4. Neutron diffraction studies of the interaction between amphotericin B and lipid-sterol model membranes

    NASA Astrophysics Data System (ADS)

    Foglia, Fabrizia; Lawrence, M. Jayne; Demeė, Bruno; Fragneto, Giovanna; Barlow, David

    2012-10-01

    Over the last 50 years or so, amphotericin has been widely employed in treating life-threatening systemic fungal infections. Its usefulness in the clinic, however, has always been circumscribed by its dose-limiting side-effects, and it is also now compromised by an increasing incidence of pathogen resistance. Combating these problems through development of new anti-fungal agents requires detailed knowledge of the drug's molecular mechanism, but unfortunately this is far from clear. Neutron diffraction studies of the drug's incorporation within lipid-sterol membranes have here been performed to shed light on this problem. The drug is shown to disturb the structures of both fungal and mammalian membranes, and co-localises with the membrane sterols in a manner consistent with trans-membrane pore formation. The differences seen in the membrane lipid ordering and in the distributions of the drug-ergosterol and drug-cholesterol complexes within the membranes are consistent with the drug's selectivity for fungal vs. human cells.

  5. Sewage influence in a macrotidal estuary: Fatty acid and sterol distributions

    NASA Astrophysics Data System (ADS)

    Quemeneur, Michelle; Marty, Yanic

    1992-04-01

    Estuarine surface sediment and suspended matter from the Morlaix River estuary were analysed for fatty acids and sterols by HPLC and GC. This estuary represents a typical example of a coastal river estuary subjected to strong tides and receiving domestic wastes in its upper reaches. Wastewater fatty acid and sterol distribution patterns have been used to estimate the anthropogenic matter influx and its behaviour as an estuarine organic matter component. The 5 β-stanols, specific to fecal material and relatively persistent in the environment, provide a spatial view of sewage dispersion in estuarine waters and sediments and are used to calculate the relative importance of anthropogenic inputs in the degradable organic matter. Their distribution at high and low water indicates that anthropogenic particles are distributed throughout the estuary and may reach the coastal areas. However, owing to the dilution and the sedimentation processes, the anthropogenic matter contribution to the total organic matter is low in the outer estuary. By contrast, sediments from the upper estuary are strongly influenced by fresh anthropogenic inputs which may be detected by fatty acid fingerprint. The 18:1( n- 7)/18:1( n- 9) ratio which indicates the ability of the sediment to degrade the anthropogenic fresh material demonstrates a perturbation all along the narrow upper estuary.

  6. Quorum-Sensing Mechanisms Mediated by Farnesol in Ophiostoma piceae: Effect on Secretion of Sterol Esterase

    PubMed Central

    de Salas, Felipe

    2015-01-01

    Ophiostoma piceae CECT 20416 is a dimorphic wood-staining fungus able to produce an extracellular sterol-esterase/lipase (OPE) that is of great biotechnological interest. In this work, we have studied the morphological change of this fungus from yeast to hyphae, which is associated with the cell density-related mechanism known as quorum sensing (QS), and how this affects the secretion of OPE. The data presented here confirm that the molecule E,E-farnesol accumulates as the cell number is growing within the population. The exogenous addition of this molecule or spent medium to the cultures increased the extracellular activity of OPE 2.5 times. This fact was related not to an increase in microbial biomass or in the expression of the gene coding for OPE but to a marked morphological transition in the cultures. Moreover, the morphological transition also occurred when a high cell density was inoculated into the medium. The results suggest that E,E-farnesol regulates through QS mechanisms the morphological transition in the dimorphic fungus O. piceae and that it is associated with a higher extracellular esterase activity. Furthermore, identification and transcriptional analysis of genes tup1 and cyr1, which are involved in the response, was carried out. Here we report enhanced production of a sterol-esterase/lipase of biotechnological interest by means of QS mechanisms. These results may be useful in increasing the production of secreted enzymes of other dimorphic fungi of biotechnological interest. PMID:25888179

  7. Acute Sterol O-Acyltransferase 2 (SOAT2) Knockdown Rapidly Mobilizes Hepatic Cholesterol for Fecal Excretion

    PubMed Central

    Marshall, Stephanie M.; Gromovsky, Anthony D.; Kelley, Kathryn L.; Davis, Matthew A.; Wilson, Martha D.; Lee, Richard G.; Crooke, Rosanne M.; Graham, Mark J.; Rudel, Lawrence L.

    2014-01-01

    The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion. PMID:24901470

  8. Alpha-amylase inhibitory activity and sterol composition of the marine algae, Sargassum glaucescens

    PubMed Central

    Payghami, Nasrin; Jamili, Shahla; Rustaiyan, Abdolhossein; Saeidnia, Soodabeh; Nikan, Marjan; Gohari, Ahmad Reza

    2015-01-01

    Background: Sargassum species (phaeophyceae) are economically important brown algae in southern parts of Iran. Sargassum is mainly harvested as a row material in alginate production industries and is a source of plant foods or plant bio-stimulants even a component of animal foods. Objective: In this study, Sargassum glaucescens, collected from the seashore of Chabahar, was employed for phytochemical and biological evaluations. Materials and Methods: For that purpose, the dried algae was extracted by methanol and subjected to different chromatographic separation methods. Results: Six sterols, fucosterol (1), 24(S)-hydroxy-24-vinylcholesterol (2), 24(R)-hydroxy-24-vinylcholesterol (3), stigmasterol (4), β-sitosterol (5) and cholesterol (6) were identified by spectroscopic methods including 1H-NMR, 13C-NMR and mass spectroscopy. In vitro alpha-amylase inhibitory test was performed on the methanolic extract and the results revealed a potent inhibition (IC50 = 8.9 ± 2.4 mg/mL) of the enzyme compared to acarbose as a positive control. Conclusion: Various biological activities and distribution of sterols in Sargassum genus have been critically reviewed here. The results concluded that these algae are a good candidate for further anti-diabetic investigations in animals and human. PMID:26692744

  9. Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bühler, Nicole; Hagiwara, Daisuke

    2015-01-01

    Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes (oshA to oshE) in the filamentous fungi Aspergillus nidulans. The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus, as well as A. nidulans. Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth. PMID:26116213

  10. Structural Insights into Inhibition of Sterol 14[alpha]-Demethylase in the Human Pathogen Trypanosoma cruzi

    SciTech Connect

    Lepesheva, Galina I.; Hargrove, Tatiana Y.; Anderson, Spencer; Kleshchenko, Yuliya; Furtak, Vyacheslav; Wawrzak, Zdzislaw; Villalta, Fernando; Waterman, Michael R.

    2010-09-02

    Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi sterol 14{alpha}-demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4{prime}-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.

  11. Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

    PubMed Central

    McCall, Laura-Isobel; El Aroussi, Amale; Choi, Jun Yong; Vieira, Debora F.; De Muylder, Geraldine; Johnston, Jonathan B.; Chen, Steven; Kellar, Danielle; Siqueira-Neto, Jair L.; Roush, William R.; Podust, Larissa M.; McKerrow, James H.

    2015-01-01

    Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis. PMID:25768284

  12. Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

    PubMed

    McCall, Laura-Isobel; El Aroussi, Amale; Choi, Jun Yong; Vieira, Debora F; De Muylder, Geraldine; Johnston, Jonathan B; Chen, Steven; Kellar, Danielle; Siqueira-Neto, Jair L; Roush, William R; Podust, Larissa M; McKerrow, James H

    2015-03-01

    Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis. PMID:25768284

  13. Common sources and estimated intake of plant sterols in the Spanish diet.

    PubMed

    Jiménez-Escrig, Antonio; Santos-Hidalgo, Ana B; Saura-Calixto, Fulgencio

    2006-05-01

    Plant sterols (PS) are minor lipid components of plants, which may have potential health benefits, mainly based in their cholesterol-lowering effect. The aim of this study was to determine the composition and content of PS in plant-based foods commonly consumed in Spain and to estimate the PS intake in the Spanish diet. For this purpose, the determination of PS content, using a modern methodology to measure free, esterified, and glycosidic sterol forms, was done. Second, an estimation of the intake of PS, using the Spanish National Food Consumption data, was made. The daily intake per person of PS--campesterol, beta-sitosterol, stigmasterol, and stigmastanol--in the Spanish diet was estimated at 276 mg, the largest component being beta-sitosterol (79.7%). Other unknown compounds, tentatively identified as PS, may constitute a considerable potential intake (99 mg). When the daily PS intake among European diets was compared in terms of campesterol, beta-sitosterol, stigmasterol, and stigmastanol, the PS intake in the Spanish diet was in the same range of other countries such as Finland (15.7% higher) or The Netherlands (equal). However, some qualitative differences in the PS sources were detected, that is, the predominant brown bread and vegetable fat consumption in the northern diets versus the white bread and vegetable oil consumption in the Spanish diet. These differences may help to provide a link between the consumption of PS and healthy effects of the diet. PMID:16637708

  14. Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols.

