WorldWideScience

Sample records for cell sphingolipid biosynthesis

  1. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

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    Fikadu G Tafesse

    2015-10-01

    Full Text Available The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  2. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

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    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D

    2016-01-29

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  3. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  4. Sphingolipid trafficking and protein sorting in epithelial cells

    NARCIS (Netherlands)

    Slimane, TA; Hoekstra, D

    2002-01-01

    Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asy

  5. Inhibition of Sphingolipid Metabolism Enhances Resveratrol Chemotherapy in Human Gastric Cancer Cells

    OpenAIRE

    Shin, Kyong-Oh; Park, Nam-Young; Seo, Cho-hee; Hong, Seon-Pyo; Oh, Ki-Wan; Hong, Jin-Tae; Han, Sang-Kil; Lee, Yong-Moon

    2012-01-01

    Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol (100 μM) for 24 hr induced...

  6. A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation.

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    Lihong Zhao

    2011-05-01

    Full Text Available Sphingolipids, lipids with a common sphingoid base (also termed long chain base backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln and toppler (to, with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydroceramide synthase 1 (CerS1, which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases.

  7. Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs.

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    Toshiyuki Yamaji

    Full Text Available Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs. A TALEN pair targeting the human CERT gene (alternative name COL4A3BP encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase, and B4GalT5 (encoding the major lactosylceramide synthase, and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

  8. Roles of Sphingolipid Metabolism in Pancreatic β Cell Dysfunction Induced by Lipotoxicity

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    Julien Véret

    2014-06-01

    Full Text Available Pancreatic β cells secrete insulin in order to maintain glucose homeostasis. However, various environmental stresses such as obesity have been shown to induce loss of secretory responsiveness in pancreatic β cells and pancreatic β cell apoptosis which can favor the development of type 2 diabetes (T2D. Indeed, elevated levels of free fatty acids (FFAs have been shown to induce β cell apoptosis. Importantly, the chronic adverse effects of FFAs on β cell function and viability are potentiated in the presence of hyperglycaemia, a phenomenon that has been termed gluco-lipotoxicity. The molecular mechanisms underlying the pathogenesis of gluco-lipotoxicity in pancreatic β cells are not completely understood. Recent studies have shown that sphingolipid metabolism plays a key role in gluco-lipotoxicity induced apoptosis and loss of function of pancreatic β cells. The present review focuses on how the two main sphingolipid mediators, ceramides and sphingoid base-1-phosphates, regulate the deleterious effects of gluco-lipotoxicity on pancreatic β cells. The review highlights the role of a sphingolipid biostat on the dysregulation of β cell fate and function induced by gluco-lipotoxicity, offering the possibility of new therapeutic targets to prevent the onset of T2D.

  9. Inhibition of sphingolipid metabolism enhances resveratrol chemotherapy in human gastric cancer cells.

    Science.gov (United States)

    Shin, Kyong-Oh; Park, Nam-Young; Seo, Cho-Hee; Hong, Seon-Pyo; Oh, Ki-Wan; Hong, Jin-Tae; Han, Sang-Kil; Lee, Yong-Moon

    2012-09-01

    Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol (100 μM) for 24 hr induced cell death and cell cycle arrest in gastric cancer cells. Exposure to the combination of resveratrol and dimethylsphingosine (DMS) increased cytotoxicity, demonstrating that sphingolipid metabolites intensify resveratrol activity. Specifically, DHCer accumulated in a resveratrol concentration-dependent manner in SNU-1 and HT-29 cells, but not in SNU-668 cells. LC-MS/MS analysis showed that specific DHCer species containing C24:0, C16:0, C24:1, and C22:0 fatty acids chain were increased by up to 30-fold by resveratrol, indicating that resveratrol may partially inhibit DHCer desaturase. Indeed, resveratrol mildly inhibited DHCer desaturase activity compared to the specific inhibitor GT-11 or to retinamide (4-HPR); however, in SNU-1 cells resveratrol alone exhibited a typical cell cycle arrest pattern, which GT-11 did not alter, indicating that inhibition of DHCer desaturase is not essential to the cytotoxicity induced by the combination of resveratrol and sphingolipid metabolites. Resveratrol-induced p53 expression strongly correlated with the enhancement of cytotoxicity observed upon combination of resveratrol with DMS or 4-HPR. Taken together, these results show that DHCer accumulation is a novel lipid biomarker of resveratrol-induced cytotoxicity in human gastric cancer cells. PMID:24009836

  10. Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease

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    Bryan, Phillips-Farfán; Edgar Alejandro, Medina-Torres; Sara Elva, Espinosa-Padilla; Gemma, Fabrias

    2016-01-01

    Inflammatory bowel disease (IBD) describes different illnesses characterized by chronic inflammation of the gastrointestinal tract. Although the pathogenic mechanisms leading to IBD are poorly understood, immune system disturbances likely underlie its development. Sphingolipids (SLs) have been identified as important players and promising therapeutic targets to control inflammation in IBD. Interestingly, it seems that microorganisms of the normal gut microbiota and probiotics are involved in sphingolipid function. However, there is a great need to investigate the role of SLs as intermediates in the crosstalk between intestinal immunity and microorganisms. This review focuses on recent investigations that describe some mechanisms involved in the regulation of cytokine profiles by SLs. We also describe the importance of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells. This, in turn, modulates the immune response in the bowel and likely in other peripheral organs. The potential of SLs and gut microbiota as targets or therapeutic agents for IBD is also discussed. PMID:27656050

  11. A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells.

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    Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle

    2016-05-01

    Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells. PMID:27036914

  12. Sphingolipids, rafts and multidrug resistance

    NARCIS (Netherlands)

    Hinrichs, Joann Wilhelm Jakob

    2004-01-01

    The main goal of the research described in this thesis was to obtain more insight into the potential role of sphingolipids in multidrug resistance (MDR) of tumor cells. The approachinvolved the monitoring of sphingolipid dynamics in terms of metabolism and localization in relation to the acquisition

  13. Plant sphingolipids: Their importance in cellular organization and adaption.

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    Michaelson, Louise V; Napier, Johnathan A; Molino, Diana; Faure, Jean-Denis

    2016-09-01

    Sphingolipids and their phosphorylated derivatives are ubiquitous bio-active components of cells. They are structural elements in the lipid bilayer and contribute to the dynamic nature of the membrane. They have been implicated in many cellular processes in yeast and animal cells, including aspects of signaling, apoptosis, and senescence. Although sphingolipids have a better defined role in animal systems, they have been shown to be central to many essential processes in plants including but not limited to, pollen development, signal transduction and in the response to biotic and abiotic stress. A fuller understanding of the roles of sphingolipids within plants has been facilitated by classical biochemical studies and the identification of mutants of model species. Recently the development of powerful mass spectrometry techniques hailed the advent of the emerging field of lipidomics enabling more accurate sphingolipid detection and quantitation. This review will consider plant sphingolipid biosynthesis and function in the context of these new developments. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27086144

  14. Sphingolipids, rafts and multidrug resistance

    OpenAIRE

    Hinrichs, Joann Wilhelm Jakob

    2004-01-01

    The main goal of the research described in this thesis was to obtain more insight into the potential role of sphingolipids in multidrug resistance (MDR) of tumor cells. The approachinvolved the monitoring of sphingolipid dynamics in terms of metabolism and localization in relation to the acquisition of MDR and the expression of ATP-binding cassette (ABC) transporters involved in drug efflux. This research was conducted in two model cell lines, both human MDR cancer cells, over expressing eith...

  15. Genetic determinants of circulating sphingolipid concentrations in European populations.

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    Andrew A Hicks

    2009-10-01

    Full Text Available Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI, cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS between 318,237 single-nucleotide polymorphisms (SNPs and levels of circulating sphingomyelin (SM, dihydrosphingomyelin (Dih-SM, ceramide (Cer, and glucosylceramide (GluCer single lipid species (33 traits; and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32 in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08x10(-66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3 associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4 or less. Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be

  16. Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins.

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    Yamaji, Toshiyuki; Hanada, Kentaro

    2015-02-01

    In recent decades, many sphingolipid enzymes, sphingolipid-metabolism regulators and sphingolipid transfer proteins have been isolated and characterized. This review will provide an overview of the intracellular localization and topology of sphingolipid enzymes in mammalian cells to highlight the locations where respective sphingolipid species are produced. Interestingly, three sphingolipids that reside or are synthesized in cytosolic leaflets of membranes (ceramide, glucosylceramide and ceramide-1-phosphate) all have cytosolic lipid transfer proteins (LTPs). These LTPs consist of ceramide transfer protein (CERT), four-phosphate adaptor protein 2 (FAPP2) and ceramide-1-phosphate transfer protein (CPTP), respectively. These LTPs execute functions that affect both the location and metabolism of the lipids they bind. Molecular details describing the mechanisms of regulation of LTPs continue to emerge and reveal a number of critical processes, including competing phosphorylation and dephosphorylation reactions and binding interactions with regulatory proteins and lipids that influence the transport, organelle distribution and metabolism of sphingolipids. PMID:25382749

  17. Assessment of crosstalks between the Snf1 kinase complex and sphingolipid metabolism in S. cerevisiae via systems biology approaches.

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    Borklu Yucel, Esra; Ulgen, Kutlu O

    2013-11-01

    Sphingolipids are essential building blocks of the plasma membranes and are highly bioactive in the regulation of diverse cellular functions and pathological processes, a fact which renders the sphingolipid metabolism an important research area. In this study, a computational framework was recruited for the reconstruction of a functional interaction network for sphingolipid metabolism in Baker's yeast, SSN. Gene Ontology (GO) annotations were integrated with functional interaction data of the BIOGRID database and the reconstructed protein interaction network was subjected to topological and descriptive analyses. SSN was of a scale-free nature, following a power law model with γ=1.41. Prominent processes of SSN revealed that the reconstructed network encapsulated the involvement of sphingolipid metabolism in vital cellular processes such as energy homeostasis, cell growth and/or death and synthesis of building blocks. To investigate the potential of SSN for predicting signal transduction pathways regulating and/or being regulated by sphingolipid biosynthesis in yeast, a case study involving the S. cerevisiae counterpart of AMP-activated protein kinase, the Snf1 kinase complex, was conducted. The mutant strain lacking the catalytic α subunit, snf1Δ/snf1Δ, had elevated inositol phosphorylceramide and mannosyl-inositol phosphorylceramide levels, and decreased mannosyl-diinositol phosphorylceramide levels compared to the wild type strain, revealing that Snf1p has a regulatory role in the sphingolipid metabolism. Transcriptome data belonging to that strain available in the literature were mapped onto SSN and the correlated SSN was further investigated to evaluate the possible crosstalk machineries where sphingolipids and Snf1p function in coordination, in other words the crosstalk points between sphingolipid-mediated and Snf1 kinase signalling. The subsequent investigation of the discovered candidate crosstalk processes by performing sensitivity experiments imply a

  18. Downregulation of the autophagy protein ATG-7 correlates with distinct sphingolipid profile in MCF-7 cells sensitized to photodamage

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    Separovic, Duska; Kelekar, Ameeta; Tarca, Adi L.; Bielawski, Jacek; Kessel, David

    2009-06-01

    The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h) and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced, whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.

  19. Targeting Cell Membrane Lipid Rafts by Stoichiometric Functionalization of Gold Nanoparticles With a Sphingolipid-Binding Domain Peptide.

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    Paramelle, David; Nieves, Daniel; Brun, Benjamin; Kraut, Rachel S; Fernig, David G

    2015-04-22

    A non-membrane protein-based nanoparticle agent for the tracking of lipid rafts on live cells is produced by stoichiometric functionalization of gold nanoparticles with a previously characterized sphingolipid- and cell membrane microdomain-binding domain peptide (SBD). The SBD peptide is inserted in a self-assembled monolayer of peptidol and alkane thiol ethylene glycol, on gold nanoparticles surface. The stoichiometric functionalization of nanoparticles with the SBD peptide, essential for single molecule tracking, is achieved by means of non-affinity nanoparticle purification. The SBD-nanoparticles have remarkable long-term resistance to electrolyte-induced aggregation and ligand-exchange and have no detectable non-specific binding to live cells. Binding and diffusion of SBD-nanoparticles bound to the membrane of live cells is measured by real-time photothermal microscopy and shows the dynamics of sphingolipid-enriched microdomains on cells membrane, with evidence for clustering, splitting, and diffusion over time of the SBD-nanoparticle labeled membrane domains. The monofunctionalized SBD-nanoparticle is a promising targeting agent for the tracking of lipid rafts independently of their protein composition and the labelling requires no prior modification of the cells. This approach has potential for further functionalization of the particles to manipulate the organization of, or targeting to microdomains that control signaling events and thereby lead to novel diagnostics and therapeutics.

  20. Sphingolipid topology and membrane protein nanoclusters

    NARCIS (Netherlands)

    Hötzl, S.

    2009-01-01

    Sphingolipids are an essential class of membrane lipids in eukaryotic cells. Due to their high packing density and their affinity for cholesterol, sphingolipids are able to promote bilayer rigidity and impermeability. Apart from its ability to maintain biomembrane integrity, sphingomyelin (SM) is al

  1. Moss cell walls: structure and biosynthesis

    OpenAIRE

    Alison W. Roberts; Eric M Roberts; Haigler, Candace H.

    2012-01-01

    The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperm...

  2. Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

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    Toume, Moeko; Tani, Motohiro

    2016-08-01

    Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells. PMID:27312128

  3. Orm family proteins mediate sphingolipid homeostasis

    DEFF Research Database (Denmark)

    Breslow, David K; Collins, Sean R; Bodenmiller, Bernd;

    2010-01-01

    Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes...... in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form...... a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression...

  4. Dictyostelium discoideum to human cells: pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance.

    Science.gov (United States)

    Alexander, Stephen; Min, Junxia; Alexander, Hannah

    2006-03-01

    Resistance to chemotherapy is a major obstacle for the treatment of cancer and a subject of extensive research. Numerous mechanisms of drug resistance have been proposed, and they differ for different drugs. Nevertheless, it is clear that our understanding of this important problem is still incomplete, and that new targets for modulating therapy still await discovery. The attractive biology and the availability of powerful molecular techniques have made the cellular slime mold Dictyostelium discoideum, a powerful non-mammalian model for drug target discovery, and the problem of drug resistance. To understand the molecular basis of chemoresistance to the widely used drug cisplatin, both genetic and pharmacological approaches have been applied to this versatile experimental system. These studies have resulted in the identification of novel molecular pathways which can be used to increase the efficacy of cisplatin, and brought attention to the role of sphingolipids in mediating the cellular response to chemotherapeutic drugs. In the following review, we will describe the history and utility of D. discoideum in pharmacogenetics, and discuss recent studies which focus attention on the role of sphingolipids in chemotherapy and chemoresistance. PMID:16403600

  5. Sphingolipids in human synovial fluid--a lipidomic study.

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    Marta Krystyna Kosinska

    Full Text Available Articular synovial fluid (SF is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides and minor glycerophospholipid species, including (lysophosphatidic acid, (lysophosphatidylglycerol, and bis(monoacylglycerophosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA and late (lOA stages of osteoarthritis (OA, and rheumatoid arthritis (RA. SF without cells and cellular debris from 9 postmortem donors (control, 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry--directly or coupled with hydrophilic interaction liquid chromatography. We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF. The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases.