    PubMed

    Lund, Eric D; Chu, Fu-Lin E; Soudant, Philippe; Harvey, Ellen

    2007-01-01

    Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host. PMID:17112755

  15. Sterols from Mytilidae Show Anti-Aging and Neuroprotective Effects via Anti-Oxidative Activity

    PubMed Central

    Sun, Yujuan; Lin, Yanfei; Cao, Xueli; Xiang, Lan; Qi, Jianhua

    2014-01-01

    For screening anti-aging samples from marine natural products, K6001 yeast strain was employed as a bioassay system. The active mussel extract was separated to give an active sterol fraction (SF). SF was further purified, and four sterol compounds were obtained. Their structures were determined to be cholesterol (CHOL), brassicasterol, crinosterol, and 24-methylenecholesterol. All compounds showed similar anti-aging activity. To understand the action mechanism involved, anti-oxidative experiments, reactive oxygen species (ROS) assays, and malondialdehyde (MDA) tests were performed on the most abundant compound, CHOL. Results indicated that treatment with CHOL increases the survival rate of yeast under oxidative stress and decreases ROS and MDA levels. In addition, mutations of uth1, skn7, sod1, and sod2, which feature a K6001 background, were employed and the lifespans of the mutations were not affected by CHOL. These results demonstrate that CHOL exerts anti-aging effects via anti-oxidative stress. Based on the connection between neuroprotection and anti-aging, neuroprotective experiments were performed in PC12 cells. Paraquat was used to induce oxidative stress and the results showed that the CHOL and SF protect the PC12 cells from the injury induced by paraquat. In addition, these substance exhibited nerve growth factor (NGF) mimic activities again confirmed their neuroprotective function. PMID:25429428

  16. A series of cationic sterol lipids with gene transfer and bactericidal activity

    PubMed Central

    Randazzo, R. A. S.; Bucki, R.; Janmey, P. A.

    2009-01-01

    A family of cationic lipids was synthesized via direct amide coupling of spermine to the C-24 position of cholic acid analogs. Four monosubstituted spermines and a bis-substituted spermine were evaluated as plasmid transfection reagents, as bacteriostatic agents, and as bactericidal agents. The incorporation of a double bond in the sterol moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine2000. Inclusion of the double bond had no effect on the general trend of increasing bactericidal activity with increasing sterol hydrophobicity. Co-formulation of the most hydrophilic of the compounds with its bis-substituted analogue led to enhancement in transfection activity. The bis-substituted compound, when tested alone, emerged as the most bacteriostatic compound in the family with minimum inhibitory concentrations (MIC) of 4 μM against B. subtilis and 16 μM against E. coli and therapeutic indexes (minimum hemolytic concentration/minimum inhibitory concentration) of 61 and 15, respectively. Cationic lipids can be optimized for both gene delivery and antibacterial applications by similar modifications. PMID:19364656

  17. Origins of suspended particulate matter based on sterol distribution in low salinity water mass observed in the offshore East China Sea.

    PubMed

    Kim, Moonkoo; Jung, Jee-Hyun; Jin, Yongnu; Han, Gi Myeong; Lee, Taehee; Hong, Sang Hee; Yim, Un Hyuk; Shim, Won Joon; Choi, Dong-Lim; Kannan, Narayanan

    2016-07-15

    The molecular composition and distribution of sterols were investigated in the East China Sea to identify the origins of suspended particulate matter (SPM) in offshore waters influenced by Changjiang River Diluted Water (CRDW). Total sterol concentrations ranged from 3200 to 31,900pgL(-1) and 663 to 5690pgL(-1) in the particulate and dissolved phases, respectively. Marine sterols dominated representing 71% and 66% in the particulate and dissolved phases, respectively. Typical sewage markers, such as coprostanol, were usually absent at ~250km offshore. However, sterols from allochthonous terrestrial plants were still detected at these sites. A negative relationship was observed between salinity and concentrations of terrestrial sterols in SPM, suggesting that significant amounts of terrestrial particulate matter traveled long distance offshore in the East China Sea, and the Changjiang River Diluted Water (CRDW) was an effective carrier of land-derived particulate organic matter to the offshore East China Sea. PMID:27167134

  18. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  19. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N; Bridges, Dave; Park, Edwards A; Cole, Robert N; Elam, Marshall B; Raghow, Rajendra

    2016-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  20. Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation

    PubMed Central

    Dong, Qingming; Giorgianni, Francesco; Beranova-Giorgianni, Sarka; Deng, Xiong; O'Meally, Robert N.; Bridges, Dave; Park, Edwards A.; Cole, Robert N.; Elam, Marshall B.; Raghow, Rajendra

    2015-01-01

    Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c–SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCFFbw7 ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated in vitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73 in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver. PMID:26589965

  1. Designing and encoding models for synthetic biology

    PubMed Central

    Endler, Lukas; Rodriguez, Nicolas; Juty, Nick; Chelliah, Vijayalakshmi; Laibe, Camille; Li, Chen; Le Novère, Nicolas

    2009-01-01

    A key component of any synthetic biology effort is the use of quantitative models. These models and their corresponding simulations allow optimization of a system design, as well as guiding their subsequent analysis. Once a domain mostly reserved for experts, dynamical modelling of gene regulatory and reaction networks has been an area of growth over the last decade. There has been a concomitant increase in the number of software tools and standards, thereby facilitating model exchange and reuse. We give here an overview of the model creation and analysis processes as well as some software tools in common use. Using markup language to encode the model and associated annotation, we describe the mining of components, their integration in relational models, formularization and parametrization. Evaluation of simulation results and validation of the model close the systems biology ‘loop’. PMID:19364720

  2. Peri-encoding predictors of memory encoding and consolidation.

    PubMed

    Cohen, Noga; Pell, Liat; Edelson, Micah G; Ben-Yakov, Aya; Pine, Alex; Dudai, Yadin

    2015-03-01

    We review reports of brain activations that occur immediately prior to the onset or following the offset of to-be-remembered information and can predict subsequent mnemonic success. Memory-predictive pre-encoding processes, occurring from fractions of a second to minutes prior to event onset, are mainly associated with activations in the medial temporal lobe (MTL), amygdala and midbrain, and with enhanced theta oscillations. These activations may be considered as the neural correlates of one or more cognitive operations, including contextual processing, attention, and the engagement of distinct computational modes associated with prior encoding or retrieval. Post-encoding activations that correlate with subsequent memory performance are mainly observed in the MTL, sensory cortices and frontal regions. These activations may reflect binding of elements of the encoded information and initiation of memory consolidation. In all, the findings reviewed here illustrate the importance of brain states in the immediate peri-encoding time windows in determining encoding success. Understanding these brain states and their specific effects on memory may lead to optimization of the encoding of desired memories and mitigation of undesired ones. PMID:25446944

  3. Ontology application and use at the ENCODE DCC

    PubMed Central

    Malladi, Venkat S.; Erickson, Drew T.; Podduturi, Nikhil R.; Rowe, Laurence D.; Chan, Esther T.; Davidson, Jean M.; Hitz, Benjamin C.; Ho, Marcus; Lee, Brian T.; Miyasato, Stuart; Roe, Gregory R.; Simison, Matt; Sloan, Cricket A.; Strattan, J. Seth; Tanaka, Forrest; Kent, W. James; Cherry, J. Michael; Hong, Eurie L.

    2015-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a catalog of genomic annotations. To date, the project has generated over 4000 experiments across more than 350 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory network and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All ENCODE experimental data, metadata and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage and distribution to community resources and the scientific community. As the volume of data increases, the organization of experimental details becomes increasingly complicated and demands careful curation to identify related experiments. Here, we describe the ENCODE DCC’s use of ontologies to standardize experimental metadata. We discuss how ontologies, when used to annotate metadata, provide improved searching capabilities and facilitate the ability to find connections within a set of experiments. Additionally, we provide examples of how ontologies are used to annotate ENCODE metadata and how the annotations can be identified via ontology-driven searches at the ENCODE portal. As genomic datasets grow larger and more interconnected, standardization of metadata becomes increasingly vital to allow for exploration and comparison of data between different scientific projects. Database URL: https://www.encodeproject.org/ PMID:25776021

  4. Ontology application and use at the ENCODE DCC.

    PubMed

    Malladi, Venkat S; Erickson, Drew T; Podduturi, Nikhil R; Rowe, Laurence D; Chan, Esther T; Davidson, Jean M; Hitz, Benjamin C; Ho, Marcus; Lee, Brian T; Miyasato, Stuart; Roe, Gregory R; Simison, Matt; Sloan, Cricket A; Strattan, J Seth; Tanaka, Forrest; Kent, W James; Cherry, J Michael; Hong, Eurie L

    2015-01-01

    The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a catalog of genomic annotations. To date, the project has generated over 4000 experiments across more than 350 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory network and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All ENCODE experimental data, metadata and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage and distribution to community resources and the scientific community. As the volume of data increases, the organization of experimental details becomes increasingly complicated and demands careful curation to identify related experiments. Here, we describe the ENCODE DCC's use of ontologies to standardize experimental metadata. We discuss how ontologies, when used to annotate metadata, provide improved searching capabilities and facilitate the ability to find connections within a set of experiments. Additionally, we provide examples of how ontologies are used to annotate ENCODE metadata and how the annotations can be identified via ontology-driven searches at the ENCODE portal. As genomic datasets grow larger and more interconnected, standardization of metadata becomes increasingly vital to allow for exploration and comparison of data between different scientific projects. PMID:25776021