  6. Yeast cells lacking all known ceramide synthases continue to make complex sphingolipids and to incorporate ceramides into glycosylphosphatidylinositol (GPI) anchors

    DEFF Research Database (Denmark)

    Vionnet, Christine; Roubaty, Carole; Ejsing, Christer S.;

    2010-01-01

    In yeast, the inositolphosphorylceramides mostly contain C26:0 fatty acids. Inositolphosphorylceramides were considered to be important for viability, since the inositolphosphorylceramide synthase AUR1 is essential. Yet, lcb1 cells, unable to make sphingoid bases and inositolphosphorylceramides, ......, are viable if they harbor SLC1-1, a gain of function mutation in the 1-acyl-glycerol-3-phosphate acyltransferase SLC1. SLC1-1 allows to incorporate C26:0 fatty acids into phosphatidylinositol (PI), thus generating PIii, an abnormal, C26-containing PI, presumably acting as surrogate...... genetic backgrounds but to still make some abnormal uncharacterized inositol-containing sphingolipids. Indeed, we find that 4 quadruple mutants make substantial amounts of unphysiological inositolphosphorylphytosphingosines but that they also still make small amounts of normal inositolphosphorylceramides...

  7. Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides

    DEFF Research Database (Denmark)

    Cerantola, Vanessa; Guillas, Isabelle; Roubaty, Carole;

    2009-01-01

    require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Delta.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells...

  8. Sphingolipid and Ceramide Homeostasis: Potential Therapeutic Targets

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    Simon A. Young

    2012-01-01

    Full Text Available Sphingolipids are ubiquitous in eukaryotic cells where they have been attributed a plethora of functions from the formation of structural domains to polarized cellular trafficking and signal transduction. Recent research has identified and characterised many of the key enzymes involved in sphingolipid metabolism and this has led to a heightened interest in the possibility of targeting these processes for therapies against cancers, Alzheimer's disease, and numerous important human pathogens. In this paper we outline the major pathways in eukaryotic sphingolipid metabolism and discuss these in relation to disease and therapy for both chronic and infectious conditions.

  9. Stoichiometric network reconstruction and analysis of yeast sphingolipid metabolism incorporating different states of hydroxylation.

    Science.gov (United States)

    Kavun Ozbayraktar, Fatma Betul; Ulgen, Kutlu O

    2011-04-01

    The first elaborate metabolic model of Saccharomyces cerevisiae sphingolipid metabolism was reconstructed in silico. The model considers five different states of sphingolipid hydroxylation, rendering it unique among other models. It is aimed to clarify the significance of hydroxylation on sphingolipids and hence to interpret the preferences of the cell between different metabolic pathway branches under different stress conditions. The newly constructed model was validated by single, double and triple gene deletions with experimentally verified phenotypes. Calcium sensitivity and deletion mutations that may suppress calcium sensitivity were examined by CSG1 and CSG2 related deletions. The model enabled the analysis of complex sphingolipid content of the plasma membrane coupled with diacylglycerol and phosphatidic acid biosynthesis and ATP consumption in in silico cell. The flux data belonging to these critically important key metabolites are integrated with the fact of phytoceramide induced cell death to propose novel potential drug targets for cancer therapeutics. In conclusion, we propose that IPT1, GDA1, CSG and AUR1 gene deletions may be novel candidates of drug targets for cancer therapy according to the results of flux balance and variability analyses coupled with robustness analysis.

  10. Isolation and Functional Characterisation of the Genes Encoding △8-Sphingolipid Desaturase from Brassica rapa

    Institute of Scientific and Technical Information of China (English)

    Shu-Fen Li; Li-Ying Song; Wei-Bo Yin; Yu-Hong Chen; Liang Chen; Ji-Lin Li; Richard R.-C. Wang; Zan-Min Hu

    2012-01-01

    △8-Sphingolipid desaturase is the key enzyme that catalyses desaturation at the C8 position of the long-chain base of sphingolipids in higher plants.There have been no previous studies on the genes encoding △8-sphingolipid desaturases in Brassica rapa.In this study,four genes encoding △8-sphingolipid desaturases from B.rapa were isolated and characterised.Phylogenetic analyses indicated that these genes could be divided into two groups:BrD8A,BrD8C and BrD8D in group Ⅰ,and BrD8B in group Ⅱ.The two groups of genes diverged before the separation of Arabidopsis and Brassica.Though the four genes shared a high sequence similarity,and their coding desaturases all located in endoplasmic reticulum,they exhibited distinct expression patterns.Heterologous expression in Saccharomyces cerevisiae revealed that BrD8A/B/C/D were functionally diverse △8-sphingolipid desaturases that catalyse different ratios of the two products 8(Z)- and 8(E)-C18-phytosphingenine.The aluminium tolerance of transgenic yeasts expressing BrD8A/B/C/D was enhanced compared with that of control cells.Expression of BrD8A in A rabidopsis changed the ratio of 8(Z):8(E)-C 18-phytosphingenine in transgenic plants.The information reported here provides new insights into the biochemical functional diversity and evolutionary relationship of △8-sphingolipid desaturase in plants and lays a foundation for further investigation of the mechanism of 8(Z)- and 8(E)-C18-phytosphingenine biosynthesis.

  11. Nuclear Sphingolipid Metabolism

    Science.gov (United States)

    Lucki, Natasha C.; Sewer, Marion B.

    2014-01-01

    Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane–associated ganglioside GM1 plays a pivotal role in Ca2+ homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes. PMID:21888508

  12. The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma.

    Science.gov (United States)

    Paulenda, T; Draber, P

    2016-07-01

    A family of widely expressed ORM-like (ORMDL) proteins has been recently linked to asthma in genomewide association studies in humans and extensively explored in in vivo studies in mice. ORMDL proteins are key regulators of serine palmitoyltransferase, an enzyme catalyzing the initial step of sphingolipid biosynthesis. Sphingolipids play prominent roles in cell signaling and response to stress, and they affect the mechanistic properties of cellular membranes. Deregulation of sphingolipid biosynthesis and their recycling has been proven to support and even cause several diseases including allergy, inflammation, and asthma. ORMDL3, the most extensively studied member of the ORMDL family, has been shown to be important for endoplasmic reticulum homeostasis by regulating the unfolded protein response and calcium response. In immune cells, ORMDL3 is involved in migration and in the production of proinflammatory cytokines. Furthermore, changes in the expression level of ORMDL3 are important in allergen-induced asthma pathologies. This review focuses on functional aspects of the ORMDL family proteins, which may serve as new therapeutic targets for the treatment of asthma and some other life-threatening diseases. PMID:26969910

  13. The GARP complex is required for cellular sphingolipid homeostasis

    DEFF Research Database (Denmark)

    Fröhlich, Florian; Petit, Constance; Kory, Nora;

    2015-01-01

    (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation...... the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2....

  14. A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

    OpenAIRE

    Lori Clay; Fabrice Caudron; Annina Denoth-Lippuner; Barbara Boettcher; St\\xfdphanie Buvelot Frei; Erik Lee Snapp; Yves Barral

    2014-01-01

    eLife digest Cell division isn't always about splitting a cell into two identical parts. The diversity of many of our own cells relies on asymmetric cell divisions. The yeast used to make bread rely on a process called ‘budding’ that involves a small daughter cell emerging from the surface of the mother cell. Mother cells can only produce around 20–50 daughter cells before dying from old age. However, their daughters are always born rejuvenated, and not aged like their mothers. Budding involv...

  15. Plant Cell Wall Matrix Polysaccharide Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Ajay Pal S. Sandhu; Gursharn S. Randhawa; Kanwarpal S. Dhugga

    2009-01-01

    The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemi-cellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysacchar-ides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrUs, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Cs/) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incre-mentally unravel the mechanisms of Golgi polysaccharide biosynthesis.

  16. Differential expression of sphingolipids in MRP1 overexpressing HT29 cells

    NARCIS (Netherlands)

    Kok, JW; Veldman, Robert; Klappe, K; Koning, H; Filipeanu, Catalin M.; Muller, Michael

    2000-01-01

    We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistan

  17. A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia▿

    Science.gov (United States)

    Sonda, Sabrina; Štefanić, Saša; Hehl, Adrian B.

    2008-01-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 μM. The inhibition of parasite replication was irreversible at 10 μM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  18. A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

    Science.gov (United States)

    Sonda, Sabrina; Stefanic, Sasa; Hehl, Adrian B

    2008-02-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  19. Polarized sphingolipid transport from the subapical compartment changes during cell polarity development

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Hoekstra, D

    2000-01-01

    The subapical compartment (SAC) plays an important role in the polarized transport of proteins and lipids. In hepatoma-derived HepG2 cells, fluorescent analogues of sphingomyelin and glucosylceramide are sorted in the SAG. Here, evidence is provided that shows that polarity development is regulated

  20. TOR Complex 2-Ypk1 Signaling Maintains Sphingolipid Homeostasis by Sensing and Regulating ROS Accumulation

    Directory of Open Access Journals (Sweden)

    Brad J. Niles

    2014-02-01

    Full Text Available Reactive oxygen species (ROS are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae. We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability.

  1. Analyzing the complex machinery of cell wall biosynthesis

    OpenAIRE

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a highly interesting target of scientific research. In this thesis a protein-protein interaction strategy was used to gain insight in the cell wall biosynthesis of Arabidopsis thaliana and to identif...

  2. Membrane domains and polarized trafficking of sphingolipids

    NARCIS (Netherlands)

    Maier, O; Slimane, TA; Hoekstra, D

    2001-01-01

    The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid-cholesterol enriched membrane microdomains, so called rafts. In

  3. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    Science.gov (United States)

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  4. Vitamin E metabolite 13'-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human cancer cells by modulating sphingolipids and suppress colon tumor development in mice.

    Science.gov (United States)

    Jang, Yumi; Park, Na-Young; Rostgaard-Hansen, Agnetha Linn; Huang, Jianjie; Jiang, Qing

    2016-06-01

    Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13'-COOH and δTE-13'-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOH-induced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents. PMID:27016075

  5. Role of sphingosine kinase localization in sphingolipid signaling

    Institute of Scientific and Technical Information of China (English)

    Binks; W; Wattenberg

    2010-01-01

    The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to theplasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.

  6. Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

    Science.gov (United States)

    Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

    2012-11-01

    Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA. PMID:22898792

  7. Beyond the Cherry-Red Spot: Ocular Manifestations of Sphingolipid-mediated Neurodegenerative and Inflammatory Disorders

    OpenAIRE

    Chen, Hui; Chan, Annie Y.; Stone, Donald U.; Nawajes A Mandal

    2013-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay Sachs or Niemann Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, in...

  8. 7-ketocholesterol incorporation into sphingolipid/cholesterol-enriched (lipid raft) domains is impaired by vitamin E: a specific role for alpha-tocopherol with consequences on cell death.

    Science.gov (United States)

    Royer, Marie-Charlotte; Lemaire-Ewing, Stéphanie; Desrumaux, Catherine; Monier, Serge; Pais de Barros, Jean-Paul; Athias, Anne; Néel, Dominique; Lagrost, Laurent

    2009-06-01

    Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment. PMID:19351882

  9. Analyzing the complex machinery of cell wall biosynthesis

    NARCIS (Netherlands)

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a

  10. The role of sphingolipids in selected cardiovascular diseases

    Directory of Open Access Journals (Sweden)

    Krzysztof Kurek

    2013-09-01

    Full Text Available Bioactive sphingolipids are engaged with numerous cellular processes such as cell differentiation, proliferation and apoptosis. Sphingolipid metabolism in heart is regulated by physical exercise and PPARs. Ceramide, the main second messenger of sphingomyelin pathway of signal transduction, was found to be involved in development of cardiac dysfunction after ischemia/reperfusion. On the other hand ceramide derivative sphingosine- 1- phosphate has been shown to exert potent cardioprotective action and guards cardiomyocytes against ischemic/reperfusion injury. Pharmacological compounds, which regulate metabolism of sphingolipids can be potentially useful in treatment of selected cardiovascular diseases. The aim of this work is critical review of physiological and pathological role of sphingolipids in circulatory system.

  11. Novel Functional Association of Serine Palmitoyltransferase Subunit 1-A Peptide in Sphingolipid Metabolism with Cytochrome P4501A1 Transactivation and Proliferative Capacity of the Human Glioma LN18 Brain Tumor Cell Line

    Directory of Open Access Journals (Sweden)

    J. Stewart

    2006-09-01

    Full Text Available Some chemical modulators of cytochrome P4501A1, Cyp1A1, expression also perturb the activity of serine palmitoyltransferase, SPT, a heterodimeric protein responsible for catalyzing the first reaction in sphingolipid biosynthesis. The effect of altered SPT activity on Cyp1A1 expression has generally been attributed to changes in the composition of bioactive sphingolipids, generated downstream in the SPT metabolic pathway, but the precise mechanism remains poorly defined. A generally accepted model for chemical-induced transactivation of the Cyp1A1 gene involves intracellular signaling mediated by proteins including the arylhydrocarbon receptor, AhR, whose interaction with the 90 kilo Dalton heat shock protein, Hsp90, is essential for maintaining a high affinity ligandbinding receptor conformation. Because ligand-induced Cyp1A1 expression is important in the bioactivation of environmentally relevant compounds to genotoxic derivatives capable of perturbing cellular processes, binding to Hsp90 represents an important regulatory point in the cytotoxicity process. In the present study, based on evidence that indicates subunit 1 of serine palmitoyltransferase, SPT1, interacts with Hsp90, both ligand-induced Cyp1A1 transactivation and capacity for proliferation were evaluated using the wild type Glioma LN18 human brain cancer cell line and its recombinant counterparts expressing green fluorescent SPT1 fusion proteins. Exposure to the prototypical Cyp1A1 inducer, 3-methylcholanthrene, 3-MC, resulted in the translocation of SPT1 from a primarily cytoplasmic domain to sites of focal adhesion complexes. Immunolabel for Hsp90, which was dispersed throughout the cell, became primarily cytoplasmic, while the distribution of AhR remained unaffected. When compared to the wild type, cells transfected with recombinant SPT1-GFP vectors had significantly attenuated levels of 3-MC-induced Cyp1A1 mRNA, as determined by quantitative reverse transcription PCR. Although

  12. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; Olsen, Ole D; Groth-Pedersen, Line;

    2013-01-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destab...

  13. Beyond the cherry-red spot: Ocular manifestations of sphingolipid-mediated neurodegenerative and inflammatory disorders.

    Science.gov (United States)

    Chen, Hui; Chan, Annie Y; Stone, Donald U; Mandal, Nawajes A

    2014-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay-Sachs or Niemann-Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases. PMID:24011710

  14. Liposomal Nanomedicine with Short Chain Sphingolipids Modulate Tumor Cell Membrane Permeability Modulate Tumor Cell Membrane Permeability and Improve Chemotherapy

    NARCIS (Netherlands)

    L.R.C. Pedrosa (Lília R. Cordeiro)

    2014-01-01

    markdownabstract__Abstract__ Chapter 6 discusses the significance of the results described in this thesis and future perspectives. The main goal of the thesis was the application of SCS enriched liposomes to improve chemotherapy outcome, by enhancing drug bioavailability in target tumor cells. De

  15. Sphingolipids: Important Players in Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Ramona Halmer

    2014-06-01

    Full Text Available Multiple Sclerosis (MS is the most common cause for permanent disability in young adults. Current pathophysiological understanding has identified an autoaggressive immune reaction with infiltration of immune cells into the central nervous system and local inflammatory and demyelinating reactions. The current therapy focuses on a modulation or suppression of immune functions. Sphingolipids, main components of nervous tissue, have been linked to MS already 60 years ago with the description of an unusual myelin lipid distribution in diseased patients. There is tremendous information developing on the role of different sphingolipids in MS. Antibodies against sphingomyelin, sulfatide or galacosylceramide have been detected in serum or CSF of MS patients, although up to now, this knowledge did not find its way into clinical use. Ceramide and the enzymes linked to its production have been described to play a pivotal role in oligendrocyte damage and demyelination. Nowadays, especially sphingosine-1-phosphate (S1P is in the focus of pathophysiological research and therapy development. A S1P analogue, FTY720, is a widely distributed therapy against relapsing-remitting MS, attenuating the emigration of activated, autoreactive lymphocytes from lymph nodes, thereby preventing new inflammatory infiltration into the central nervous system. Beside, there is more and more evidence, that especially S1P receptors on oligodendrocytes and astrocytes are involved in demyelination processes and subsequent axonal degeneration, important features of chonic progressive MS disease course. Further information and research on the manifold role of sphingolipids are needed to prepare the ground for further clinical trials. This review focuses on the current knowledge of the role of sphingolipids in MS and describes the current therapeutical implications.