  5. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  6. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  7. Evaluating standard terminologies for encoding allergy information

    PubMed Central

    Goss, Foster R; Zhou, Li; Plasek, Joseph M; Broverman, Carol; Robinson, George; Middleton, Blackford; Rocha, Roberto A

    2013-01-01

    Objective Allergy documentation and exchange are vital to ensuring patient safety. This study aims to analyze and compare various existing standard terminologies for representing allergy information. Methods Five terminologies were identified, including the Systemized Nomenclature of Medical Clinical Terms (SNOMED CT), National Drug File–Reference Terminology (NDF-RT), Medication Dictionary for Regulatory Activities (MedDRA), Unique Ingredient Identifier (UNII), and RxNorm. A qualitative analysis was conducted to compare desirable characteristics of each terminology, including content coverage, concept orientation, formal definitions, multiple granularities, vocabulary structure, subset capability, and maintainability. A quantitative analysis was also performed to compare the content coverage of each terminology for (1) common food, drug, and environmental allergens and (2) descriptive concepts for common drug allergies, adverse reactions (AR), and no known allergies. Results Our qualitative results show that SNOMED CT fulfilled the greatest number of desirable characteristics, followed by NDF-RT, RxNorm, UNII, and MedDRA. Our quantitative results demonstrate that RxNorm had the highest concept coverage for representing drug allergens, followed by UNII, SNOMED CT, NDF-RT, and MedDRA. For food and environmental allergens, UNII demonstrated the highest concept coverage, followed by SNOMED CT. For representing descriptive allergy concepts and adverse reactions, SNOMED CT and NDF-RT showed the highest coverage. Only SNOMED CT was capable of representing unique concepts for encoding no known allergies. Conclusions The proper terminology for encoding a patient's allergy is complex, as multiple elements need to be captured to form a fully structured clinical finding. Our results suggest that while gaps still exist, a combination of SNOMED CT and RxNorm can satisfy most criteria for encoding common allergies and provide sufficient content coverage. PMID:23396542

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Use of Enterococcus, BST and sterols as indicators for poultry pollution source tracking in surface and groundwater

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study has applied Enterococcus, Bacterial Source Tracking (BST) and sterol analysis for pollution source identification from poultry sources. Fecal contamination was detected in 100% of surface water and 15% of groundwater sites tested. E. faecium was the dominant species in aged litter sampl...

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

    PubMed

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

    2013-12-01

    White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS. PMID:24327437

  11. SHORT-TERM EFFICACY OF PLANT STEROLS CONSUMED AT BREAKFAST OR AT EACH MEAL IN LOWERING BLOOD CHOLESTEROL LEVELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: To compare under controlled conditions the effect of plant sterol consumed as a single morning dose or divided through the day on blood lipid profile. Method: A randomized, placebo-controlled, crossover-feeding, single blind trial was conducted in 19 subjects with LDL- cholesterol level...

  12. Hair sterol signatures coupled to multivariate data analysis reveal an increased 7β-hydroxycholesterol production in cognitive impairment.

    PubMed

    Son, Hyun-Hwa; Lee, Do-Yup; Seo, Hong Seog; Jeong, Jihyeon; Moon, Ju-Yeon; Lee, Jung-Eun; Chung, Bong Chul; Kim, Eosu; Choi, Man Ho

    2016-01-01

    Altered cholesterol metabolism could be associated with cognitive impairment. The quantitative profiling of 19 hair sterols was developed using gas chromatography-mass spectrometry coupled to multivariate data analysis. The limit of quantification of all sterols ranged from 5 to 20 ng/g, while the calibration linearity was higher than 0.98. The precision (% CV) and accuracy (% bias) ranged from 3.2% to 9.8% and from 83.2% to 119.4%, respectively. Among the sterols examined, 8 were quantitatively detected from two strands of 3-cm-long scalp hair samples of female participants, including mild cognitive impairment (MCI, n=15), Alzheimer's disease (AD, n=31), and healthy controls (HC, n=36). The cognitive impairment (MCI or AD) was correlated with a higher metabolic rate than that of HCs based on 7β-hydroxycholesterol (P<0.005). Significant negative correlations (r=-0.822) were detected between Mini-Mental State Examination (MMSE) scores and hair sample metabolic ratios of 7β-hydroxycholesterol to cholesterol, which is an accepted, sensitive, and specific tool for discriminating HCs from individuals with MCI or AD. In conclusion, improved diagnostic values can be obtained using hair sterol signatures coupled with MMSE scores. This method may prove useful for predictive diagnosis in population screening of cognitive impairment. PMID:26385606

  13. Online LC-GC analysis of free sterols/stanols and intact steryl/stanyl esters in cereals.

    PubMed

    Esche, Rebecca; Scholz, Birgit; Engel, Karl-Heinz

    2013-11-20

    The suitability of online liquid chromatography-gas chromatography for the analysis of free sterols/stanols, steryl/stanyl fatty acid esters, and trans-steryl/stanyl ferulic acid esters in cereals is demonstrated. The silylated lipid extracts were fractionated via liquid chromatography on a normal phase, and the fractions containing the sterol classes were transferred online to the gas chromatograph for the analysis of their individual compositions. The study provides for the first time data on free sterols/stanols and intact steryl/stanyl esters in sweet corn, popcorn, and proso millet. Sweet corn revealed the highest contents of free sterols/stanols and steryl/stanyl fatty acid esters, and popcorn, in turn, the highest amounts of trans-steryl/stanyl ferulic acid esters. The distribution patterns of the proso millet samples revealed pronounced differences from those of sweet corn and popcorn as they particularly exhibited high proportions of free cholesterol and cholesteryl fatty acid esters. Furthermore, no trans-steryl/stanyl ferulic acid esters could be detected. PMID:24117337

  14. BIOCHEMISTRY OF DINOFLAGELLATE LIPIDS, WITH PARTICULAR REFERENCE TO THE FATTY ACID AND STEROL COMPOSITION OF A KARENIA BREVIS BLOOM

    EPA Science Inventory

    Leblond, Jeffrey D., Terence J. Evens and Peter J. Chapman. 2003. Biochemistry of Dinoflagellate Lipids, with Particular Reference to the Fatty Acid and Sterol Composition of a Karenia brevis Bloom. Phycologia. 42(4):324-331. (ERL,GB 1160).

    The harmful marine dinoflagella...

  15. Sterol-inhibiting fungicide impacts on soil microbial ecology in Atlantic Coastal Plain soils

    NASA Astrophysics Data System (ADS)

    White, P. M.; Potter, T. L.; Strickland, T. C.

    2008-12-01

    Seventy-five percent of the peanuts (Arachus hypogaia) produced in the United States are grown in the Atlantic Coastal Plain region. Portions of this area, including Alabama and Georgia, exhibit a subtropical climate that promotes soil-borne plant fungal diseases. Most fields receive repeated fungicide applications during the growing season to suppress the disease causing organisms, such as Sclerotium rolfsii, Rhizoctonia solani, and Cylindrocladium parasiticum. Information regarding fungicide effects on the soil microbial community, with components principally responsible for transformation and fate of fungicides and other soil-applied pesticides, is limited. The objectives of the study were to assess soil microbial community response to (1) varying rates of the sterol-inhibiting fungicide tebuconazole (0, single application, season max, 2x season max), and (2) field rates of the sterol-inhibitors cyproconazole, prothioconazole, tebuconazole, and flutriafol, and thiol-competitor chlorothalonil. The sterol-inhibitors exhibited different half lives, as listed in the FOOTPRINT database, ranging from <1 day to >1300 d. Chlorothalonil was chosen because it is the most frequently applied fungicide to peanut. Shifts in the fungi, gram positive and gram negative bacteria, were monitored during the experiments using phospholipid fatty acid (PLFA) profiles. Ergosterol levels and pesticide decay rates were also monitored to evaluate the effectiveness of the fungicide and soil residence time, respectively. In the rate study, the highest rate of tebuconazole reduced the fungal biomarker 18:2ω6,9c to 2.6 nmol g-1 dry soil at 17 d, as compared to the control (4.1 nmol g-1 dry soil). However, levels of the fungal PLFA biomarker were similar regardless of rate at 0 and 32 d. The gram negative bacterial PLFA mole percent was greater at 17 d for the two highest rates of tebuconazole, but was similar at 0 and 32 d. Gram positive and fungal mole percents were not affected at any time

  16. Two digital video encoder circuits

    NASA Astrophysics Data System (ADS)

    Eldon, John A.

    1992-11-01

    Central to `multimedia' image processing is the desire to encode computer graphics data into a standard television signal, complete with line, field, and color subcarrier synchronizing information. The numerous incompatibilities between television and computer display standards render this operation far less trivial than it sounds to anyone who hasn't worked with both types of signals. To simplify the task of encoding computer graphics signals into standard NTSC (North America and Japan) or PAL (most of Europe) television format for display, broadcast, or recording, TRW LSI Products Inc. has introduced the two newest members of it multimedia integrated circuit family, the TMC22090 and TMC22190 digital video encoders.

  17. Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

    SciTech Connect

    Xu,S.; Benoff, B.; Liou, H.; Lobel, P.; Stock, A.

    2007-01-01

    NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two {beta}-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the {beta}-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.

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

    PubMed

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

    2011-08-30

    Niemann--Pick C disease is an inherited disorder in which cholesterol and other lipids accumulate in the late endosomal/lysosomal compartment. Recently, cyclodextrins (CD) have been shown to reduce symptoms and extend lifespan in animal models of the disease. In the present studies we examined the mechanism of sterol transport by CD using in vitro model systems and fluorescence spectroscopy and NPC2-deficient fibroblasts. We demonstrate that cholesterol transport from the lysosomal cholesterol-binding protein NPC2 to CD occurs via aqueous diffusional transfer and is very slow; the rate-limiting step appears to be dissociation of cholesterol from NPC2, suggesting that specific interactions between NPC2 and CD do not occur. In contrast, the transfer rate of the fluorescent cholesterol analogue dehydroergosterol (DHE) from CD to phospholipid membranes is very rapid and is directly proportional to the acceptor membrane concentration, as is DHE transfer from membranes to CD. Moreover, CD dramatically increases the rate of sterol transfer between membranes, with rates that can approach those mediated by NPC2. The results suggest that sterol transfer from CD to membranes occurs by a collisional transfer mechanism involving direct interaction of CD with membranes, similar to that shown previously for NPC2. For CD, however, absolute rates are slower compared to NPC2 for a given concentration, and the lysosomal phospholipid lysobisphosphatidic acid (LBPA) does not stimulate rates of sterol transfer between membranes and CD. As expected from the apparent absence of interaction between CD and NPC2, the addition of CD to NPC2-deficient fibroblasts rapidly rescued the cholesterol accumulation phenotype. Thus, the recent observations of CD efficacy in mouse models of NPC disease are likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD--membrane interactions. PMID:21740003

  19. The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants.

    PubMed

    Mangas, Susana; Bonfill, Mercè; Osuna, Lidia; Moyano, Elisabeth; Tortoriello, Jaime; Cusido, Rosa M; Piñol, M Teresa; Palazón, Javier

    2006-09-01

    Considering that exogenously applied methyl jasmonate can enhance secondary metabolite production in a variety of plant species and that 2,3-oxidosqualene is a common precursor of triterpenes and sterols in plants, we have studied Centella asiatica and Galphimia glauca (both synthesizing triterpenoid secondary compounds) and Ruscus aculeatus (which synthesizes steroidal secondary compounds) for their growth rate and content of free sterols and respective secondary compounds, after culturing with or without 100 microM methyl jasmonate. Our results show that elicited plantlets of G. glauca and to a higher degree C. asiatica (up to 152-times more) increased their content of triterpenoids directly synthesized from 2,3-oxidosqualene (ursane saponins and nor-seco-friedelane galphimines, respectively) at the same time as growth decreased. In contrast, the free sterol content of C. asiatica decreased notably, and remained practically unaltered in G. glauca. However, in the case of R. aculeatus, which synthesizes steroidal saponins (mainly spirostane type) indirectly from 2,3-oxidosqualene after the latter is converted to the plant phytosterol-precursor cycloartenol, while the growth rate and free sterol content clearly decreased, the spirostane saponine content was virtually unchanged (aerial part) or somewhat lower (roots) in presence of the same elicitor concentration. Our results suggest that while methyl jasmonate may be used as an inducer of enzymes involved in the triterpenoid synthesis downstream from 2,3-oxidosqualene in both C. asiatica and G. glauca plantlets, in those of C. asiatica and R. aculeatus it inhibited the enzymes involved in sterol synthesis downstream from cycloartenol. PMID:16876832

  20. Topsensterols A–C, Cytotoxic Polyhydroxylated Sterol Derivatives from a Marine Sponge Topsentia sp.

    PubMed Central

    Chen, Min; Wu, Xu-Dong; Zhao, Qing; Wang, Chang-Yun

    2016-01-01

    Three new polyhydroxylated sterol derivatives topsensterols A–C (1–3) have been isolated from a marine sponge Topsentia sp. collected from the South China Sea. Their structures were elucidated by detailed analysis of the spectroscopic data, especially the NOESY spectra. Topsensterols A–C (l–3) possess novel 2β,3α,4β,6α-tetrahydroxy-14α-methyl Δ9(11) steroidal nuclei with unusual side chains. Compound 2 exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC50 value of 8.0 μM. Compound 3 displayed cytotoxicity against human erythroleukemia cell line K562 with an IC50 value of 6.0 μM. PMID:27490555

  1. Fatty acids and sterols composition, and antioxidant activity of oils extracted from plant seeds.

    PubMed

    Kozłowska, Mariola; Gruczyńska, Eliza; Ścibisz, Iwona; Rudzińska, Magdalena

    2016-12-15

    This study determined and compared the contents of bioactive components in plant seed oils extracted with n-hexane (Soxhlet method) and chloroform/methanol (Folch method) from coriander, caraway, anise, nutmeg and white mustard seeds. Oleic acid dominated among unsaturated fatty acids in nutmeg and anise seed oils while petroselinic acid was present in coriander and caraway oils. Concerning sterols, β-sitosterol was the main component in seed oils extracted with both methods. The content of total phenolics in nutmeg, white mustard and coriander seed oils extracted with chloroform/methanol was higher than in their counterparts prepared with n-hexane. The seed oil samples extracted according to the Folch method exhibited a higher ability to scavenge DPPH radicals compared to the oil samples prepared with the Soxhlet method. DPPH values of the methanolic extracts derived from oils produced with the Folch method were also higher than in the oils extracted with n-hexane. PMID:27451203

  2. A sterol binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing

    PubMed Central

    Mousley, Carl J.; Yuan, Peihua; Gaur, Naseem A.; Trettin, Kyle D.; Nile, Aaron H.; Deminoff, Stephen J.; Dewar, Brian J.; Wolpert, Max; Macdonald, Jeffrey M.; Herman, Paul K.; Hinnebusch, Alan G.; Bankaitis, Vytas A.

    2012-01-01

    SUMMARY Kes1, and other oxysterol binding protein (OSBP) superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids, and dampens gene expression driven by Gcn4; the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic ‘large Mediator’ complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing. PMID:22341443

  3. Topsensterols A-C, Cytotoxic Polyhydroxylated Sterol Derivatives from a Marine Sponge Topsentia sp.

    PubMed

    Chen, Min; Wu, Xu-Dong; Zhao, Qing; Wang, Chang-Yun

    2016-01-01

    Three new polyhydroxylated sterol derivatives topsensterols A-C (1-3) have been isolated from a marine sponge Topsentia sp. collected from the South China Sea. Their structures were elucidated by detailed analysis of the spectroscopic data, especially the NOESY spectra. Topsensterols A-C (l-3) possess novel 2β,3α,4β,6α-tetrahydroxy-14α-methyl Δ(9(11)) steroidal nuclei with unusual side chains. Compound 2 exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC50 value of 8.0 μM. Compound 3 displayed cytotoxicity against human erythroleukemia cell line K562 with an IC50 value of 6.0 μM. PMID:27490555

  4. Regulatory polymorphisms underlying complex disease traits.

    PubMed

    Knight, Julian C

    2005-02-01

    There is growing evidence that genetic variation plays an important role in the determination of individual susceptibility to complex disease traits. In contrast to coding sequence polymorphisms, where the consequences of non-synonymous variation may be resolved at the level of the protein phenotype, defining specific functional regulatory polymorphisms has proved problematic. This has arisen for a number of reasons, including difficulties with fine mapping due to linkage disequilibrium, together with a paucity of experimental tools to resolve the effects of non-coding sequence variation on gene expression. Recent studies have shown that variation in gene expression is heritable and can be mapped as a quantitative trait. Allele-specific effects on gene expression appear relatively common, typically of modest magnitude and context specific. The role of regulatory polymorphisms in determining susceptibility to a number of complex disease traits is discussed, including variation at the VNTR of INS, encoding insulin, in type 1 diabetes and polymorphism of CTLA4, encoding cytotoxic T lymphocyte antigen, in autoimmune disease. Examples where regulatory polymorphisms have been found to play a role in mongenic traits such as factor VII deficiency are discussed, and contrasted with those polymorphisms associated with ischaemic heart disease at the same gene locus. Molecular mechanisms operating in an allele-specific manner at the level of transcription are illustrated, with examples including the role of Duffy binding protein in malaria. The difficulty of resolving specific functional regulatory variants arising from linkage disequilibrium is demonstrated using a number of examples including polymorphism of CCR5, encoding CC chemokine receptor 5, and HIV-1 infection. The importance of understanding haplotypic structure to the design and interpretation of functional assays of putative regulatory variation is highlighted, together with discussion of the strategic use of

  5. Peroxisome proliferators and fatty acids negatively regulate liver X receptor-mediated activity and sterol biosynthesis.