  16. Function of laccases in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Larsen, Anders; Holm, Preben Bach; Andersen, Jeppe Reitan

    2011-01-01

    substrate specificities and expression patterns. As part of the strategic research centre Bio4Bio, the present project deals with laccase functions in relation to cell wall formation in grasses based on a study of the model species Brachypodium distachyon. Thirty-one isozymes have been retrieved from......Laccases are multicopper oxidases capable of polymerizing monolignols. Histochemical assays have shown temporal and spatial correlation with secondary cell wall formation in both herbs and woody perennials. However, in plants laccases constitutes a relatively large group of isoenzymes with unique...... hybridization. Specific isozymes that show high correlation with the process of secondary cell wall formation will be further studied in a reverse genetic study in which candidates will be knocked out using RNA interference. Phenotypes of knock-out mutants are to be described in relation to cell wall...

  17. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  18. Podocyte pathology and nephropathy - sphingolipids in glomerular diseases.

    Science.gov (United States)

    Merscher, Sandra; Fornoni, Alessia

    2014-01-01

    Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay-Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann-Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in post-reperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed. PMID:25126087

  19. The GARP complex is required for cellular sphingolipid homeostasis

    Science.gov (United States)

    Fröhlich, Florian; Petit, Constance; Kory, Nora; Christiano, Romain; Hannibal-Bach, Hans-Kristian; Graham, Morven; Liu, Xinran; Ejsing, Christer S; Farese, Robert V; Walther, Tobias C

    2015-01-01

    Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells. Their levels and localization are tightly regulated. However, the mechanisms underlying this regulation remain largely unknown. In this study, we identify the Golgi-associated retrograde protein (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2. DOI: http://dx.doi.org/10.7554/eLife.08712.001 PMID:26357016

  20. Structure, function, and biosynthesis of plant cell walls: proceedings of the seventh annual symposium in botany

    Energy Technology Data Exchange (ETDEWEB)

    Dugger, W.M.; Bartnicki-Garcia, S. (eds.)

    1984-01-01

    Papers in the following areas were included in these symposium proceedings: (1) cell wall chemistry and biosynthesis; (2) cell wall hydrolysis and associated physiology; (3) cellular events associated with cell wall biosynthesis; and (4) interactions of plant cell walls with pathogens and related responses. Papers have been individually abstracted for the data base. (ACR)

  1. On-Off Switches for Secondary Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Huan-Zhong Wang; Richard A.Dixon

    2012-01-01

    Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport.They also provide textiles,timber,and potentially second-generation biofuels for human use.Genes responsible for synthesis of the different cell wall components,namely cellulose,hemicelluloses,and lignin,are coordinately expressed and under transcriptional regulation.In the past several years,cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis.Positive and negative regulators,which function upstream of NAC master switches,have also been identified in different plant tissues.Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production.

  2. On-off switches for secondary cell wall biosynthesis.

    Science.gov (United States)

    Wang, Huan-Zhong; Dixon, Richard A

    2012-03-01

    Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport. They also provide textiles, timber, and potentially second-generation biofuels for human use. Genes responsible for synthesis of the different cell wall components, namely cellulose, hemicelluloses, and lignin, are coordinately expressed and under transcriptional regulation. In the past several years, cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis. Positive and negative regulators, which function upstream of NAC master switches, have also been identified in different plant tissues. Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production. PMID:22138968

  3. Stereoselective total synthesis of sphingolipids

    Indian Academy of Sciences (India)

    PARAMESH JANGILI; PERLA RAMESH; BISWANATH DAS

    2016-11-01

    A novel sphingosine, 1,2-diacetyl D-erythro-sphinganine having a characteristic almond flavour was isolated from the edible mushroom Grifola gargal. We have synthesized this sphinganine along with the three other sphingolipids, such as 1,2-diacetyl L-threo-sphinganine, D-erythro-sphinganine triacetateand L-threo-sphinganine triacetate using Garner aldehyde as the starting material involving the Grignard reaction and Mitsunobu inversion. The sphingolipids 1,2-diacetyl D-erythro-sphinganine and 1,2-diacetyl L-threo-sphinganine have been synthesized for the first time.

  4. Genetic determinants of circulating sphingolipid concentrations in European populations

    NARCIS (Netherlands)

    A.A. Hicks (Andrew); P.P. Pramstaller (Peter Paul); A. Johansson (Åsa); V. Vitart (Veronique); I. Rudan (Igor); P. Ugocsai (Peter); Y.S. Aulchenko (Yurii); C.S. Franklin (Christopher); G. Liebisch (Gerhard); I. Jonasson (Inger); I.V. Zorkoltseva (Irina); C. Pattaro (Cristian); C. Hayward (Caroline); A.J. Isaacs (Aaron); C. Hengstenberg (Christian); S. Campbell (Susan); C. Gnewuch (Carsten); A.C.J.W. Janssens (Cécile); A.V. Kirichenko (Anatoly); I.R. König (Inke); F. Marroni (Fabio); O. Polasek (Ozren); A. Demirkan (Ayşe); I. Kolcic (Ivana); C. Schwienbacher (Christine); W. Igl (Wilmar); Z. Biloglav (Zrinka); J.C.M. Witteman (Jacqueline); I. Pichler (Irene); G. Zaboli (Ghazal); T.I. Axenovich (Tatiana); A. Peters (Annette); S. Schreiber (Stefan); H.E. Wichmann (Heinz Erich); J. Erdmann (Jeanette); H. Schunkert (Heribert); N. Hastie (Nick); B.A. Oostra (Ben); S.H. Wild (Sarah); T. Meitinger (Thomas); U. Gyllensten (Ulf); P. Tikka-Kleemola (Päivi); J.F. Wilson (James); A.F. Wright (Alan); G. Schmitz (Gerd)

    2009-01-01

    textabstractSphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that enco

  5. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  6. Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism.

    Science.gov (United States)

    Rolando, Monica; Escoll, Pedro; Buchrieser, Carmen

    2016-06-01

    Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the host's sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival. PMID:27191778

  7. Another brick in the cell wall: biosynthesis dependent growth model.

    Directory of Open Access Journals (Sweden)

    Adelin Barbacci

    Full Text Available Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper.

  8. Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

    Science.gov (United States)

    Meshcheryakova, Anastasia; Köfeler, Harald C.; Triebl, Alexander; Mungenast, Felicitas; Heinze, Georg; Gerner, Christopher; Zimmermann, Philip; Jaritz, Markus; Mechtcheriakova, Diana

    2016-01-01

    The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery

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

    Science.gov (United States)

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

    2016-08-01

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

  10. Sphingolipids and plant defense/disease: the "death" connection and beyond

    Directory of Open Access Journals (Sweden)

    Robert eBerkey

    2012-04-01

    Full Text Available Sphingolipids comprise a major class of structural materials and lipid signaling molecules in all eukaryotic cells. Over the past two decades, there has been a phenomenal growth in the study of sphingolipids (i.e. sphingobiology at an average rate of >1000 research articles per year. Sphingolipid studies in plants, though accounting for only a small fraction (~6% of the total number of publications, have also enjoyed proportionally rapid growth in the past decade. Concomitant with the growth of sphingobiology, there has also been tremendous progress in our understanding of the molecular mechanisms of plant innate immunity. In this review, we (i cross examine and analyze the major findings that establish and strengthen the intimate connections between sphingolipid metabolism and plant programmed cell death (PCD associated with plant defense or disease; (ii highlight and compare key bioactive sphingolipids involved in the regulation of plant PCD and possibly defense; (iii discuss the potential role of sphingolipids in polarized membrane/protein trafficking and formation of lipid rafts as subdomains of cell membranes in relation to plant defense; and (iv where possible, attempt to identify potential parallels for immunity-related mechanisms involving sphingolipids across kingdoms.

  11. Structure of Sphingolipids From Sea Cucumber Cucumaria frondosa and Structure-Specific Cytotoxicity Against Human HepG2 Cells.

    Science.gov (United States)

    Jia, Zicai; Song, Yu; Tao, Suyuan; Cong, Peixu; Wang, Xiaoxu; Xue, Changhu; Xu, Jie

    2016-03-01

    To investigate the relationship between structure and activity, three glucocerebroside series (CFC-1, CFC-2 and CFC-3), ceramides (CF-Cer) and long-chain bases (CF-LCB) of sea cucumber Cucumaria frondosa (C. frondosa) were isolated and evaluated in HepG2 cells. The molecular species of CFC-1, CFC-2 and CFC-3 and CF-Cer were identified using reversed-phase liquid chromatography with heated electrospray ionization coupled to high-resolution mass spectrometry (RPLC-HESI-HRMS), and determined on the basis of chemical and spectroscopic evidence: For the three glucocerebroside series, fatty acids (FA) were mainly saturated (18:0 and 22:0), monounsaturated (22:1, 23:1 and 24:1) and 2-hydroxyl FA (2-HFA) (23:1 h and 24:1 h), the structure of long-chain bases (LCB) were dihydroxy (d17:1, d18:1 and d18:2) and trihydroxy (t16:0 and t17:0), and the glycosylation was glucose; For CF-Cer, FA were primarily saturated (17:0) and monounsaturated (16:1 and 19:1), the structure of LCB were dihydroxy (d17:1 and d18:1), and trihydroxy (t16:0). The results of cell experiment indicated that all of three glucocerebroside series, CF-Cer and CF-LCB exhibited an inhibitory effects on cell proliferation. Moreover, CFC-3 was most effective in three glucocerebrosides to HepG-2 cell viability. The inhibition effect of CF-LCB was the strongest, and the inhibition effect of CF-Cer was much stronger than glucocerebrosides. PMID:26861868

  12. Stress-induced sphingolipid signaling: role of type-2 neutral sphingomyelinase in murine cell apoptosis and proliferation.

    Directory of Open Access Journals (Sweden)

    Raphael Devillard

    Full Text Available BACKGROUND: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+-dependent neutral sphingomyelinase-2 (nSMase2 plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. METHODOLOGY AND PRINCIPAL FINDINGS: For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum requires nSMase2 activation. CONCLUSION AND SIGNIFICANCE: nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

  13. Plant sphingolipids today - Are they still enigmatic?

    NARCIS (Netherlands)

    Spassieva, S; Hille, J; Voesenek, L.A.C.J.

    2003-01-01

    Sphingolipids are a diverse group of lipids found in all eukaryotes and some bacteria, consisting of a hydrophobic ceramide and a hydrophilic head group. We have summarised the contemporary understanding of the structure of plant sphingolipids with an emphasis on glucosylceramides and inositolphosph

  14. Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

    DEFF Research Database (Denmark)

    Nybroe, O; Albrechtsen, M; Dahlin, J;

    1985-01-01

    The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B...

  15. Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells.

    Science.gov (United States)

    Pomara, Cristoforo; Barone, Rosario; Marino Gammazza, Antonella; Sangiorgi, Claudia; Barone, Fulvio; Pitruzzella, Alessandro; Locorotondo, Nicola; Di Gaudio, Francesca; Salerno, Monica; Maglietta, Francesca; Sarni, Antonio Luciano; Di Felice, Valentina; Cappello, Francesco; Turillazzi, Emanuela

    2016-06-01

    Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real-time PCR. The results obtained show that testosterone levels increase at a 3.9 μM concentration of nandrolone and return to the basal level a 15.6 μM dose of nandrolone. Nandrolone-induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a-hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down-regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects. PMID:26626779

  16. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

    Science.gov (United States)

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C; Suttorp, Norbert; Proia, Richard L; Witzenrath, Martin; Kuebler, Wolfgang M

    2015-03-31

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  17. A New Sphingolipid from the Fungus Paxillus panuoides

    Institute of Scientific and Technical Information of China (English)

    GAO Jin-Ming; ZHANG Zeng-Qiang; LIU Ji-Kai

    2003-01-01

    @@ The ceramides, cleavage products of various sphingolipids, including gangliosides and cerebrosides, are in volved in various signal transduction pathways. Many extracellular stresses, such as tumor necrosis factors-α (TNFα) and human immunodeficiency virus (HIV) have been shown to activate sphingomyelinases that release ceramides which inhibit cell growth and induce apoptosis. Because of the importance of ceramides, chemistry and biology of ceramides have been the vital subject of the latest research in recent years. [1~4

  18. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii.

    Science.gov (United States)

    Lindahl, Lina; Genheden, Samuel; Eriksson, Leif A; Olsson, Lisbeth; Bettiga, Maurizio

    2016-04-01

    Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo-lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress.

  19. Endocytosis of NBD-sphingolipids in neurons : Exclusion from degradative compartments and transport to the Golgi complex

    NARCIS (Netherlands)

    Babia, T; Ledesma, MD; Saffrich, R; Kok, JW; Dotti, CG; Egea, G

    2001-01-01

    Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell li

  20. Rice Brittleness Mutants: A Way to Open the 'Black Box' of Monocot Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Baocai Zhang; Yihua Zhou

    2011-01-01

    Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae.Mechanical strength is an important agronomy trait of rice(Oryza sativa L.)plants that affects crop lodging and grain yield.As a prominent physical property of cell walls,mechanical strength reflects upon the structure of different wall polymers and how they interact.Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling.Our group focuses on the study of isolation of brittle culm(bc)mutants and characterization of their corresponding genes.To date,several bc mutants have been reported.The identified genes have covered several pathways of cell wall biosynthesis,revealing many secrets of monocot cell wall biosynthesis.Here,we review the progress achieved in this research field and also highlight the perspectives in expectancy.All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.

  1. Vascular sphingolipids in physiological and pathological adaptation.

    Science.gov (United States)

    Bao, Jun-Xiang; Su, Yu-Ting; Cheng, Yao-Ping; Zhang, Hai-Jun; Xie, Xiao-Ping; Chang, Yao-Ming

    2016-01-01

    Sphingolipids (SLs) are compounds containing a long-chain fatty alcohol amine called sphingosine which exists in cellular membranes, cytoplasm, nucleus, interstitial fluid, blood and lymphatic circulation. SLs act as essential constituents of membranes of eukaryotic cells, so the seesaw of SLs will lead to structural alteration of membranes instigating cellular functional change. SLs also act as crucial signaling molecules taking effect intracellularly or extracellularly which regulates activity of downstream molecules determining cellular adaptation to numerous stimulus. This review aims to highlight the contribution of SLs to physiological and pathophysiological remodeling of vasculature. We will first provide a short overview on metabolism, trafficking and compartmentalization of SLs. Then the regulation of SLs on reactive oxygen species (ROS) formation, vascular tone modulation, endothelial barrier integrity, apoptosis and autophagy are summarized. Finally, we will discuss how the SLs are modulated contributing to vascular development, angiogenesis and vascular remodeling in pathological situations as hypertension, atherosclerosis, and aging. The compellingly regulative actions of SLs bring about copious therapeutic targets for potential pharmacological intervention on the diseases involving vascular maladaptation. PMID:27100498

  2. Overexpression of Patatin-Related Phospholipase AIIIβ Altered the Content and Composition of Sphingolipids in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Maoyin eLi

    2014-10-01

    Full Text Available In plants, fatty acids are primarily synthesized in plastids and then transported to the endoplasmic reticulum for synthesis of most of the complex membrane lipids, including glycerolipids and sphingolipids. The first step of sphingolipid synthesis, which uses a fatty acid and a serine as substrates, is critical for sphingolipid homeostasis; its disruption leads to an altered plant growth. Phospholipase As (PLAs have been implicated in the trafficking of fatty acids from plastids to the endoplasmic reticulum. Previously, we found that overexpression of a patatin-related phospholipase, pPLAIIIβ, resulted in a smaller plant size and altered anisotropic cell expansion. Here, we determined the content and composition of sphingolipids in pPLAIIIβ-knockout and overexpression plants (pPLAIIIβ-KO and -OE. 3-keto-sphinganine, the product of the first step of sphingolipid synthesis, had a 26% decrease in leaves of pPLAIIIβ-KO while a 52% increase in pPLAIIIβ-OE compared to wild type (WT. The levels of free long-chain base species, dihydroxy-C18:0 and trihydroxy-18:0 (d18:0 and t18:0, were 38% and 97% higher, respectively, in pPLAIIIβ-OE than in WT. The level of complex sphingolipids ceramide d18:0-16:0 and t18:1-16:0 had a 2-fold increase in pPLAIIIβ-OE. The level of hydroxyceramide d18:0-h16:0 was 72% higher in pPLAIIIβ-OE compared to WT. The levels of several species of glucosylceramide and glycosylinositolphosphoceramide tended to be higher in pPLAIIIβ-OE than in WT. The total content of the complex sphingolipids showed a slightly higher in pPLAIIIβ-OE than in WT. These results revealed an involvement of phospholipase-mediated lipid homeostasis in plant growth.