    PubMed

    Johnson, T E; Ledwith, B J

    2001-04-01

    Peroxisome proliferators (PPs) are potent tumor promoters in rodents. The mechanism of hepatocarcinogenesis requires the nuclear receptor peroxisome proliferator activated receptor-alpha (PPARalpha), but might also involve the PPARalpha independent alteration of signaling pathways that regulate cell growth. Here, we studied the effects of PPs on the mevalonate pathway, a critical pathway that controls cell proliferation. Liver X receptors (LXRs) are nuclear receptors that act as sterol sensors in the mevalonate pathway. In gene reporter assays in COS-7 cells, the basal activity of the LXR responsive reporter gene (LXRE-luc) was suppressed by 10 microM lovastatin and zaragozic acid A, suggesting that this activity was attributed to the activation of native LXRs, by endogenously produced mevalonate products. The potent PP and rodent tumor promoter, pirinixic acid (WY-14643) also inhibited LXR-mediated transcription in a dose related manner (approximate IC(50) of 100 microM). As did several other PPs including ciprofibric acid and mono-ethylhexylphthalate. Polyunsaturated and medium to long chain fatty acids at 100 microM were also potent inhibitors; the arachidonic acid analogue eicosatetraynoic acid being the most active (approximate IC(50) of 10 microM). Of the PPs and fatty acids tested, there was a strong correlation between the ability of these agents to suppress de novo sterol synthesis in a rat hepatoma cell line, H4IIEC3, and inhibit LXR-mediated transcription in COS-7 cells, but a discordance between these endpoints and PPARalpha activation and fatty acid acyl-CoA oxidase induction. Taken together, these results suggest that PPs and fatty acids negatively regulate the mevalonate pathway through a mechanism that is not entirely dependent on PPARalpha activation. Because of the importance of the mevalonate pathway in regulating cell proliferation, the modulation of this pathway by PPs and fatty acids might contribute to their actions on cell growth

  6. Conversion of Exogenous Cholesterol into Glycoalkaloids in Potato Shoots, Using Two Methods for Sterol Solubilisation

    PubMed Central

    Petersson, Erik V.; Nahar, Nurun; Dahlin, Paul; Broberg, Anders; Tröger, Rikard; Dutta, Paresh C.; Jonsson, Lisbeth; Sitbon, Folke

    2013-01-01

    Steroidal glycoalkaloids (SGA) are toxic secondary metabolites naturally occurring in the potato, as well as in certain other Solanaceous plant species, such as tomato, eggplant and pepper. To investigate the steroidal origin of SGA biosynthesis, cut potato shoots were fed cholesterol labelled with deuterium (D) in the sterol ring structure (D5- or D6-labelled), or side chain (D7-labelled), and analysed after three or five weeks. The labelled cholesterol and presence of D-labelled SGA were analysed by GC-MS and LC-MS/MS, respectively. When feeding D-labelled cholesterol solubilised in Tween-80, labelled cholesterol in free form became present in both leaves and stems, although the major part was recovered as steryl esters. Minor amounts of D-labelled SGA (α-solanine and α-chaconine) were identified in cholesterol-treated shoots, but not in blank controls, or in shoots fed D6-27-hydroxycholesterol. Solubilising the labelled cholesterol in methyl-β-cyclodextrin instead of Tween-80 increased the levels of labelled SGA up to 100-fold, and about 1 mole% of the labelled cholesterol was recovered as labelled SGA in potato leaves. Both side chain and ring structure D labels were retained in SGA, showing that the entire cholesterol molecule is converted to SGA. However, feeding side chain D7-labelled cholesterol resulted in D5-labelled SGA, indicating that two hydrogen atoms were released during formation of the SGA nitrogen-containing ring system. Feeding with D7-sitosterol did not produce any labelled SGA, indicating that cholesterol is a specific SGA precursor. In conclusion, we have demonstrated a superior performance of methyl-β-cyclodextrin for delivery of cholesterol in plant tissue feeding experiments, and given firm evidence for cholesterol as a specific sterol precursor of SGA in potato. PMID:24349406

  7. Ezetimibe Reduces Plant Sterol Accumulation and Favorably Increases Platelet Count in Sitosterolemia

    PubMed Central

    Othman, Rgia A.; Myrie, Semone B.; Mymin, David; Merkens, Louise S.; Roullet, Jean-Baptiste; Steiner, Robert D.; Jones, Peter J.H.

    2014-01-01

    Objective To assess if ezetimibe (EZE), a sterol-absorption inhibitor, improves platelet (PLT) count and size relative to its effect on plasma plant sterol (PS) in patients with sitosterolemia (STSL). Study design Patients with STSL (5 males, 3 females, 16 to 56 years of age) receiving EZE intervention as part of their routine care participated in this study. EZE was discontinued for 14 weeks (off) and then resumed for another 14 weeks (on). Hematology variables along with plasma and red blood cells (RBC) PS and total cholesterol (TC) levels were measured at the end of each phase. Results EZE increased PLT count (23 ± 9%) and decreased mean PLT volume (MPV; 10 ± 3%, all P < .05). In patients off EZE, PLT counts inversely correlated (r = − 0.96 and r = − 0.91, all P < .01) with plasma and RBC PS to TC ratio (PS/TC), and MPV positively correlated (r = 0.91, P = .03 and r = 0.93, P = .02) with plasma and RBC PS/TC. EZE reduced plasma and RBC sitosterol (−35 ± 4 and −28 ± 3%), total PS (−37 ± 4 and −28 ± 3%, all P < .0001) levels and PS/TC (−27 ± 4 and −28 ± 4%, P < .01). Conclusion EZE reduces plasma and RBC PS levels, and increasing PLT count and decreasing MPV, and thereby may reduce the risk for bleeding in STSL. Plasma PS levels and ABCG5/ABCG8 genes should be analyzed in patients with unexplained hematologic abnormalities. PMID:25444527

  8. Comparison of sterols and fatty acids in two species of Ganoderma

    PubMed Central

    2012-01-01

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

  9. Serial position encoding of signs.

    PubMed

    Miozzo, Michele; Petrova, Anna; Fischer-Baum, Simon; Peressotti, Francesca

    2016-09-01

    Reduced short-term memory (STM) capacity has been reported for sign as compared to speech when items have to be recalled in a specific order. This difference has been attributed to a more precise and efficient serial position encoding in verbal STM (used for speech) than visuo-spatial STM (used for sign). We tested in the present investigation whether the reduced STM capacity with signs stems from a lack of positional encoding available in verbal STM. Error analyses reported in prior studies have revealed that positions are defined in verbal STM by distance from both the start and the end of the sequence (both-edges positional encoding scheme). Our analyses of the errors made by deaf participants with finger-spelled letters revealed that the both-edges positional encoding scheme underlies the STM representation of signs. These results indicate that the cause of the STM disadvantage is not the type of positional encoding but rather the difficulties in binding an item in visuo-spatial STM to its specific position in the sequence. Both-edges positional encoding scheme could be specific of sign, since it has not been found in visuo-spatial STM tasks conducted with hearing participants. PMID:27244095

  10. High-throughput functional testing of ENCODE segmentation predictions

    PubMed Central

    Kwasnieski, Jamie C.; Fiore, Christopher; Chaudhari, Hemangi G.

    2014-01-01

    The histone modification state of genomic regions is hypothesized to reflect the regulatory activity of the underlying genomic DNA. Based on this hypothesis, the ENCODE Project Consortium measured the status of multiple histone modifications across the genome in several cell types and used these data to segment the genome into regions with different predicted regulatory activities. We measured the cis-regulatory activity of more than 2000 of these predictions in the K562 leukemia cell line. We tested genomic segments predicted to be Enhancers, Weak Enhancers, or Repressed elements in K562 cells, along with other sequences predicted to be Enhancers specific to the H1 human embryonic stem cell line (H1-hESC). Both Enhancer and Weak Enhancer sequences in K562 cells were more active than negative controls, although surprisingly, Weak Enhancer segmentations drove expression higher than did Enhancer segmentations. Lower levels of the covalent histone modifications H3K36me3 and H3K27ac, thought to mark active enhancers and transcribed gene bodies, associate with higher expression and partly explain the higher activity of Weak Enhancers over Enhancer predictions. While DNase I hypersensitivity (HS) is a good predictor of active sequences in our assay, transcription factor (TF) binding models need to be included in order to accurately identify highly expressed sequences. Overall, our results show that a significant fraction (∼26%) of the ENCODE enhancer predictions have regulatory activity, suggesting that histone modification states can reflect the cis-regulatory activity of sequences in the genome, but that specific sequence preferences, such as TF-binding sites, are the causal determinants of cis-regulatory activity. PMID:25035418

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

    PubMed

    Lin, Yuguang; Knol, Diny; Menéndez-Carreño, María; Blom, Wendy A M; Matthee, Joep; Janssen, Hans-Gerd; Trautwein, Elke A

    2016-01-27

    Plant sterols (PS) in foods are subject to thermal oxidation to form PS oxidation products (POP). This study measured POP contents of 19 foods prepared by typical household baking and cooking methods using margarines without (control) and with 7.5% added PS (as 12.5% PS-esters, PS-margarine). Median POP contents per portion size of cooked foods were 0.57 mg (range 0.05-1.11 mg) with control margarine versus 1.42 mg (range 0.08-20.5 mg) with PS-margarine. The oxidation rate of PS (ORP) was 0.50% (median) with the PS-margarine and 3.66% with the control margarine. Using the PS-margarine, microwave-cooked codfish had the lowest POP content, with 0.08 mg per portion, while shallow-fried potatoes had the highest POP content, 20.5 mg per portion. Median POP contents in cookies, muffins, banana bread, and sponge cake baked with the control or PS-margarine were 0.12 mg (range 0.11-0.21 mg) and 0.24 mg (range 0.19-0.60 mg) per portion, with a corresponding ORP of 1.38% and 0.06%, respectively. POP contents in all the cooked and baked foods did not exceed 20.5 mg per typical portion size. A wide variation in the distribution of individual POP among different foods existed, with 7-keto-PS and 5,6-epoxy-PS being the major oxidation products. PMID:26697919

  12. Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

    PubMed Central

    Lopez, Adam M.; Posey, Kenneth S.; Turley, Stephen D.

    2014-01-01

    Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal−/−:Soat2+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs. 1.9 mg in Lal+/+:Soat2+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal−/−:Soat2+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal−/−:Soat2−/− littermates. The level of EC accumulation in the SI of the Lal−/−:Soat2−/− mice was also much less than in their Lal−/−:Soat2+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal−/−:Soat2−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function. PMID:25450374

  13. Sterol biosynthesis: strong inhibition of maize delta 5,7-sterol delta 7-reductase by novel 6-aza-B-homosteroids and other analogs of a presumptive carbocationic intermediate of the reduction reaction.

    PubMed

    Rahier, A; Taton, M

    1996-06-01

    A series of mono- and diazasteroids have been synthesized as analogs of a predicted carbocationic intermediate of delta 5,7-sterol delta 7-reductase (delta 7-SR). 6-Aza-B-homo-5 alpha-cholest-7-en-3 beta-ol (4), a novel compound whose synthesis is described for the first time, and 6,7-diaza-5 alpha-cholest-8(14)-en-3 beta-ol (6) were shown to be very powerful inhibitors of delta 7-SR in a preparation isolated from maize (Zea mays) (K(i),app = 50-70 nM, Ki,app/Km,app = 1.0 x 10(-4) to 1.3 x 10(-4). The data are consistent with a carbonium ion mechanism for the reduction; compounds 4 and 6 probably act as reaction intermediate analogs. Compound 4, in contrast to compound 6, displayed in the same microsomal preparation more than 50-fold selectivity for inhibition of the delta 7-SR versus delta 8-delta 7-sterol isomerase, cycloeucalenol isomerase, and delta 8,14-sterol delta 14-reductase, the mechanism of these four enzymes involving presumptive cationic intermediates centered respectively at C7, C8, C9, and C14. These observations highlight the paramount importance of the location of the positively charged nitrogen atom(s) in the B-ring structure for selectivity among these enzymes involving structurally close cationic reaction intermediates. Efficient in vivo inhibition of sterol biosynthesis in bramble cell suspension cultures by a low concentration of compound 4 was demonstrated and confirmed the in vitro properties of this derivative.) PMID:8679532

  14. Gravity referenced elevation encoder development

    NASA Astrophysics Data System (ADS)

    Goddard, R. E.

    1993-05-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  15. Gravity referenced elevation encoder development

    NASA Technical Reports Server (NTRS)

    Goddard, R. E.

    1993-01-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  16. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  17. Fly Photoreceptors Encode Phase Congruency.

    PubMed

    Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  18. Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages.

    PubMed Central

    Jepson, C A; Harrison, J A; Kraemer, F B; Yeaman, S J

    1996-01-01

    The development of atherosclerotic plaques in arteries is a key step in atherogenesis, with cholesterol ester accumulation in macrophage-derived foam cells being recognized as a major pathogenic event in this process. In this study, the mouse macrophage cell line J774.2 was induced to accumulate intracellular sterol esters by incubation with 25-hydroxycholesterol in the presence of oleic acid. The accumulation of sterol esters in these cells was found to be accompanied by a marked decrease in the activity of the enzyme responsible for their hydrolysis, namely hormone-sensitive lipase (HSL); Western blotting studies revealed a corresponding decrease in the levels of the HSL polypeptide. Similar findings were obtained after incubation with oxidized low-density lipoprotein or very-low-density lipoprotein. These findings suggest that down-regulation of the expression of HSL is important in cholesterol ester accumulation in macrophages. PMID:8761468

  19. Sterol 14α-demethylase as a potential target for antitrypanosomal therapy: enzyme inhibition and parasite cell growth

    PubMed Central

    Lepesheva, Galina I.; Ott, Robert D.; Hargrove, Tatiana Y.; Kleshchenko, Yuliya Y.; Schuster, Inge; Nes, W. David; Hill, George C.; Villalta, Fernando; Waterman, Michael R.

    2008-01-01

    Summary Sterol 14α-demethylases (CYP51) serve as primary targets for antifungal drugs and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands which demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a novel lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors. PMID:18022567

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

    PubMed

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

    2014-01-22

    Peppers (Capsicum spp.) are consumed worldwide, imparting flavor, aroma, and color to foods, additionally containing high concentrations of biofunctional compounds. This is the first report about the effect of the inoculation of two Rhizobium strains on sterols, triterpenes, fatty acids, and volatile compounds of leaves and fruits of pepper (Capsicum annuum L.) plants. Generally, inoculation with strain TVP08 led to the major changes, being observed a decrease of sterols and triterpenes and an increase of fatty acids, which are related to higher biomass, growth, and ripening of pepper fruits. The increase of volatile compounds may reflect the elicitation of plant defense after inoculation, since the content on methyl salicylate was significantly increased in inoculated material. The findings suggest that inoculation with Rhizobium strains may be employed to manipulate the content of interesting metabolites in pepper leaves and fruits, increasing potential health benefits and defense abilities of inoculated plants. PMID:24405510

  1. [Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM As-1656 strain].

    PubMed

    Molchanova, M A; Andriushina, V A; Savinova, T S; Stytsenko, T S; Rodina, N V; Voĭshvillo, N E

    2007-01-01

    A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ. PMID:17682396

  2. The influence of amphotericin B on the molecular organization and structural properties of DPPC lipid membranes modified by sterols

    NASA Astrophysics Data System (ADS)

    Kamiński, Daniel M.; Pociecha, Damian; Górecka, Ewa; Gagoś, Mariusz

    2015-02-01

    In this work, we studied the influence of the polyene antibiotic amphotericin B (AmB) on dipalmitoylphosphatidylcholine (DPPC) multibilayers modified by cholesterol and ergosterol investigated by means of X-ray diffraction. The periodic structures related to AmB-DPPC-sterol complexes in multibilayers are completely created in temperatures above the main lipid phase transition. The differences between all observed multilayer structures are related to the thickness. Multibilayers composed of lipid and sterols have similar thickness of 51 Å. Addition of amphotericin B leads to creation of new periodic structures. Multibilayers composed of lipid-ergosterol-AmB have thickness of 44 Å which does not change with temperature. Multibilayers composed of lipid-cholesterol-AmB at 20 °C are 43 Å and gets thicker at 50 °C. Most probably this is caused by weaker van der Vaals cholesterol-amphotericin B interactions.

  3. Towards squalamine mimics: synthesis and antibacterial activities of head-to-tail dimeric sterol-polyamine conjugates.