  3. Maternal and embryonic control of uterine sphingolipid-metabolizing enzymes during murine embryo implantation.

    Science.gov (United States)

    Kaneko-Tarui, Tomoko; Zhang, Ling; Austin, Kathleen J; Henkes, Luiz E; Johnson, Joshua; Hansen, Thomas R; Pru, James K

    2007-10-01

    During early gestation in invasively implanting species, the uterine stromal compartment undergoes dramatic remodeling, defined by the differentiation of stromal fibroblast cells into decidual cells. Lipid signaling molecules from a number of pathways are well-established functional components of this decidualization reaction. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of bioactive sphingolipid metabolites established from studies in other organ systems, we hypothesized that uterine sphingolipid metabolism would change during implantation. By a combination of Northern blot, Western blot, and immunohistochemical analyses, we establish that enzymes at each of the major catalytic steps in the sphingolipid cascade become transcriptionally up-regulated in the uterus during decidualization. Each of the enzymes analyzed was up-regulated from Days of Pregnancy (DOP) 4.5-7.5. When comparing embryo-induced decidualization (decidual) with mechanically induced decidualization (deciduomal), sphingomyelin phosphodiesterase 1 (Smpd1) mRNA and sphingosine kinase 1 (SPHK1) protein were shown to be dually regulated in the endometrium by both maternal and embryonic factors. As measured by the diacyl glycerol kinase assay, ceramide levels rose in parallel with Smpd1 gene expression, suggesting that elevated transcription of sphingolipid enzymes results in heightened catalytic activity of the pathway. Altogether, these findings place sphingolipids on a growing list of lipid signaling molecules that become increasingly present at the maternal-embryonic interface.

  4. Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells

    International Nuclear Information System (INIS)

    The effect of dexamethasone on the incorporation and conversion of [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10(-6) M) also caused a marked decrease in the biosynthesis of this polyunsaturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the delta 5 desaturation of labeled 20:3 omega 6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells

  5. The role of sphingolipids in neuronal plasticity of the brain.

    Science.gov (United States)

    Sonnino, Sandro; Prinetti, Alessandro

    2016-05-01

    This Editorial highlights a study by Müller et al. in which the authors suggest a new sphingolipid-dependent mechanism for behavioral extinction. Their study should be considered in the broad perspective of sphingolipid metabolic pathways and traffic (depicted in the graphic). Read the highlighted article 'A sphingolipid mechanism for behavioral extinction' on page 589. PMID:26990419

  6. Importance of hyaluronan biosynthesis and degradation in cell differentiation and tumor formation

    OpenAIRE

    Heldin P.

    2003-01-01

    Hyaluronan is an important connective tissue glycosaminoglycan. Elevated hyaluronan biosynthesis is a common feature during tissue remodeling under both physiological and pathological conditions. Through its interactions with hyaladherins, hyaluronan affects several cellular functions such as cell migration and differentiation. The activities of hyaluronan-synthesizing and -degrading enzymes have been shown to be regulated in response to growth factors. During tumor progression hyaluronan sti...

  7. [Biosynthesis of enniatin by washed cells of Fusarium sambucinum].

    Science.gov (United States)

    Minasian, A E; Chermenskĭ, D N; Bezborodov, A M

    1979-01-01

    Biosynthesis of the depsipeptide membrane ionophore--enniatin B by the washed mycelium Fusarium sambucinum Fuck 52 377 was studied. Metabolic precursors of enniatin B, alpha-ketovaleric acid, 14C-L-valine, and 14CH3-methionine, were added to the system after starvation. The amino acid content in the metabolic pool increased 1.5 times after addition of alpha-ketovaleric acid, 2.2 times after that of valine, and 2.5 times after addition of methionine. 14C-L-valine and 14CH3-methionine were incorporated into the molecule of enniatin B. Valine methylation in the molecule occurred at the level of synthesized depsipeptide. Amino acids of the metabolic pool performed the regulatory function in the synthesis. PMID:583180

  8. Breakdown of the regulatory control of pyrimidine biosynthesis in human breast cancer cells.

    Science.gov (United States)

    Sigoillot, Frederic D; Sigoillot, Severine M; Guy, Hedeel I

    2004-04-20

    The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.

  9. Sphingolipids in High Fat Diet and Obesity-Related Diseases

    Directory of Open Access Journals (Sweden)

    Songhwa Choi

    2015-01-01

    Full Text Available Nutrient oversupply associated with a high fat diet (HFD significantly alters cellular metabolism, and specifically including sphingolipid metabolism. Sphingolipids are emerging as bioactive lipids that play key roles in regulating functions, in addition to their traditional roles as membrane structure. HFD enhances de novo sphingolipid synthesis and turnover of sphingolipids via the salvage pathway, resulting in the generation of ceramide, and more specifically long chain ceramide species. Additionally, HFD elevates sphingomyelin and sphingosine-1 phosphate (S1P levels in several tissues including liver, skeletal muscle, adipose tissue, and cardiovascular tissues. HFD-stimulated sphingolipid generation contributes to systemic insulin resistance, dysregulated lipid accumulation, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related conditions. Furthermore, altered sphingolipid levels, particularly ceramide and sphingomyelin, are involved in obesity-induced endothelial dysfunction and atherosclerosis. In this review, HFD-mediated sphingolipid metabolism and its impact on HFD-induced biology and pathobiology will be discussed.

  10. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

    Science.gov (United States)

    Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

    2016-08-01

    GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. PMID:27382114

  11. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

    Science.gov (United States)

    Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

    2016-08-01

    GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.

  12. Biosynthesis and release of proteins by isolated pulmonary Clara cells

    International Nuclear Information System (INIS)

    The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14C-glucosamine indicating that it was not a glycoprotein. Data demonstrate the release of several proteins from isolated Clara cells but the major protein had a M.W. of 6 Kd

  13. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    DEFF Research Database (Denmark)

    Klemm, Robin W; Ejsing, Christer S.; Surma, Michal A;

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane...

  14. Unraveling the dynamics of sphingolipid metabolism by mass spectrometry based (phospho)proteomics

    NARCIS (Netherlands)

    Lebesgue, N.F.M.

    2016-01-01

    Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules with imbalances affecting cellular function and contributing to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is m

  15. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures. 

  16. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    International Nuclear Information System (INIS)

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well

  17. Sphingolipids and mitochondrial function, lessons learned from yeast

    Directory of Open Access Journals (Sweden)

    Pieter Spincemaille

    2014-06-01

    Full Text Available Mitochondrial dysfunction is a hallmark of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also of cancer, diabetes and rare diseases such as Wilson’s disease (WD and Niemann Pick type C1 (NPC. Mitochondrial dysfunction underlying human pathologies has often been associated with an aberrant cellular sphingolipid metabolism. Sphingolipids (SLs are important membrane constituents that also act as signaling molecules. The yeast Saccharomyces cerevisiae has been pivotal in unraveling mammalian SL metabolism, mainly due to the high degree of conservation of SL metabolic pathways. In this review we will first provide a brief overview of the major differences in SL metabolism between yeast and mammalian cells and the use of SL biosynthetic inhibitors to elucidate the contribution of specific parts of the SL metabolic pathway in response to for instance stress. Next, we will discuss recent findings in yeast SL research concerning a crucial signaling role for SLs in orchestrating mitochondrial function, and translate these findings to relevant disease settings such as WD and NPC. In summary, recent research shows that S. cerevisiae is an invaluable model to investigate SLs as signaling molecules in modulating mitochondrial function, but can also be used as a tool to further enhance our current knowledge on SLs and mitochondria in mammalian cells.

  18. Hemicellulose biosynthesis and degradation in tobacco cell walls

    NARCIS (Netherlands)

    Compier, M.G.M.

    2005-01-01

    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose

  19. Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

    Science.gov (United States)

    Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

    2015-09-01

    Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. PMID:26057225

  20. Endothelium in brain: Receptors, mitogenesis, and biosynthesis in glial cells

    Energy Technology Data Exchange (ETDEWEB)

    MacCumber, M.W.; Ross, C.A.; Snyder, S.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1990-03-01

    The authors have explored the cellular loci of endothelin (ET) actions and formation in the brain, using cerebellar mutant mice was well as primary and continuous cell cultures. A glial role is favored by several observations: (1) mutant mice lacking neuronal Purkinje cells display normal ET receptor binding and enhanced stimulation by ET of inositolphospholipid turnover; (ii) in weaver mice lacking neuronal granule cells, ET stimulation of inositolphospholipid turnover is not significantly diminished; (iii) C{sub 6} glioma cells and primary cultures of cerebellar astroglia exhibit substantial ET receptor binding and ET-induced stimulation of inositolphospholipid turnover; (iv) ET promotes mitogenesis of C{sub 6} glioma cells and primary cerebellar astroglia; and (v) primary cultures of cerebellar astroglia contain ET mRNA. ET also appears to have a neuronal role, since it stimulates inositolphospholipid turnover in primary cultures of cerebellar granule cells, and ET binding declines in granule cell-deficient mice. Thus, ET can be produced by glia and act upon both glia and neurons in a paracrine fashion.

  1. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    Science.gov (United States)

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

  2. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor

    Science.gov (United States)

    The phenylpropanoid biosynthesis pathway that generates lignin subunits represents a significant target to alter the abundance and composition of lignin. The major regulators of phenylpropanoid metabolism are myb transcription factors, which have been shown to modulate secondary cell wall compositi...

  3. Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

    OpenAIRE

    1984-01-01

    Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer c...

  4. CjbHLH1 homologs regulate sanguinarine biosynthesis in Eschscholzia californica cells.

    Science.gov (United States)

    Yamada, Yasuyuki; Motomura, Yukiya; Sato, Fumihiko

    2015-05-01

    Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica. PMID:25713177

  5. Mechanisms and impact of ceramide phosphoethanolamine biosynthesis

    OpenAIRE

    Hentes Vacaru, A.M.

    2009-01-01

    Sphingolipids represent an essential class of membrane molecules in eukaryotic cells. They are primarily found in the outer leaflet of the plasma membrane where they help create a rigid and impermeable barrier to the extracellular environment. While sphingomyelin (SM) is the most abundant sphingolipid in mammals and nematodes, insects produce the SM analogue ceramide phosphoethanolamine (CPE) as a major plasma membrane constituent. Little is known about the biological role of CPE or about the...

  6. Inborn errors of metabolism in the biosynthesis and remodelling of phospholipids

    NARCIS (Netherlands)

    Wortmann, S.B.; Espeel, M.; Almeida, L.; Reimer, A.; Bosboom, D.G.; Roels, F.; Brouwer, A.P.M. de; Wevers, R.A.

    2015-01-01

    Since the proposal to define a separate subgroup of inborn errors of metabolism involved in the biosynthesis and remodelling of phospholipids, sphingolipids and long chain fatty acids in 2013, this group is rapidly expanding. This review focuses on the disorders involved in the biosynthesis of phosp

  7. There is more to a lipid than just being a fat: sphingolipid-guided differentiation of oligodendroglial lineage from embryonic stem cells

    OpenAIRE

    Bieberich, Erhard

    2010-01-01

    Dr. Robert K. Yu's research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells...

  8. Biosynthesis and biotransformation of lipids in plant cell cultures and algae

    International Nuclear Information System (INIS)

    The biosynthesis and metabolism of lipids in plant cell cultures grown photoautotrophically, has been studied since 1970. The most prominently occuring lipids in cell cultures and whole plants are phospholipids, glycolipids, triglycerides and glycosides. Radioactively labelled lipids have been produced from soybean cell cultures incubated with 14C-linoleic acid, and the fate of the phospholipid formed was investigated. Freshwater and marine algae cultured under different conditions of light, temperature and nutrient media have also been investigated for their lipid and fatty acid content. The exploitation of biotechnological processes for producing valuable lipids is encouraged. (U.K.)

  9. Localization and biosynthesis of polyamines in insulin-producing cells

    DEFF Research Database (Denmark)

    Hougaard, D M; Larsson, L I; Nielsen, Jens Høiriis

    1986-01-01

    Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine...

  10. Arabinogalactan biosynthesis

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Geshi, Naomi

    2015-01-01

    Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize...

  11. A sphingolipid mechanism for behavioral extinction.

    Science.gov (United States)

    Huston, Joseph P; Kornhuber, Johannes; Mühle, Christiane; Japtok, Lukasz; Komorowski, Mara; Mattern, Claudia; Reichel, Martin; Gulbins, Erich; Kleuser, Burkhard; Topic, Bianca; De Souza Silva, Maria A; Müller, Christian P

    2016-05-01

    Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a

  12. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

    OpenAIRE

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M.; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael,; Arenz, Christoph; Hocke, Andreas C.; Suttorp, Norbert; Proia, Richard L.

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism that protects against systemic hypoxemia by redistributing blood flow from poorly to better ventilated areas of the lung, thereby minimizing ventilation-perfusion mismatch. However, in chronic hypoxemia-associated lung disease, HPV contributes to pulmonary hypertension. In this study, we provide novel evidence for a dual role of sphingolipids as important signal mediators in HPV, which critically depends on the presence of ...

  13. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids...... to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present...... and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate...

  14. Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.

    Science.gov (United States)

    Lange, B M; Ketchum, R E; Croteau, R B

    2001-09-01

    Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.

  15. Importance of hyaluronan biosynthesis and degradation in cell differentiation and tumor formation

    Directory of Open Access Journals (Sweden)

    Heldin P.

    2003-01-01

    Full Text Available Hyaluronan is an important connective tissue glycosaminoglycan. Elevated hyaluronan biosynthesis is a common feature during tissue remodeling under both physiological and pathological conditions. Through its interactions with hyaladherins, hyaluronan affects several cellular functions such as cell migration and differentiation. The activities of hyaluronan-synthesizing and -degrading enzymes have been shown to be regulated in response to growth factors. During tumor progression hyaluronan stimulates tumor cell growth and invasiveness. Thus, elucidation of the molecular mechanisms which regulate the activities of hyaluronan-synthesizing and -degrading enzymes during tumor progression is highly desired.

  16. Biosynthesis of psoralens. Psoralen 5-monooxygenase activity from elicitor-treated Ammi majus cells.

    Science.gov (United States)

    Hamerski, D; Matern, U

    1988-11-01

    Microsomes prepared from cultured Ammi majus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) converted psoralen to bergaptol (5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5-methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.

  17. Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis.

    Science.gov (United States)

    Zhao, Qiao; Zeng, Yining; Yin, Yanbin; Pu, Yunqiao; Jackson, Lisa A; Engle, Nancy L; Martin, Madhavi Z; Tschaplinski, Timothy J; Ding, Shi-You; Ragauskas, Arthur J; Dixon, Richard A

    2015-04-01

    Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.

  18. Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells

    International Nuclear Information System (INIS)

    A green and efficient biosynthesis method to prepare fluorescence-tunable biocompatible cadmium selenide quantum dots using Escherichia coli cells as biological matrix was proposed. Decisive factors in biosynthesis of cadmium selenide quantum dots in a designed route in Escherichia coli cells were elaborately investigated, including the influence of the biological matrix growth stage, the working concentration of inorganic reactants, and the co-incubation duration of inorganic metals to biomatrix. Ultraviolet-visible, photoluminescence, and inverted fluorescence microscope analysis confirmed the unique optical properties of the biosynthesized cadmium selenide quantum dots. The size distribution of the nanocrystals extracted from cells and the location of nanocrystals foci in vivo were also detected seriously by transmission electron microscopy. A surface protein capping layer outside the nanocrystals was confirmed by Fourier transform infrared spectroscopy measurements, which were supposed to contribute to reducing cytotoxicity and maintain a high viability of cells when incubating with quantum dots at concentrations as high as 2 μM. Cell morphology observation indicated an effective labeling of living cells by the biosynthesized quantum dots after a 48 h co-incubation. The present work demonstrated an economical and environmentally friendly approach to fabricating highly fluorescent quantum dots which were expected to be an excellent fluorescent dye for broad bio-imaging and labeling. (papers)

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

    Science.gov (United States)

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

    2002-12-01

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

  20. Localization of sesquiterpene lactone biosynthesis in cells of capitate glandular trichomes of Helianthus annuus (Asteraceae).

    Science.gov (United States)

    Amrehn, Evelyn; Aschenbrenner, Anna-Katharina; Heller, Annerose; Spring, Otmar

    2016-03-01

    Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid. PMID:25956500

  1. Early evolution of polyisoprenol biosynthesis and the origin of cell walls

    Science.gov (United States)

    2016-01-01

    After being a matter of hot debate for years, the presence of lipid membranes in the last common ancestor of extant organisms (i.e., the cenancestor) now begins to be generally accepted. By contrast, cenancestral cell walls have attracted less attention, probably owing to the large diversity of cell walls that exist in the three domains of life. Many prokaryotic cell walls, however, are synthesized using glycosylation pathways with similar polyisoprenol lipid carriers and topology (i.e., orientation across the cell membranes). Here, we provide the first systematic phylogenomic report on the polyisoprenol biosynthesis pathways in the three domains of life. This study shows that, whereas the last steps of the polyisoprenol biosynthesis are unique to the respective domain of life of which they are characteristic, the enzymes required for basic unsaturated polyisoprenol synthesis can be traced back to the respective last common ancestor of each of the three domains of life. As a result, regardless of the topology of the tree of life that may be considered, the most parsimonious hypothesis is that these enzymes were inherited in modern lineages from the cenancestor. This observation supports the presence of an enzymatic mechanism to synthesize unsaturated polyisoprenols in the cenancestor and, since these molecules are notorious lipid carriers in glycosylation pathways involved in the synthesis of a wide diversity of prokaryotic cell walls, it provides the first indirect evidence of the existence of a hypothetical unknown cell wall synthesis mechanism in the cenancestor.

  2. Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells

    DEFF Research Database (Denmark)

    Billestrup, N; Møldrup, A; Serup, P;

    1990-01-01

    The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, we have transfected...... of GH receptors in one clone (1.24) selected for detailed analysis was increased 2.6-fold compared to untransfected cells. The increased GH receptor expression was accompanied by an increased responsiveness to GH. Thus, the maximal GH-stimulated increase of insulin biosynthesis was 4.1-fold in 1...... magnitude of GH-stimulated insulin biosynthesis. A close stoichiometric relationship between the level of receptor expression and the level of GH-stimulated insulin biosynthesis was observed. We conclude from these results that the hepatic GH receptor is able to mediate the effect of GH on insulin...

  3. IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model.

    Science.gov (United States)

    von Bismarck, Philipp; Winoto-Morbach, Supandi; Herzberg, Mona; Uhlig, Ulrike; Schütze, Stefan; Lucius, Ralph; Krause, Martin F

    2012-06-01

    Airway epithelial NF-κB is a key regulator of host defence in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (aSMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defence as well as in pathologic processes of acute lung injury. Little is known about the regulatory mechanisms of pulmonary sphingolipid metabolism in bacterial infections of the lung. The objective of this study was to evaluate the influence of NF-κB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation. In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS we investigated TNF-α, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to aSMase activity and ceramide and S1-P lung tissue concentrations. Airway epithelial NF-κB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of aSMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-κB translocation. This confirms the critical role of airway epithelial NF-κB pathway for the inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defence mechanisms. PMID:22469869

  4. Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

    International Nuclear Information System (INIS)

    Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m2 g−1. -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N2 adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity, respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m2 g−1.

  5. Biosynthesis of amorphous mesoporous aluminophosphates using yeast cells as templates

    Energy Technology Data Exchange (ETDEWEB)

    Sifontes, Ángela B., E-mail: asifonte@ivic.gob.ve [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); González, Gema [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Tovar, Leidy M.; Méndez, Franklin J. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Gomes, Maria E. [Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Cañizales, Edgar [Área de Análisis Químico Inorgánico, PDVSA, INTEVEP, Los Teques 1070-A (Venezuela, Bolivarian Republic of); Niño-Vega, Gustavo; Villalobos, Hector [Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Brito, Joaquin L. [Centro de Química, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    2013-02-15

    Graphical abstract: Display Omitted Highlights: ► Amorphous aluminophosphates can take place using yeast as template. ► A mesoporous material was obtained. ► The specific surface area after calcinations ranged between 176 and 214 m{sup 2} g{sup −1}. -- Abstract: In this study aluminophosphates have been synthesized from aluminum isopropoxide and phosphoric acid solutions using yeast cells as template. The physicochemical characterization was carried out by thermogravimetric analysis; X-ray diffraction; Fourier transform infrared; N{sub 2} adsorption–desorption isotherms; scanning electron microscopy; transmission electron microscopy and potentiometric titration with N-butylamine for determination of: thermal stability; crystalline structure; textural properties; morphology and surface acidity, respectively. The calcined powders consisted of an intimate mixture of amorphous and crystallized AlPO particles with sizes between 23 and 30 nm. The average pore size observed is 13–16 nm and the specific surface area after calcinations (at 650 °C) ranged between 176 and 214 m{sup 2} g{sup −1}.

  6. Sphingolipids: promising lipid-class molecules with potential applications for industry. A review

    Directory of Open Access Journals (Sweden)

    Miazek, K.

    2016-01-01

    Full Text Available Introduction. Sphingolipids are a group of lipid molecules, the focus on which has been gradually increasing during recent years. This review presents sphingolipids, as valuable compounds with a high potential for industry. Literature. Structures of sphingolipids are described and their natural sources are presented. Different methods for extraction, purification and structural characterization of sphingolipids are evaluated. Activity of sphingolipids towards various microorganisms is discussed and methods for chemical modifications of natural sphingolipids to obtain novel properties are depicted. Finally, applications for implementing sphingolipid molecules in food, cosmetic, pharmaceutical or medical industry are proposed. Conclusions. Sphingolipids are molecules of high impact and their importance will inevitably increase in the future.

  7. Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi.

    Science.gov (United States)

    Abreu, I N; Andreazza, N L; Sawaya, A C H F; Eberlin, M N; Mazzafera, P

    2007-11-01

    Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus. PMID:17682964

  8. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells

    Science.gov (United States)

    Rischer, Heiko; Orešič, Matej; Seppänen-Laakso, Tuulikki; Katajamaa, Mikko; Lammertyn, Freya; Ardiles-Diaz, Wilson; Van Montagu, Marc C. E.; Inzé, Dirk; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2006-01-01

    Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant. PMID:16565214

  9. A non-target chemometric strategy applied to UPLC-MS sphingolipid analysis of a cell line exposed to chlorpyrifos pesticide: a feasibility study

    OpenAIRE

    Kássio M. G. Lima; Bedia, Carmen; Tauler, Romá

    2014-01-01

    A non-target chemometrics study based on the application of Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method to a data set obtained by ultra-performance liquid chromatographic coupled to mass spectrometry (UPLC-MS) has been applied to the study of human prostate cancer (DU145) cell line samples treated with the organophosphate pesticide chlorpyrifos (CPF). Full scan UPLC-MS data sets were segmented in 17 different chromatographic windows and submitted to a non-target d...

  10. Mutations in the Lipopolysaccharide biosynthesis pathway interfere with crescentin-mediated cell curvature in Caulobacter crescentus.

    Science.gov (United States)

    Cabeen, Matthew T; Murolo, Michelle A; Briegel, Ariane; Bui, N Khai; Vollmer, Waldemar; Ausmees, Nora; Jensen, Grant J; Jacobs-Wagner, Christine

    2010-07-01

    Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.

  11. Interaction between abscisic acid and nitric oxide in PB90-induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures.

    Science.gov (United States)

    Chen, Qian; Chen, Zunwei; Lu, Li; Jin, Haihong; Sun, Lina; Yu, Qin; Xu, Hongke; Yang, Fengxia; Fu, Mengna; Li, Shengchao; Wang, Huizhong; Xu, Maojun

    2013-01-01

    Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells. PMID:23554409

  12. Biosynthesis of Bacterial Cellulose/Carboxylic Multi-Walled Carbon Nanotubes for Enzymatic Biofuel Cell Application

    Directory of Open Access Journals (Sweden)

    Pengfei Lv

    2016-03-01

    Full Text Available Novel nanocomposites comprised of bacterial cellulose (BC with carboxylic multi-walled carbon nanotubes (c-MWCNTs incorporated into the BC matrix were prepared through a simple method of biosynthesis. The biocathode and bioanode for the enzyme biological fuel cell (EBFC were prepared using BC/c-MWCNTs composite injected by laccase (Lac and glucose oxidase (GOD with the aid of glutaraldehyde (GA crosslinking. Biosynthesis of BC/c-MWCNTs composite was characterized by digital photos, scanning electron microscope (SEM, and Fourier Transform Infrared (FTIR. The experimental results indicated the successful incorporation of c-MWCNTs into the BC. The electrochemical and biofuel performance were evaluated by cyclic voltammetry (CV and linear sweep voltammetry (LSV. The power density and current density of EBFCs were recorded at 32.98 µW/cm3 and 0.29 mA/cm3, respectively. Additionally, the EBFCs also showed acceptable stability. Preliminary tests on double cells indicated that renewable BC have great potential in the application field of EBFCs.

  13. Progesterone-specific stimulation of triglyceride biosynthesis in a breast cancer cell line (T-47D)

    Energy Technology Data Exchange (ETDEWEB)

    Judge, S.M.; Chatterton, R.T. Jr.

    1983-09-01

    The purpose of this study was to examine the lactogenic response of human mammary cancer cell lines to hormones in vitro. Progesterone was found to stimulate the incorporation of 14C from (14C)acetate into triglycerides (TG) and to promote accumulation of TG with a fatty acid composition similar to that of human milk fat in T-47D cells. Lipid droplets were observed in larger numbers without concomitant accumulation of casein granules in cells incubated with progesterone, but secretion of lipid into the medium did not occur. An effect of progesterone on TG accumulation was detectable after 12 hr and was maximal at 72 hr. Increasing doses of progesterone (10(-9) to 10(-5) M) caused a progressive increase in TG accumulation. The presence of cortisol and/or prolactin did not alter TG formation nor the dose response of the cells to progesterone. The growth rate of T-47D cells was not altered by the presence of progesterone in the medium. Neither of the human mammary cancer cell lines, MCF-7 and HBL-100, nor the human fibroblast cell lines, 28 and 857, responded to progesterone. The data indicate that, while the normally lactogenic hormones do not stimulate milk product biosynthesis in the cell lines tested, progesterone specifically stimulated synthesis and accumulation of TG in the T-47D cells.

  14. Stereo and region-selective biosynthesis of two new dihydroartemisinic acid glycosides by suspension-cultured cells of Artemisia annua

    Directory of Open Access Journals (Sweden)

    Jianhua Zhu

    2014-01-01

    Full Text Available Background: The system of plant-cultured cells is one of the optimal systems to investigate biosynthesis pathway and their bioactive intermediates. Objective: To study the biosynthesis of dihydroartemisinic acid (1 by suspension-cultured cells of Artemisia annua. Materials and Methods: Substrate (compound 1 was administered into the suspension-cultured cells of A. annua and co-cultured for 2 days. The methanol extract was separated on various column chromatography methods and the structures of two biosynthesis products were elucidated based on the analysis of 1 H NMR, 13 C NMR, 2D NMR, and ESI-MS. Time-course curve was also established. Furthermore, in vitro antitumor activities of compounds 1-3 against HepG2, K562, and A549 cell lines were evaluated by MTT assay. Results: Two new compounds were obtained, namely 3α-hydroxy-dihydroartemisinic acid-α-D - glucopyranosyl ester (2 and 15-hydroxy-cadin-4-en-12-oic acid-β-d - glucopyranosyl ester (3. The results demonstrated that the cultured cells of A. annua possessed the abilities to stereo-selective hydroxylate and region-selective glycosylate sesquiterpene compounds in a highly efficient manner. Inhibitory effects of compounds 1-3 on proliferation of HepG2, K562, and A549 cell lines in vitro were also investigated. Conclusion: Two new dihydroartemisinic acid glycosides were obtained by stereo- and region-selective biosynthesis with cultured cells of A. annua.

  15. Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

    Directory of Open Access Journals (Sweden)

    Anna P Lucarelli

    Full Text Available The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB and extensively drug-resistant strains (XDR-TB is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+ or Mn(2+ and inhibited by Ca(2+ and Cu(2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+ and Cu(2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

  16. Cholesterol, sphingolipids, and glycolipids: What do we know about their role in raft-like membranes?

    DEFF Research Database (Denmark)

    Rog, T.; Vattulainen, I.

    2014-01-01

    Lipids rafts are considered to be functional nanoscale membrane domains enriched in cholesterol and sphingolipids, characteristic in particular of the external leaflet of cell membranes. Lipids, together with membrane-associated proteins, are therefore considered to form nanoscale units...... emerged based on recent atomistic and coarse-grained molecular dynamics simulation studies on the key lipid raft components, which include cholesterol, sphingolipids, glycolipids, and the proteins interacting with these classes of lipids. The simulation results are compared to experiments when possible...... with potential specific functions. Although the understanding of the structure of rafts in living cells is quite limited, the possible functions of rafts are widely discussed in the literature, highlighting their importance in cellular functions. In this review, we discuss the understanding of rafts that has...

  17. New insights into sphingolipid metabolism and functions by using chemical tools

    OpenAIRE

    Cingolani, Francesca

    2015-01-01

    Tesi realitzada a l'Institut de Química Avançada de Catalunya (IQAC-CSIC) Sphingolipids (SLs), essential components of cell membranes, are bioactive molecules involved in the regulation of a variety of cellular processes. Ceramide and sphingosine generally mediate anti-proliferative stimuli, while sphingosine-1-phospate plays an opposite role. The bioactive role of dihydroceramide has also been recently described, showing its involvement in processes like apoptosis and autophagy. Alteratio...