    PubMed

    Chen, Wen-Hua; Wennersten, Christine; Moellering, Robert C; Regen, Steven L

    2013-03-01

    Four dimeric sterol-polyamine conjugates have been synthesized from the homo- and hetero-connection of monomeric sterol-polyamine analogs in a head-to-tail manner. These dimeric conjugates show strong antibacterial activity against a broad spectrum of Gram-positive bacteria, whereas their corresponding activities against Gram-negative bacteria are relatively moderate. Though no significant difference was observed in the activities of these conjugates, cholic acid-containing dimeric conjugates generally exhibit higher activities than the corresponding deoxycholic acid-derived analogs. This is in contrast to the finding that a monomeric deoxycholic acid-spermine conjugate was more active than the corresponding cholic acid-derived analog. PMID:23495155

  4. Regulatory analysis of the C. elegans genome with spatiotemporal resolution

    PubMed Central

    Araya, Carlos L.; Kawli, Trupti; Kundaje, Anshul; Jiang, Lixia; Wu, Beijing; Vafeados, Dionne; Terrell, Robert; Weissdepp, Peter; Gevirtzman, Louis; Mace, Daniel; Niu, Wei; Boyle, Alan P.; Xie, Dan; Ma, Lijia; Murray, John I.; Reinke, Valerie; Waterston, Robert H.; Snyder, Michael

    2015-01-01

    Summary Discovering the structure and dynamics of transcriptional regulatory events in the genome with cellular and temporal resolution is crucial to understanding the regulatory underpinnings of development and disease. We determined the genomic distribution of binding sites for 92 transcription factors (TFs) and regulatory proteins across multiple stages of C. elegans development by performing 241 ChIP-seq experiments. Integrating regulatory binding and cellular-resolution expression data yielded a spatiotemporally-resolved metazoan TF binding map. Using this map, we explore developmental regulatory circuits that encode combinatorial logic at the levels of co-binding and co-expression of TFs, characterizing (1) the genomic coverage and clustering of regulatory binding, (2) the binding preferences of and biological processes regulated by TFs, (3) the global TF co-associations and genomic subdomains that suggest shared patterns of regulation, and (4) key TFs and TF co-associations for fate specification of individual lineages and cell-types. PMID:25164749

  5. A search for mosquito larvicidal compounds by blocking the sterol carrying protein, AeSCP-2, through computational screening and docking strategies

    PubMed Central

    Kumar, R. Barani; Shanmugapriya, B.; Thiyagesan, K.; Kumar, S. Raj; Xavier, Suresh M.

    2010-01-01

    Background: Sterol is a very vital compound for most of the insects and mosquitoes to complete their life cycle. Unfortunately mosquitoes cannot synthesize the sterol, it depends on mammals for the same. Mosquitoes take the sterol from the plant decays during their larval stage in the form of phytosterol, which is then converted to cholesterol for further growth and reproduction. This conversion occurs with the help of the sterol carrier protein 2(SCP2). Methods: Mosquito populations are controlled by plant-based inhibitors, which inhibit sterol carrier protein (SCPI-Sterol carrier protein inhibitor) activity. In this article, we explain the methods of inhibiting Aedes aegypti SCP2 by insilico methods including natural inhibitor selection and filtrations by virtual screening and interaction studies. Results: In this study protein-ligand interactions were carried out with various phytochemicals, as a result of virtual screening Alpha-mangostin and Panthenol were found to be good analogs, and were allowed to dock with the mosquito cholesterol carrier protein AeSCP-2. Conclusion: Computational selections of SCPIs are highly reliable and novel methods for discovering new and more effective compounds to control mosquitoes. PMID:21808576

  6. Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

    PubMed

    Khadka, Manoj; Salem, Mohamed; Leblond, Jeffrey D

    2015-01-01

    Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3β-ol, and a minor unknown sterol which appeared to be a C(28:4) compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted. PMID:25996517

  7. Assessment of anthropogenic contamination with sterol markers in surface sediments of a tropical estuary (Itajaí-Açu, Brazil).

    PubMed

    Frena, Morgana; Bataglion, Giovana A; Tonietto, Alessandra E; Eberlin, Marcos N; Alexandre, Marcelo R; Madureira, Luiz A S

    2016-02-15

    The Itajaí-Açu estuarine region is one of the most important estuarine systems of south Brazil, due to the location of the Itajaí Harbor, which is the major route of international trading of the state and the largest national fishing pole landing. In addition, industries as well as urban and tourism activities are potential sources of pollution in this area. In the present study, sediment samples from 12 stations along the estuarine system were collected and extracted followed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis. Eight sterols were identified and quantified, indicating natural and anthropogenic sources. Coprostanol concentrations ranged from <4 up to 8930 ng g(-1) of dry weight sediment with higher values being observed in the area next to the Itajaí Harbor and under influence of Itajaí-Mirim River flow, which receives wastewater from several cities. Concentrations and selected sterol ratios were useful tools used to distinguish anthropogenic and biogenic organic matter (OM) sources in the studied area, where coprostanol concentrations higher than 500 ng g(-1) were observed in 42% of the stations analyzed, indicating strong sewage contamination. Factor analysis with principal component analysis (FA/PCA) has distinguished two different groups of samples, with high and low total sterol concentrations. FA/PCA results revealed that the stations located in the estuary were separated by PC1 because they are clearly contaminated by sewage, also pointed by coprostanol/(coprostanol+cholestanol) and coprostanol/cholesterol ratios and by the higher concentrations of fecal sterols. PMID:26657388

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

    PubMed Central

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

    2013-01-01

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

  9. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  10. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  11. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  12. The Role of fadD19 and echA19 in Sterol Side Chain Degradation by Mycobacterium smegmatis.

    PubMed

    Wrońska, Natalia; Brzostek, Anna; Szewczyk, Rafał; Soboń, Adrian; Dziadek, Jarosław; Lisowska, Katarzyna

    2016-01-01

    Mycobacteria are able to degrade natural sterols and use them as a source of carbon and energy. Several genes which play an important role in cholesterol ring degradation have been described in Mycobacterium smegmatis. However, there are limited data describing the molecular mechanism of the aliphatic side chain degradation by Mycobacterium spp. In this paper, we analyzed the role of the echA19 and fadD19 genes in the degradation process of the side chain of cholesterol and β-sitosterol. We demonstrated that the M. smegmatis fadD19 and echA19 genes are not essential for viability. FadD19 is required in the initial step of the biodegradation of C-24 branched sterol side chains in Mycobacterium smegmatis mc²155, but not those carrying a straight chain like cholesterol. Additionally, we have shown that echA19 is not essential in the degradation of either substrate. This is the first report, to our knowledge, on the molecular characterization of the genes playing an essential role in C-24 branched side chain sterol degradation in M. smegmatis mc²155. PMID:27164074

  13. [Determination of β-sitosterol and total sterols content and antioxidant activity of oil in acai (Euterpe oleracea)].

    PubMed

    He, Cheng; Li, Wei; Zhang, Jian-Jun; Qu, Sheng-Sheng; Li, Jia-Jing; Wang, Lin-Yuan

    2014-12-01

    In order to establish a method for the determination of the sterols of the oil in the freeze-dried acai (Euterpe oleracea Mart.) and to evaluate its antioxidant activities, a saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for the analysis of phytosterols in PEE (Petroleum ether extract). Separation was achieved on a Purosper STAR LP C18 column with a binary, gradient solvent system of acetonitrile and isopropanol. Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol and the total sterols. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (r = 0.999 2) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. The highest content of total sterols is β-sitosterol. The antioxidant activities of the extracts were evaluated using the total oxyradical scavenging capacity assay (TOSC assay). The result showed that the PEE exhibited significant antioxidant properties, sample concentration and the antioxidant capacity had a certain relevance. PMID:25911812

  14. Application of pressurized fluid extraction technique in the gas chromatography-mass spectrometry determination of sterols from marine sediment samples.

    PubMed

    Li, Donghao; Dong, Meihua; Shim, Won Joon; Kannan, Narayanan

    2007-08-10

    In order to determine steroid compounds in GC/MS an analytical method using pressurized fluid extraction (PFE) was developed. While extracting in-house reference material (coastal sediment) typical recovery in PFE ranged from 80 to 120% (+/-2.5-14.5) and the average extraction yield in PFE in comparison to conventional soxhlet extraction was 115%. In particular, the PFE showed higher extraction efficiency for C29 and dien sterols. Optimizing parameters such as temperature and pressure is critical in achieving this efficiency. Sterols in the sediment were derivatized with silyl reagent BSTFA in acetone for the final determination. A short column florisil cleanup offered the best separation of the GC/MS sensitive derivatives from co-contaminants. Thirty-three coastal sediment samples were analyzed using PFE and Soxhlet extraction methods. The results on extraction efficiency, silyl derivatization kinetics and purification efficiency demonstrated that PFE is far superior in extracting sterols from sediment samples. It is simple, fast, efficient and amenable for automation. PMID:17540389

  15. Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport

    PubMed Central

    Georgiev, Alexander G.; Johansen, Jesper; Ramanathan, Vidhya D.; Sere, Yves Y.; Beh, Christopher T.; Menon, Anant K.

    2013-01-01

    The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis. PMID:23668914

  16. Inhibitory effects of various oxygenated sterols on the differentiation and function of tumor-specific cytotoxic T lymphocytes

    SciTech Connect

    Spangrude, G.J.; Sherris, D.; Daynes, R.A.

    1982-05-01

    Irradiation of skin with ultraviolet light (UVL) is capable of causing many biological and biochemical changes in this complex organ. One early consequence is the oxidation of epidermal plasma membrane cholesterol, causing the induction of a wide variety of photoproducts. It is well recognized that some oxygenated sterols possess potent biological activity on mammalian cells by their ability to inhibit endogeneous mevalonate and cholesterol biosynthesis. In the few immunological systems that have been studied, there is general agreement that lymphocyte function is altered in the presence of certain oxygenated sterols. Insight into the biochemical basis for altered lymphocyte function is lacking, as both afferent and efferent blockades have been suggested. These studies were undertaken to determine the effect of various oxygenated sterols (representing a number of known cholesterol-derived photoproducts) on the generation (afferent) and function (efferent) of cytotoxic T lymphocytes (CTLs). Cell-mediated immune responses which result in the generation of both alloantigen-specific and syngeneic tumor-specific CTLs were evaluated. (JMT)

  17. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  18. Shaft encoder presents digital output

    NASA Technical Reports Server (NTRS)

    Hillis, D. A.