  18. Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells

    International Nuclear Information System (INIS)

    The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, the authors have transfected a GH receptor cDNA under the transcriptional control of the human metallothionein promoter into RIN5-AH cells. The transfected cells were found to exhibit an increased expression of GH receptors and to contain a specific GH receptor mRNA that was not expressed in the parent cell line. The expression of GH receptors in one clone (1.24) selected for detailed analysis was increased 2.6-fold compared to untransfected cells. The increased GH receptor expression was accompanied by an increased responsiveness to GH. Thus, the maximal GH-stimulated increase of insulin biosynthesis was 4.1-fold in 1.24 cells compared to 1.9-fold in the nontransfected RIN5-AH cells. The expression of the transfected receptor was stimulated 1.6- and 2.3-fold when cells were cultured in the presence of 25 or 50 μM Zn2+ was associated with an increased magnitude of GH-stimulated insulin biosynthesis. A close stoichiometric relationship between the level of receptor expression and the level of GH-stimulated insulin biosynthesis was observed. They conclude from these results that the hepatic GH receptor is able to mediate the effect of GH on insulin biosynthesis in RIN5-AH cells

  19. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells

    Science.gov (United States)

    Augustin, Megan M.; Ruzicka, Dan R.; Shukla, Ashutosh K.; Augustin, Jörg M.; Starks, Courtney M.; O’Neil-Johnson, Mark; McKain, Michael R.; Evans, Bradley S.; Barrett, Matt D.; Smithson, Ann; Wong, Gane Ka-Shu; Deyholos, Michael K.; Edger, Patrick P.; Pires, J. Chris; Leebens-Mack, James H.; Mann, David A.; Kutchan, Toni M.

    2015-01-01

    Summary Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol. PMID:25939370

  20. Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Larios-Rodriguez, E; Rangel-Ayon, C; Herrera-Urbina, R [Departamento de Ingenieria Quimica y Metalurgia, Universidad de Sonora, Rosales y Luis Encinas S/N, Hermosillo, Sonora, C.P. 83000 (Mexico); Castillo, S J [Departamento de Investigacion en Fisica, Universidad de Sonora, Rosales y Luis Encinas S/N, Hermosillo, Sonora, C.P. 83000 (Mexico); Zavala, G, E-mail: elarios@polimeros.uson.mx [Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos (Mexico)

    2011-09-02

    Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.

  1. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    Science.gov (United States)

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  2. Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.

    Directory of Open Access Journals (Sweden)

    Ayşe Demirkan

    Full Text Available Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034 on plasma levels of 24 sphingomyelins (SPM, 9 ceramides (CER, 57 phosphatidylcholines (PC, 20 lysophosphatidylcholines (LPC, 27 phosphatidylethanolamines (PE, and 16 PE-based plasmalogens (PLPE, as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204 and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57. After a correction for multiple comparisons (P-value<2.2×10(-9, we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1 and two with sphingolipids (PLD2 and APOE explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3 suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2 to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our

  3. Sphingolipid and cholesterol dependence of alphavirus membrane fusion - Lack of correlation with lipid raft formation in target liposomes

    NARCIS (Netherlands)

    Waarts, BL; Bittman, R; Wilschut, J

    2002-01-01

    Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane.

  4. Biosynthesis of sphinganine-analog mycotoxins.

    Science.gov (United States)

    Du, L; Zhu, X; Gerber, R; Huffman, J; Lou, L; Jorgenson, J; Yu, F; Zaleta-Rivera, K; Wang, Q

    2008-06-01

    Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.

  5. Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells.

    OpenAIRE

    Billestrup, N; Møldrup, A; Serup, P.; Mathews, L S; Norstedt, G; Nielsen, J H

    1990-01-01

    The stimulation of insulin biosynthesis in the pancreatic insulinoma cell line RIN5-AH by growth hormone (GH) is initiated by GH binding to specific receptors. To determine whether the recently cloned rat hepatic GH receptor is able to mediate the insulinotropic effect of GH, we have transfected a GH receptor cDNA under the transcriptional control of the human metallothionein promoter into RIN5-AH cells. The transfected cells were found to exhibit an increased expression of GH receptors and t...

  6. Identification of candidate genes in Populus cell wall biosynthesis using text-mining, co-expression network and comparative genomics

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaohan [ORNL; Ye, Chuyu [ORNL; Bisaria, Anjali [ORNL; Tuskan, Gerald A [ORNL; Kalluri, Udaya C [ORNL

    2011-01-01

    Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of ethanol from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidences supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional genomics in relation to cell wall biosynthesis.

  7. Systems Level Engineering of Plant Cell Wall Biosynthesis to Improve Biofuel Feedstock Quality

    Energy Technology Data Exchange (ETDEWEB)

    Hazen, Samuel

    2013-09-27

    Our new regulatory model of cell wall biosynthesis proposes original network architecture with several newly incorporated components. The mapped set of protein-DNA interactions will serve as a foundation for 1) understanding the regulation of a complex and integral plant component and 2) the manipulation of crop species for biofuel and biotechnology purposes. This study revealed interesting and novel aspects of grass growth and development and further enforce the importance of a grass model system. By functionally characterizing a suite of genes, we have begun to improve the sparse model for transcription regulation of biomass accumulation in grasses. In the process, we have advanced methodology and brachy molecular genetic tools that will serve as valuable community resource.

  8. Combining polysaccharide biosynthesis and transport in a single enzyme: dual-function cell wall glycan synthases.

    Directory of Open Access Journals (Sweden)

    Jonathan Kent Davis

    2012-06-01

    Full Text Available Extracellular polysaccharides are synthesized by a wide variety of species, from unicellular bacteria and Archaea to the largest multicellular plants and animals in the biosphere. In every case, the biosynthesis of these polymers requires transport across a membrane, from the cytosol to either the lumen of secretory pathway organelles or directly into the extracellular space. Although some polysaccharide biosynthetic substrates are moved across the membrane to sites of polysaccharide synthesis by separate transporter proteins before being incorporated into polymers by glycosyltransferase proteins, many polysaccharide biosynthetic enzymes appear to have both transporter and transferase activities. In these cases, the biosynthetic enzymes utilize substrate on one side of the membrane and deposit the polymer product on the other side. This review discusses structural characteristics of plant cell wall glycan synthases that couple synthesis with transport, drawing on what is known about such dual-function enzymes in other species.

  9. Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

    Science.gov (United States)

    Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.

  10. The consequences of genetic and pharmacologic reduction in sphingolipid synthesis.

    Science.gov (United States)

    Schiffmann, Raphael

    2015-01-01

    A new therapy based on substrate synthesis reduction in sphingolipidoses is showing promise. The consequences of decreasing sphingolipid synthesis depend on the level at which synthetic blockage occurs and on the extent of the blockage. Complete synthetic blockage may be lethal if it includes all sphingolipids, such as in a global knockout of serine palmitoyltransferase. Partial inhibition of sphingolipid synthetic pathways is usually benign and may have beneficial effects in a number of lysosomal diseases and in more common pathologies, as seen in animal models for atherosclerosis, polycystic kidney disease, diabetes, and asthma. Studies of various forms of sphingolipid synthesis reduction serve to highlight not only the cellular role of these lipids but also the potential risks and therapeutic benefits of pharmacological agents to be used in therapy for human diseases.

  11. Increase in sphingolipid catabolic enzyme activity during aging

    OpenAIRE

    Sacket, Santosh J; Chung, Hae-young; Okajima, Fumikazu; Im, Dong-Soon

    2009-01-01

    Aim: To understand the contribution of sphingolipid metabolism and its metabolites to development and aging. Methods: A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats. Results: Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic en...

  12. Marine Bifunctional Sphingolipids from the Sponge Oceanapia ramsayi

    Directory of Open Access Journals (Sweden)

    Emile M. Gaydou

    2008-04-01

    Full Text Available During the course of our continuing studies on marine natural lipid products,two known sphingolipids have been isolated for the first time from a specimen of themarine sponge Oceanapia ramsayi collected at Itampolo on the west coast of Madagascarin the Indian Ocean. The structures were elucidated using NMR data and by comparisonwith literature data. The occurrence of these sphingolipids within other Oceanapia spp. isdiscussed.

  13. 8th Annual Glycoscience Symposium: Integrating Models of Plant Cell Wall Structure, Biosynthesis and Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Azadi, Paratoo [Univ. of Georgia, Athens, GA (United States)

    2015-09-24

    The Complex Carbohydrate Research Center (CCRC) of the University of Georgia holds a symposium yearly that highlights a broad range of carbohydrate research topics. The 8th Annual Georgia Glycoscience Symposium entitled “Integrating Models of Plant Cell Wall Structure, Biosynthesis and Assembly” was held on April 7, 2014 at the CCRC. The focus of symposium was on the role of glycans in plant cell wall structure and synthesis. The goal was to have world leaders in conjunction with graduate students, postdoctoral fellows and research scientists to propose the newest plant cell wall models. The symposium program closely followed the DOE’s mission and was specifically designed to highlight chemical and biochemical structures and processes important for the formation and modification of renewable plant cell walls which serve as the basis for biomaterial and biofuels. The symposium was attended by both senior investigators in the field as well as students including a total attendance of 103, which included 80 faculty/research scientists, 11 graduate students and 12 Postdoctoral students.

  14. Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

    Science.gov (United States)

    Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

    2008-12-01

    The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

  15. Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Stepp, M.A.

    1986-01-01

    Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of (/sup 3/H)-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the ..cap alpha..1 and ..cap alpha..2 chains of type I and the ..cap alpha..1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells.

  16. Xyloglucan biosynthesis by Golgi membranes from suspension-cultured sycamore (Acer pseudoplatanus) cells

    International Nuclear Information System (INIS)

    Xyloglucan is a major hemicellulose polysaccharide in plant cell walls. Biosynthesis of such cell wall polysaccharides is closely linked to the process of plant cell growth and development. Xyloglucan polysaccharides consist of a β-1,4 glucan backbone synthesized by xyloglucan synthase and sidechains of xylose, galactose, and fucose added by other transferase enzymes. Most plant Golgi and plasma membranes also contain glucan synthases I ampersand II, which make β-1,4 and β-1,3 glucans, respectively. All of these enzymes have very similar activities. Cell walls on suspension-cultured cells from Acer pseudoplatanus (sycamore maple) were enzymatically softened prior to cell disruption by passing through a 30 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on top-loaded or flotation sucrose density gradients. Samples were collected by gradient fractionation and assayed for membrane markers and xyloglucan and glucan synthase activities. Standard marker assays (cyt. c reductase for eR, IDPase ampersand UDPase for Golgi, and eosin 5'-malelmide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of 14C-labeled sugars from UDP-glucose and UDP-xylose was used to detect xyloglucan synthase, glucan synthases I ampersand II, and xylosyl transferase in Golgi membrane fractions. These activities overlapped, although distinct peaks of xyloglucan synthase and xylosyl transferase were found. Ca++ had a stimulatory effect on glucan synthases I ampersand II, while Mn++ had an inhibitory effect on glucan synthase I in the presence of Ca++. The similarity of these various synthase activities demonstrates the need for careful structural characterization of newly synthesized polysaccharides

  17. Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

    Directory of Open Access Journals (Sweden)

    Blachutzik Jörg O

    2012-08-01

    Full Text Available Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

  18. Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone.

    Science.gov (United States)

    Welle, R; Grisebach, H

    1989-07-01

    Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone. PMID:2500065

  19. Mechanisms and impact of ceramide phosphoethanolamine biosynthesis

    NARCIS (Netherlands)

    Hentes Vacaru, A.M.

    2009-01-01

    Sphingolipids represent an essential class of membrane molecules in eukaryotic cells. They are primarily found in the outer leaflet of the plasma membrane where they help create a rigid and impermeable barrier to the extracellular environment. While sphingomyelin (SM) is the most abundant sphingolip

  20. Increase in sphingolipid catabolic enzyme activity during aging

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Hae-young CHUNG; Fumikazu OKAJIMA; Dong-soon IM

    2009-01-01

    Aim:To understand the contribution of sphingolipid metabolism and its metabolites to development and aging.Methods: A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats.Results: Catabolic enzyme activities as well as the level of sphingomyelinase (SMase) and ceramidase (CDase) were higher than that of anabolic enzyme activities, sphingomyelin synthase and ceramide synthase. This suggested an accumulation of ceramide and sphingosine during development and aging. The liver showed the highest neutral-SMase activity among the tested enzymes while the kidney and brain exhibited higher neutral-SMase and ceramidase activities, indicating a high production of ceramide in liver and ceramide/sphingosine in the kidney and brain. The activities of sphingolipid metabolic enzymes were significantly elevated in all tested tissues during development and aging, although the onset of significant increase in activity varied on the tissue and enzyme type. During aging, 18 out of 21 enzyme activities were further increased on day 720 compared to day 180.Conclusion: Differential increases in sphingolipid metabolic enzyme activities suggest that sphingolipids including ceramide and sphingosine might play important and dynamic roles in proliferation, differentiation and apoptosis during development and aging.

  1. Delayed Biosynthesis of Varicella-Zoster Virus Glycoprotein C: Upregulation by Hexamethylene Bisacetamide and Retinoic Acid Treatment of Infected Cells

    OpenAIRE

    Storlie, Johnathan; Jackson, Wallen; Hutchinson, Jennifer; Grose, Charles

    2006-01-01

    In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new...

  2. Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Tani, Motohiro; Kuge, Osamu

    2012-12-01

    Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. PMID:23062277

  3. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

    Science.gov (United States)

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  4. Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

    NARCIS (Netherlands)

    Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; van Dam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

    2010-01-01

    We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal

  5. Revealing fosfomycin primary effect on Staphylococcus aureus transcriptome: modulation of cell envelope biosynthesis and phosphoenolpyruvate induced starvation

    Directory of Open Access Journals (Sweden)

    Gruden Kristina

    2010-06-01

    Full Text Available Abstract Background Staphylococcus aureus is a highly adaptable human pathogen and there is a constant search for effective antibiotics. Fosfomycin is a potent irreversible inhibitor of MurA, an enolpyruvyl transferase that uses phosphoenolpyruvate as substrate. The goal of this study was to identify the pathways and processes primarily affected by fosfomycin at the genome-wide transcriptome level to aid development of new drugs. Results S. aureus ATCC 29213 cells were treated with sub-MIC concentrations of fosfomycin and harvested at 10, 20 and 40 minutes after treatment. S. aureus GeneChip statistical data analysis was complemented by gene set enrichment analysis. A visualization tool for mapping gene expression data into biological pathways was developed in order to identify the metabolic processes affected by fosfomycin. We have shown that the number of significantly differentially expressed genes in treated cultures increased with time and with increasing fosfomycin concentration. The target pathway - peptidoglycan biosynthesis - was upregulated following fosfomycin treatment. Modulation of transport processes, cofactor biosynthesis, energy metabolism and nucleic acid biosynthesis was also observed. Conclusions Several pathways and genes downregulated by fosfomycin have been identified, in contrast to previously described cell wall active antibiotics, and was explained by starvation response induced by phosphoenolpyruvate accumulation. Transcriptomic profiling, in combination with meta-analysis, has been shown to be a valuable tool in determining bacterial response to a specific antibiotic.

  6. Isoprenoid Biosynthesis. Metabolite Profiling of Peppermint Oil Gland Secretory Cells and Application to Herbicide Target Analysis1

    Science.gov (United States)

    Lange, B. Markus; Ketchum, Raymond E.B.; Croteau, Rodney B.

    2001-01-01

    Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides. PMID:11553758

  7. Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana

    Directory of Open Access Journals (Sweden)

    Jones A Maxwell P

    2012-05-01

    Full Text Available Abstract Background Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L. was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. Results This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L. leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM, an inhibitor of phenylalanine ammonia lyase (PAL, reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27 in controls to 65.3% (±4.60. Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59 by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. Conclusions This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived

  8. Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

    Science.gov (United States)

    Thomas, T; Shah, N; Klinge, C M; Faaland, C A; Adihkarakunnathu, S; Gallo, M A; Thomas, T J

    1999-04-01

    We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells. PMID:10194516

  9. Engineering Microbial Cells for the Biosynthesis of Natural Compounds of Pharmaceutical Significance

    Directory of Open Access Journals (Sweden)

    Philippe Jeandet

    2013-01-01

    Full Text Available Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumor, anticancer, antiviral, antihypertensive, antiparasitic, antioxidant, immunological agents, and antibiotics as well as hormones, belonging to various chemical families, for instance, terpenoids, alkaloids, polyphenols, polyketides, amines, and proteins. Engineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents. Recent progresses that have been made since the millennium beginning with metabolic engineering of microorganisms for the biosynthesis of natural products of pharmaceutical significance will be reviewed.

  10. Network-based analysis of the sphingolipid metabolism in hypertension

    DEFF Research Database (Denmark)

    Fenger, Mogens; Linneberg, Allan; Jeppesen, Jørgen

    2015-01-01

    -step procedure is presented in which physiological heterogeneity is disentangled and genetic effects are analyzed by variance decomposition of genetic interactions and by an information theoretical approach including 162 single nucleotide polymorphisms (SNP) in 84 genes in the sphingolipid metabolism and related...

  11. Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway

    Institute of Scientific and Technical Information of China (English)

    XU; Maojun; DONG; Jufang; ZHU; Muyuan

    2006-01-01

    Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal

  12. Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

    Directory of Open Access Journals (Sweden)

    Blewett Ann

    2008-12-01

    Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

  13. Biological Effects of Naturally Occurring Sphingolipids, Uncommon Variants, and Their Analogs.

    Science.gov (United States)

    Lai, Mitchell K P; Chew, Wee Siong; Torta, Federico; Rao, Angad; Harris, Greg L; Chun, Jerold; Herr, Deron R

    2016-09-01

    Sphingolipids (SPs) comprise a highly diverse class of lipids that serve biological roles both as structural components of cell membranes and as mediators of cell signaling. Pharmacologic and genetic manipulation of SPs and their signaling systems have underscored their importance in most biological processes, including central nervous system development and function. Likewise, perturbations of SP accumulation or signaling have been associated with a number of disease states, such as neural tube defects, neuroinflammation, stroke, and dementia. SPs can be endogenously synthesized de novo, and their metabolism is a well-regulated process, so their value as nutraceuticals has not been scrutinized. However, there is evidence that sphingolipid-rich diets can affect lipid homeostasis, and several mycotoxins are SP analogs that are known to cause profound derangement of SP metabolism or signaling. Furthermore, plants and invertebrates have SP species that are not present in mammals. Several of these have been shown to induce biological responses in mammalian cells. These findings suggest that dietary intake of SPs or SP analogs may have significant effects on human health or disease outcome. This manuscript provides an overview of SP metabolism and signaling, their perturbations in neurological diseases, as well as potential impacts of modulating this system in the brain. PMID:27393119

  14. Biosynthesis of ascites sialoglycoprotein-1, the major O-linked glycoprotein of 13762 rat mammary adenocarcinoma ascites cells

    Energy Technology Data Exchange (ETDEWEB)

    Spielman, J.

    1987-01-01

    The present studies were undertaken to determine the timing of the major events in biosynthesis, and to characterize the contributions of chain initiation and elongation in maturation of the glycoprotein. Initiation of the earliest O-linked chains was detected by analysis of conversion of {sup 3}H-thr to {sup 3}H 2-aminobutyrate following mild alkaline borohydride elimination of O-linked sugars from peanut lectin-precipitated ASGP-1. Initiation was detected within 5 min of translation; amino sugar analysis of GlcNH{sub 2}-labeled, trypsinized cells also showed that GalNAc was added as late as 5 min prior to arrival of ASGP-1 at the cell surface. Thus initiation occurs throughout biosynthesis. Maturation of the glycoprotein from a lightly-glycosylated immature form to the heavily-glycosylated mature from involved both continued initiation of new chains and chain elongation, and occurred with a half-time of about 30 min. Analysis of labeled ASGP-1 released from the cell surface by trypsinization showed that although some newly-synthesized ASGP-1 reached the cell surface within 70-80 min of protein synthesis, the half-time for appearance of mature glycoprotein was in excess of 4 hr, indicating that most molecules reside in an intracellular compartment(s) for a considerable time.

  15. RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

    Science.gov (United States)

    Cascajo, María V; Abdelmohsen, Kotb; Noh, Ji Heon; Fernández-Ayala, Daniel J M; Willers, Imke M; Brea, Gloria; López-Lluch, Guillermo; Valenzuela-Villatoro, Marina; Cuezva, José M; Gorospe, Myriam; Siendones, Emilio; Navas, Plácido

    2016-07-01

    Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

  16. Fumonisin B₁ (FB₁) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

    Science.gov (United States)

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-03-24

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B₁ (FB₁) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB₁ at any concentration on dermal or epidermal cells. However, FB₁ significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB₁ (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB₁ impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB₁ might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB₁ on the equine hoof in more detail.

  17. Fumonisin B₁ (FB₁) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

    Science.gov (United States)

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-04-01

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B₁ (FB₁) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB₁ at any concentration on dermal or epidermal cells. However, FB₁ significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB₁ (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB₁ impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB₁ might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB₁ on the equine hoof in more detail. PMID:27023602

  18. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Aoki, Takashi [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  19. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    International Nuclear Information System (INIS)

    Highlights: ► We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. ► Disruption of the cpsII gene significantly reduced the growth of epimastigotes. ► In particular, the CPSII-null mutant severely retarded intracellular growth. ► The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes—an insect form—possess both activities, amastigotes—an intracellular replicating form of T. cruzi—are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  20. Melanin biosynthesis inhibitory activity of a compound isolated from young green barley (Hordeum vulgare L.) in B16 melanoma cells.

    Science.gov (United States)

    Meng, Tian Xiao; Irino, Nobuto; Kondo, Ryuichiro

    2015-07-01

    In the course to find compounds that inhibit melanin biosynthesis (i.e., whitening agents), we evaluated the effects of the methanol-soluble fraction (i.e., the water-soluble portion of methanol extracts-CHP20P-MeOH eluted fraction) from young green barley leaves on melanin production in B16 melanoma cells. Activity-guided fractionation led to an isolate called tricin (compound 1) as an inhibitory compound of melanin production in B16 melanoma cells. Furthermore, tricin analogs such as tricetin, tricetin trimethyl ether, luteolin, and apigenin were used for analyzing the structure-activity relationships (SAR) of 5,7-dihydroxyflavones studies. Tricin demonstrated stronger inhibitory activity compared to three other compounds. The results suggest that a hydroxyl group at the C-4' position and methoxy groups at the C-3',5' positions of the tricin skeleton may have important roles in this inhibitory activity in B16 melanoma cells. Our results suggest that tricin inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent. PMID:25827948

  1. A method for analysis and design of metabolism using metabolomics data and kinetic models: Application on lipidomics using a novel kinetic model of sphingolipid metabolism.

    Science.gov (United States)

    Savoglidis, Georgios; da Silveira Dos Santos, Aline Xavier; Riezman, Isabelle; Angelino, Paolo; Riezman, Howard; Hatzimanikatis, Vassily

    2016-09-01

    We present a model-based method, designated Inverse Metabolic Control Analysis (IMCA), which can be used in conjunction with classical Metabolic Control Analysis for the analysis and design of cellular metabolism. We demonstrate the capabilities of the method by first developing a comprehensively curated kinetic model of sphingolipid biosynthesis in the yeast Saccharomyces cerevisiae. Next we apply IMCA using the model and integrating lipidomics data. The combinatorial complexity of the synthesis of sphingolipid molecules, along with the operational complexity of the participating enzymes of the pathway, presents an excellent case study for testing the capabilities of the IMCA. The exceptional agreement of the predictions of the method with genome-wide data highlights the importance and value of a comprehensive and consistent engineering approach for the development of such methods and models. Based on the analysis, we identified the class of enzymes regulating the distribution of sphingolipids among species and hydroxylation states, with the D-phospholipase SPO14 being one of the most prominent. The method and the applications presented here can be used for a broader, model-based inverse metabolic engineering approach.

  2. Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Murat Balaban

    2011-12-01

    Full Text Available Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that

  3. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells

    OpenAIRE

    Rischer, Heiko; Orešič, Matej; Seppänen-Laakso, Tuulikki; Katajamaa, Mikko; Lammertyn, Freya; Ardiles-Diaz, Wilson; Van Montagu, Marc C. E.; Inzé, Dirk; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2006-01-01

    Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most,...

  4. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  5. A quinazoline-based HDAC inhibitor affects gene expression pathways involved in cholesterol biosynthesis and mevalonate in prostate cancer cells.

    Science.gov (United States)

    Lin, Z; Bishop, K S; Sutherland, H; Marlow, G; Murray, P; Denny, W A; Ferguson, L R

    2016-03-01

    Chronic inflammation can lead to the development of cancers and resolution of inflammation is an ongoing challenge. Inflammation can result from dysregulation of the epigenome and a number of compounds that modify the epigenome are in clinical use. In this study the anti-inflammatory and anti-cancer effects of a quinazoline epigenetic-modulator compound were determined in prostate cancer cell lines using a non-hypothesis driven transcriptomics strategy utilising the Affymetrix PrimeView® Human Gene Expression microarray. GATHER and IPA software were used to analyse the data and to provide information on significantly modified biological processes, pathways and networks. A number of genes were differentially expressed in both PC3 and DU145 prostate cancer cell lines. The top canonical pathways that frequently arose across both cell lines at a number of time points included cholesterol biosynthesis and metabolism, and the mevalonate pathway. Targeting of sterol and mevalonate pathways may be a powerful anticancer approach. PMID:26759180

  6. Nitric oxide mediates the fungal elicitor-induced Taxol biosynthesis of Taxus chinensis suspension cells through the reactive oxygen species-dependent and-independent signal pathways

    Institute of Scientific and Technical Information of China (English)

    XU Maojun; DONG Jufang

    2006-01-01

    Nitric oxide and reactive oxygen species are two important signal molecules that play key roles in plant defense responses. Nitric oxide generation and oxidative burst and accumulation of reactive oxygen species are the early reactions of Taxus chinensis suspension cells to fungal elicitor prepared from the cell walls of Penicillium citrinum. In order to investigate the relationship and/or interactions of nitric oxide and reactive oxygen species in the elicitor-induced Taxol biosynthesis of T. chinensis suspension cells, we treated the cells with nitric oxide specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetra- methylimidazoline-1-oxyl-3-oxide (cPITO), nitric oxide synthase inhibitor S,S(-1,3-phenylene-bis(1,2-eth- anediyl)-bis-isothiourea (PBITU), membrane NAD(P) H oxidase inhibitor diphenylene iodonium (DPI), superoxide dismutases (SOD) and catalase. The results show that pretreatment of T. chinensis cells with cPITO and DPI inhibited not only the elicitor-induced nitric oxide biosynthesis and oxidative burst, but also the elicitor-induced Taxol production, suggesting that both nitric oxide and reactive oxygen species are involved in elicitor-induced Taxol biosynthesis. Furthermore, pretreatment of the cells with cPITO and PBITU suppressed the elicitor-induced oxidative burst, indicating that the oxidative burst might be dependent on NO. Application of nitric oxide via its donor sodium nitroprusside (SNP) triggered Taxol biosynthesis of T. chinensis cells. The nitric oxide-induced Taxol production was suppressed by DPI, showing that the oxidative burst is involved in NO-triggered Taxol biosynthesis. However, nitric oxide and the fungal elicitor induced Taxol biosynthesis even though the accumulation of reactive oxygen species wass completely abolished in T. chinensis cells. Our data show that nitric oxide may mediate the elicitor-induced Taxol biosynthesis of T. chinensis suspension cells through both reactive oxygen species-dependent and -independent signal

  7. Exploring the use of cDNA-AFLP with leaf protoplasts as a tool to study primary cell wall biosynthesis in potato

    NARCIS (Netherlands)

    Oomen, R.J.F.J.; Bergervoet-van Deelen, J.E.M.; Bachem, C.W.B.; Visser, R.G.F.; Vincken, J.P.

    2003-01-01

    An RNA fingerprinting study of potato leaf protoplasts was performed to explore its suitability for identifying candidate genes involved in primary cell wall biosynthesis. Microscopic analysis, using calcofluor white to stain cellulose, showed that the protoplasts generated a new cell wall in the fi

  8. Expression, biosynthesis and release of preadipocyte factor-1/ delta-like protein/fetal antigen-1 in pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, B N; Carlsson, C; Møldrup, Annette;

    2003-01-01

    the biosynthesis and processing of Pref-1 to the soluble form, FA1, in pancreatic islets and insulinoma cells transfected with Pref-1 cDNA. We measured the release of FA1 by ELISA and the possible effect of FA1 in GH-stimulated beta-cell proliferation by incorporation of bromodeoxyuridine (BrdU) in insulin...

  9. Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow.

    Science.gov (United States)

    Watanabe, Hiroshi; Okahara, Kyohei; Naito-Matsui, Yuko; Abe, Mitsuhiro; Go, Shinji; Inokuchi, Jinichi; Okazaki, Toshiro; Kobayashi, Toshihide; Kozutsumi, Yasunori; Oka, Shogo; Takematsu, Hiromu

    2016-07-01

    Endomitosis is a special type of mitosis in which only cytokinesis-the final step of the cell division cycle-is defective, resulting in polyploid cells. Although endomitosis is biologically important, its regulatory aspects remain elusive. Psychosine, a lysogalactosylceramide, prevents proper cytokinesis when supplemented to proliferating cells. Cytokinetic inhibition by psychosine does not inhibit genome duplication. Consequently cells undergo multiple rounds of endomitotic cell cycles, resulting in the formation of giant multiploid cells. Here we successfully quantified psychosine-triggered multiploid cell formation, showing that membrane sphingolipids ratios modulate psychosine-triggered polyploidy in Namalwa cells. Among enzymes that experimentally remodel cellular sphingolipids, overexpression of glucosylceramide synthase to biosynthesize glycosylsphingolipids (GSLs) and neutral sphingomyelinase 2 to hydrolyze sphingomyelin (SM) additively enhanced psychosine-triggered multiploidy; almost all of the cells became polyploid. In the presence of psychosine, Namalwa cells showed attenuated cell surface SM clustering and suppression of phosphatidylinositol 4,5-bisphosphate production at the cleavage furrow, both important processes for cytokinesis. Depending on the sphingolipid balance between GSLs and SM, Namalwa cells could be effectively converted to viable multiploid cells with psychosine. PMID:27170180

  10. Auxin Biosynthesis

    OpenAIRE

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then unde...

  11. Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.

    Science.gov (United States)

    Sperling, P; Libisch, B; Zähringer, U; Napier, J A; Heinz, E

    2001-04-15

    The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves. PMID:11368168

  12. Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration

    Science.gov (United States)

    Chen, Kuchuan; Lin, Guang; Haelterman, Nele A; Ho, Tammy Szu-Yu; Li, Tongchao; Li, Zhihong; Duraine, Lita; Graham, Brett H; Jaiswal, Manish; Yamamoto, Shinya; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Mutations in Frataxin (FXN) cause Friedreich’s ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. We show that loss of frataxin homolog (fh) in Drosophila leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA. DOI: http://dx.doi.org/10.7554/eLife.16043.001 PMID:27343351

  13. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS

    OpenAIRE

    Jia-Ning Mi; Jing-Rong Wang; Zhi-Hong Jiang

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the d...