    1966-01-01

    Circuits that include compensation circuitry time a capacitance relative to a reference voltage so that a digital presentation occurs that is representative of the positional condition of the mechanical shaft being monitored. This circuitry may be employed in multiples to furnish binary encoding of a number of rotating devices simultaneously.

  19. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  20. Transcriptional Regulatory Elements in Fungal Secondary Metabolism

    PubMed Central

    Yin, Wenbing; Keller, Nancy P.

    2013-01-01

    Filamentous fungi produce a variety of secondary metabolites of diverse beneficial and detrimental activities to humankind. The genes encoding the enzymatic machinery required to make these metabolites are typically clustered in fungal genomes. There is considerable evidence that secondary metabolite gene regulation is, in part, by transcriptional control through hierarchical levels of transcriptional regulatory elements involved in secondary metabolite cluster regulation. Identification of secondary metabolism regulatory elements could potentially provide a means of increasing production of beneficial metabolites, decreasing production of detrimental metabolites, aid in the identification of ‘silent’ natural products and also contribute to a broader understanding of molecular mechanisms by which secondary metabolites are produced. This review summarizes regulation of secondary metabolism associated on transcriptional regulatory elements from a broad view as well as tremendous advances in discovery of cryptic or novel secondary metabolites by genomic mining in the basis of this knowledge. PMID:21717315

  1. Sterol and steroid catabolites from cholesterol produced by the psychrophile Pseudoalteromonas haloplanktis.

    PubMed

    Gelzo, Monica; Lamberti, Anna; Spano, Giuseppe; Dello Russo, Antonio; Corso, Gaetano; Masullo, Mariorosario

    2014-09-01

    Pseudoalteromonas haloplanktis, a psychrotrophilic marine bacterium of biotechnological interest, shows anti-biofilm properties and is particularly relevant for cold storage of vacuum packed seafood. We focused our interest on the activation of cholesterol metabolism in this bacterium as the presence in its genome of a putative 3-ketosteroid-Δ(1) -dehydrogenase. This study reports GC-MS and LC-MS/MS profiles of sterols/steroids and their derivatives found in cell extracts of P. haloplanktis grown in a medium with a low content of cholesterol. Here, for the first time, we suggest that P. haloplanktis produces some intermediates of cholesterol catabolism, putatively identified as 24-hydroxycholest-1,4-dien-3-one-26-oic acid, chol-1,4-dien-3-one-24-oic acid, 26-hydroxycholest-4-en-3-one, and pregn-4-en-3-one-20-carboxylic acid, a finding already reported in other microorganisms. The presence of these compounds, also considered steroid precursors, produced by P. haloplanktis in vacuum packed seafood could be of interest for healthy of consumers, as well as, for biotechnological applications in pharmaceutical industry. PMID:25230192

  2. Dynamics of sterols and fatty acids during UV-B treatment of oyster mushroom.

    PubMed

    Krings, Ulrich; Berger, Ralf G

    2014-04-15

    Fruiting bodies of the oyster mushroom Pleurotus ostreatus were illuminated with UV-B with a light intensity maximum at 310-320 nm and 11.5 W/m² for 60 min at 20 °C. Changes of the sterol and fatty acid spectrum were quantified. The onset of ergocalciferol (vitamin D₂) formation was immediate in fruiting bodies illuminated from the lamella side, in sliced fruiting bodies, and in the stipes. Saturation concentrations above 100 μg/g of dry matter were reached after 1h. At the same time, the concentrations of the photo-isomers lumisterol₂, tachysterol₂ and previtamin D₂ increased in this order. 22-Dihydroergocalciferol (vitamin D₄), showed the same course of increase and reached a maximum concentration of around 20 μg/g dry matter. With the exception of linoleic acid in cut fruiting bodies, fatty acid concentrations remained almost constant. One serving of UV-B pretreated sliced oyster mushroom covered the weekly demand of vitamin D of an adult. PMID:24295670

  3. Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.

    PubMed

    Zhang, M; Dwyer, N K; Neufeld, E B; Love, D C; Cooney, A; Comly, M; Patel, S; Watari, H; Strauss, J F; Pentchev, P G; Hanover, J A; Blanchette-Mackie, E J

    2001-02-01

    The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol. PMID:11032830

  4. Effects of heme oxygenase-1 expression on sterol homeostasis in rat astroglia.

    PubMed

    Vaya, Jacob; Song, Wei; Khatib, Soliman; Geng, Guoyan; Schipper, Hyman M

    2007-03-15

    Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Central oxidation of cholesterol to oxysterols has been implicated in neuroembryogenesis, synaptic plasticity, and membrane repair. In the current study, we demonstrated that transient transfection of rat astroglia with human (h)ho-1 cDNA for 3 days significantly decreased intracellular cholesterol concentrations and increased levels of four oxysterol species (measured by GC/MS) compared to untreated control cultures and HO-1-transfected cells exposed to the HO inhibitor, tin mesoporphyrin (SnMP). Relative to control preparations, oxidative stress was augmented in mitochondria (isolated by subcellular fractionation) and culture media derived from HO-1-transfected astrocytes, as evidenced by enhanced oxidation of the synthetic reporter molecules, linoleoyl tyrosine (LT), linoleoyl tyrosine cholesterol ester (LTC), or linoleoyl tyrosine deoxyguanosyl ester (LTG; measured by GC/MS and LC/MS/MS). We also observed enhanced oxidation of exogenous LTC in human neuroblastoma (M17) cells exposed for 18 h to conditioned media collected from HO-1-transfected astrocytes relative to control media. In AD and other pathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g., beta-amyloid) into altered patterns of glial sterol metabolism which, in turn, may affect neuronal membrane turnover, survival, and adaptability. PMID:17320768

  5. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    SciTech Connect

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  6. Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Stan-Lotter, Helga; Kato, Katharine; Hochstein, Lawrence I.

    1992-01-01

    Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.18 g ml (exp -1), respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23-1.24 g ml (exp -1)) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in borh the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

  7. Bending elasticities of model membranes: influences of temperature and sterol content.

    PubMed Central

    Méléard, P; Gerbeaud, C; Pott, T; Fernandez-Puente, L; Bivas, I; Mitov, M D; Dufourcq, J; Bothorel, P

    1997-01-01

    Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes. Images FIGURE 1 FIGURE 7 PMID:9168037

  8. A reanalysis of mouse ENCODE comparative gene expression data

    PubMed Central

    Gilad, Yoav; Mizrahi-Man, Orna

    2015-01-01

    Recently, the Mouse ENCODE Consortium reported that comparative gene expression data from human and mouse tend to cluster more by species rather than by tissue. This observation was surprising, as it contradicted much of the comparative gene regulatory data collected previously, as well as the common notion that major developmental pathways are highly conserved across a wide range of species, in particular across mammals. Here we show that the Mouse ENCODE gene expression data were collected using a flawed study design, which confounded sequencing batch (namely, the assignment of samples to sequencing flowcells and lanes) with species. When we account for the batch effect, the corrected comparative gene expression data from human and mouse tend to cluster by tissue, not by species. PMID:26236466

  9. 3 CFR - Regulatory Review

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 3 The President 1 2010-01-01 2010-01-01 false Regulatory Review Presidential Documents Other Presidential Documents Memorandum of January 30, 2009 Regulatory Review Memorandum for the Heads of Executive Departments and Agencies For well over two decades, the Office of Information and Regulatory Affairs (OIRA) at the Office of Management...

  10. Regulatory affairs administration as regulatory policy determinant

    SciTech Connect

    Forcier, J.R.

    1984-05-10

    It is the thesis of this article that the processing of a utility company's regulation-related work, the supporting tasks and the manner in which they are completed, can and does have a significant impact on the final results or work product of the regulatory affairs function, including even, potentially, the action of the regulatory agency. The article is therefore full of practical pointers on how the interface with the regulatory authority can best be organized, managed, and carried through to the attainment of optimum results for the utility. 2 references.

  11. Monolithic-integrated microlaser encoder.

    PubMed

    Sawada, R; Higurashi, E; Ito, T; Ohguchi, O; Tsubamoto, M

    1999-11-20

    We have developed an extremely small integrated microencoder whose sides are less than 1 mm long. It is 1/100 the size of conventional encoders. This microencoder consists of a laser diode, monolithic photodiodes, and fluorinated polyimide waveguides with total internal reflection mirrors. The instrument can measure the relative displacement between a grating scale and the encoder with a resolution of the order of 0.01 microm; it can also determine the direction in which the scale is moving. By using the two beams that were emitted from the two etched mirrors of the laser diode, by monolithic integration of the waveguide and photodiodes, and by fabrication of a step at the edge of the waveguide, we were able to eliminate conventional bulky optical components such as the beam splitter, the quarter-wavelength plate, bulky mirrors, and bulky photodetectors. PMID:18324228