  14. CCR1, an enzyme required for lignin biosynthesis in Arabidopsis, mediates cell proliferation exit for leaf development

    DEFF Research Database (Denmark)

    Xue, Jingshi; Luo, Dexian; Xu, Deyang;

    2015-01-01

    exit in leaves. CCR1 is expressed basipetally in the leaf, and ccr1 mutants exhibited multiple abnormalities, including increased cell proliferation. The ccr1 phenotypes are not due to the reduced lignin content, but instead are due to the dramatically increased level of ferulic acid (FeA......), an intermediate in lignin biosynthesis. FeA is known to have antioxidant activity, and the levels of reactive oxygen species (ROS) in ccr1 were markedly reduced. We also characterized another double mutant in CAFFEIC ACID O-METHYLTRANSFERASE (comt) and CAFFEOYL CoA 3-O-METHYLTRANSFERASE (ccoaomt), in which the FeA...... level was dramatically reduced. Cell proliferation in comt ccoaomt leaves was decreased, accompanied by elevated ROS levels, and the mutant phenotypes were partially rescued by treatment with FeA or another antioxidant (N-acetyl-L-cysteine). Taken together, our results suggest that CCR1, FeA and ROS...

  15. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays

    DEFF Research Database (Denmark)

    Henriksen, Signe Teuber; Liu, J.; Estiu, G.;

    2010-01-01

    The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use...... in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking...

  16. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets

    Science.gov (United States)

    Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Leme, Adriana Franco Paes; Colleoni, Gisele Wally Braga

    2015-01-01

    Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings. PMID:26807199

  17. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia.

    Science.gov (United States)

    Melland-Smith, Megan; Ermini, Leonardo; Chauvin, Sarah; Craig-Barnes, Hayley; Tagliaferro, Andrea; Todros, Tullia; Post, Martin; Caniggia, Isabella

    2015-04-01

    Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.

  18. The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly.

    Science.gov (United States)

    Lashbrooke, Justin; Adato, Avital; Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Granell, Antonio; Costa, Fabrizio; Aharoni, Asaph

    2015-12-01

    The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.

  19. Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients

    Directory of Open Access Journals (Sweden)

    Gallagher Michael F

    2009-12-01

    Full Text Available Abstract Background Tumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA regulation in two populations of malignant Embryonal Carcinoma (EC stem cell, which differentiate (NTera2 or remain undifferentiated (2102Ep during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC patient samples. Methods miRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR. Results Our analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples. Conclusion miRNA biosynthesis and

  20. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    Science.gov (United States)

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants. PMID:27294968

  1. Autophagy in the light of sphingolipid metabolism

    DEFF Research Database (Denmark)

    Harvald, Eva Bang; Olsen, Anne Sofie Braun; Færgeman, Nils J.

    2015-01-01

    , has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural......Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components...

  2. Radiometric investigation of biosynthesis of certain macromolecules in cell cultures inoculated with intracellularly multiplied Yersinia pseudotuberculosis and Listeria monocytogenes

    International Nuclear Information System (INIS)

    The study was conducted to investigate the changes in the biosynthesis of DNA, RNA and protein in epithelian cells from monkey kidney in the case of invasion by initial and passage Yersinia and Listeria strains, multiplied intracellularly. The Yersinia pseudotuberculosis strains IB 373 and IB 373-GP and Listeria monocytogenes strains 760 1,2a and 383-4b obtained by several passages through guinea pigs and their passage subculture were used. Inoculated cultures were labelled with 3H-thymidine, 3H uridine and 14C-valine. The results showed that in all cases 3H-thymidine and 3H-uridine inclusions into cell cultures, invaded with intracellularly multiplied Yersinia strains showed statistically higher values as compared to those from cell cultures invaded with the initial strain. 14C-valine inclusion in cells, invaded by passage strains, decreased reaching its lowest values in cell populations invaded by Yersiniae isolated during the 9th passage. In cell cultures, invaded by passage L. monocytogenes strains, a rise in the levels of 3H-thymidine and 3H-uridine inclusions was observed. Inclusion values of the precursors were higher in cell populations invaded by the monocytogenes 760-1,2a initial strain and by passage strains obtained from it. The 14C-valine inclusion varied in epithelial cells invaded by Listeriae of sero-type 1,2a and by those of sero type 4b. In the first group the highest inclusion values were recorded in epithelian cells invaded by Listeriae of the 1st passage and in the second in those invaded by Listerae isolated during the 3rd and 4th passages when their most active multiplication accured

  3. Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

    Science.gov (United States)

    Gomes, Melissa M; Coimbra, Janine B; Clara, Renan O; Dörr, Felipe A; Moreno, Ana Carolina R; Chagas, Jair R; Tufik, Sérgio; Pinto, Ernani; Catalani, Luiz H; Campa, Ana

    2014-04-01

    Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies.

  4. Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

    Science.gov (United States)

    Gomes, Melissa M; Coimbra, Janine B; Clara, Renan O; Dörr, Felipe A; Moreno, Ana Carolina R; Chagas, Jair R; Tufik, Sérgio; Pinto, Ernani; Catalani, Luiz H; Campa, Ana

    2014-04-01

    Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies. PMID:24508833

  5. Characterization of AnNce102 and its role in eisosome stability and sphingolipid biosynthesis

    OpenAIRE

    Alexandros Athanasopoulos; Christos Gournas; Sotiris Amillis; Vicky Sophianopoulou

    2015-01-01

    The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstra...

  6. Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.

    Science.gov (United States)

    Monette, Jeffrey S; Gómez, Luis A; Moreau, Régis F; Bemer, Brett A; Taylor, Alan W; Hagen, Tory M

    2010-07-23

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

  7. Ceramide signalling: regulatory role in cell proliferation, differentiation and apoptosis in human epidermis.

    Science.gov (United States)

    Geilen, C C; Wieder, T; Orfanos, C E

    1997-09-01

    The stratum corneum of vertebrates is a major structural compartment that provides mechanical protection and prevents skin desiccation. The water barrier function of the stratum corneum was first reported in 1944, and this was shown later to be associated with multilayered lipid lamellae localized in the extracellular spaces. The major lipid components isolated from the cornified epidermal layers are ceramides, which belong to the class of sphingolipids, cholesterol and free fatty acids; their biosynthesis is in tight relationship with the cutaneous barrier function. In studies in which the barrier is artificially disturbed, lipid biosynthesis is found to be directly regulated by barrier permeability. As mentioned above, the ceramides involved in this process are located in the extracellular spaces of the upper epidermal layers, whereas sphingomyelin, the most common sphingolipid, is an integral part of the bilayer plasma membrane of the keratinocytes. During the last few years, however, increasing evidence has shown that sphingolipids may also take part in cell signalling, and the term 'sphingomyelin cycle' has been coined to describe this novel path-way of signal transduction. Intracellular messengers of the sphingomyelin cycle are ceramides as the products of an agonist-stimulated sphingomyelin hydrolysis. Increased levels of intracellular ceramides induce cell differentiation and/or apoptosis and reduce cell proliferation. In contrast to the extracellular barrier-forming ceramides which are complex partly O-acylated species containing long-chain fatty acids, intracellular signal-transducing ceramides are not O-acylated and have acyl chain lengths of 16 and 18 carbon atoms. We present here a review of our present knowledge on the sphingomyelin cycle as a possible signal transduction pathway in the human epidermis. We discuss the common origin of extracellular ceramides constituting the lipid barrier and of intracellular ceramides generated by agonist

  8. Influenza Viral Manipulation of Sphingolipid Metabolism and Signaling to Modulate Host Defense System

    Directory of Open Access Journals (Sweden)

    Madhuvanthi Vijayan

    2014-01-01

    Full Text Available Viruses attempt to create a distinctive cellular environment to favor viral replication and spread. Recent studies uncovered new functions of the sphingolipid signaling/metabolism during pathogenic virus infections. While sphingolipids such as sphingomyelin and ceramide were reported to influence the entry step of several viruses, sphingolipid-metabolizing enzymes could directly alter viral replication processes. Influenza virus was shown to increase the level of sphingosine kinase (SK 1 to promote virus propagation. The mechanism involves regulation of intracellular signaling pathways, leading to the amplification of influenza viral RNA synthesis and nuclear export of viral ribonucleoprotein (RNP complex. However, bovine viral diarrhea virus inhibits SK1 to enhance the efficacy of virus replication, demonstrating the presence of virus-specific strategies for modulation of the sphingolipid system. Therefore, investigating the sphingolipid metabolism and signaling in the context of virus replication could help us design innovative therapeutic approaches to improve human health.

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

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  10. Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells

    OpenAIRE

    1987-01-01

    The tumor-promoting phorbol ester PMA induces changes in the histiocytic human lymphoma cell line U-937 akin to cellular differentiation (Ralph, P., N. Williams, M. A. S. Moore, and P. B. Litcofsky, 1982, Cell. Immunol., 71:215-223) and concomitantly stimulates the biosynthesis of plasminogen activator inhibitor 2 (PAI 2) and of urokinase-type plasminogen activator (u-PA). PAI 2 is found in a nonglycosylated intracellular and a glycosylated secreted form. The former appears to be identical to...

  11. NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants

    OpenAIRE

    Nakano, Yoshimi; Yamaguchi, Masatoshi; Endo, Hitoshi; Rejab, Nur Ardiyana; Ohtani, Misato

    2015-01-01

    Plant cells biosynthesize primary cell walls (PCW) in all cells and produce secondary cell walls (SCWs) in specific cell types that conduct water and/or provide mechanical support, such as xylem vessels and fibers. The characteristic mechanical stiffness, chemical recalcitrance, and hydrophobic nature of SCWs result from the organization of SCW-specific biopolymers, i.e., highly ordered cellulose, hemicellulose, and lignin. Synthesis of these SCW-specific biopolymers requires SCW-specific enz...

  12. 7-Ketocholesterol Incorporation into Sphingolipid/Cholesterol-enriched (Lipid Raft) Domains Is Impaired by Vitamin E

    Science.gov (United States)

    Royer, Marie-Charlotte; Lemaire-Ewing, Stéphanie; Desrumaux, Catherine; Monier, Serge; Pais de Barros, Jean-Paul; Athias, Anne; Néel, Dominique; Lagrost, Laurent

    2009-01-01

    Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike γ-tocopherol, the α-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, α-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with α-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of α-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by α-tocopherol, but not γ-tocopherol pretreatment. PMID:19351882

  13. Sphingolipid symmetry governs membrane lipid raft structure.

    Science.gov (United States)

    Quinn, Peter J

    2014-07-01

    Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37°C in mixtures containing up to 20mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.

  14. Sphingolipids: A Potential Molecular Approach to Treat Allergic Inflammation

    Directory of Open Access Journals (Sweden)

    Wai Y. Sun

    2012-01-01

    Full Text Available Allergic inflammation is an immune response to foreign antigens, which begins within minutes of exposure to the allergen followed by a late phase leading to chronic inflammation. Prolonged allergic inflammation manifests in diseases such as urticaria and rhino-conjunctivitis, as well as chronic asthma and life-threatening anaphylaxis. The prevalence of allergic diseases is profound with 25% of the worldwide population affected and a rising trend across all ages, gender, and racial groups. The identification and avoidance of allergens can manage this disease, but this is not always possible with triggers being common foods, prevalent air-borne particles and only extremely low levels of allergen exposure required for sensitization. Patients who are sensitive to multiple allergens require prophylactic and symptomatic treatments. Current treatments are often suboptimal and associated with adverse effects, such as the interruption of cognition, sleep cycles, and endocrine homeostasis, all of which affect quality of life and are a financial burden to society. Clearly, a better therapeutic approach for allergic diseases is required. Herein, we review the current knowledge of allergic inflammation and discuss the role of sphingolipids as potential targets to regulate inflammatory development in vivo and in humans. We also discuss the benefits and risks of using sphingolipid inhibitors.

  15. Xyloglucan and its biosynthesis

    Directory of Open Access Journals (Sweden)

    Olga A Zabotina

    2012-06-01

    Full Text Available The hemicellulosic polysaccharide xyloglucan (XyG, found in the primary cell walls of most plant tissues, is important for structural organization of the cell wall and regulation of growth and development. Significant recent progress in structural characterization of XyGs from different plant species has shed light on the diversification of XyG during plant evolution. Also, identification of XyG biosynthetic enzymes and examination of their interactions suggests the involvement of a multiprotein complex in XyG biosynthesis. This mini-review presents an updated overview of the diversity of XyG structures in plant taxa and recent findings on XyG biosynthesis.

  16. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS.

    Science.gov (United States)

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. PMID:26868933

  17. Engineering PQS biosynthesis pathway for enhancement of bioelectricity production in Pseudomonas aeruginosa microbial fuel cells

    DEFF Research Database (Denmark)

    Wang, Victor Bochuan; Chua, Song-Lin; Cao, Bin;

    2013-01-01

    cells with an approximate five-fold increase of maximum current density relative to the parent strain. Electrochemical analysis showed that the current increase correlates with an over-synthesis of phenazines. These results therefore demonstrate that targeting microbial cell-to-cell communication....... aeruginosa strain that produces higher concentrations of phenazines under anaerobic conditions by over-expressing the PqsE effector in a PQS negative ΔpqsC mutant. The engineered strain exhibited an improved electrical performance in microbial fuel cells (MFCs) and potentiostat-controlled electrochemical...

  18. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shucai [Northeast Normal Univ., Changchun (China); Univ. of British Columbia, Vancouver, BC (Canada); Li, Eryang [Univ. of British Columbia, Vancouver, BC (Canada); Porth, Ilga [Univ. of British Columbia, Vancouver, BC (Canada); Chen, Jin-Gui [Univ. of British Columbia, Vancouver, BC (Canada); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mansfield, Shawn D. [Univ. of British Columbia, Vancouver, BC (Canada); Douglas, Carl [Univ. of British Columbia, Vancouver, BC (Canada)

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  19. Cell wall composition and candidate biosynthesis gene expression during rice development

    DEFF Research Database (Denmark)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra;

    2016-01-01

    strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically...

  20. Biosynthesis of Monascus pigments by resting cell submerged culture in nonionic surfactant micelle aqueous solution.

    Science.gov (United States)

    Wang, Bo; Zhang, Xuehong; Wu, Zhenqiang; Wang, Zhilong

    2016-08-01

    Growing cell submerged culture is usually applied for fermentative production of intracellular orange Monascus pigments, in which accumulation of Monascus pigments is at least partially associated to cell growth. In the present work, extractive fermentation in a nonionic surfactant micelle aqueous solution was utilized as a strategy for releasing of intracellular Monascus pigments. Those mycelia with low content of intracellular Monascus pigments were utilized as biocatalyst in resting cell submerged culture. By this means, resting cell submerged culture for production of orange Monascus pigments was carried out successfully in the nonionic surfactant micelle aqueous solution, which exhibited some advantages comparing with the corresponding conventional growing cell submerged culture, such as non-sterilization operation, high cell density (24 g/l DCW) leading to high productivity (14 AU of orange Monascus pigments at 470 nm per day), and recycling of cells as biocatalyst leading to high product yield (approximately 1 AU of orange Monascus pigments at 470 nm per gram of glucose) based on energy metabolism. PMID:26971494

  1. Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

    Science.gov (United States)

    Alban, C; Joyard, J; Douce, R

    1989-05-01

    The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3

  2. (p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis.

    Science.gov (United States)

    Stott, Kristina V; Wood, Shannon M; Blair, Jimmy A; Nguyen, Bao T; Herrera, Anabel; Mora, Yannet G Perez; Cuajungco, Math P; Murray, Sean R

    2015-03-01

    Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated β-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus' re-entry into the cell cycle after termination of fatty acid starvation.

  3. Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis

    OpenAIRE

    Jennewein, Stefan; Wildung, Mark R.; Chau, MyDoanh; Walker, Kevin; Croteau, Rodney

    2004-01-01

    Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus...

  4. Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants

    Science.gov (United States)

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5–10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail. PMID:27023602

  5. Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells.

    Science.gov (United States)

    Hu, Shenqiang; Gan, Chao; Wen, Rui; Xiao, Qihai; Gou, Hua; Liu, Hehe; Zhang, Yingying; Li, Liang; Wang, Jiwen

    2014-09-15

    Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues, with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4 to 6, 6 to 8, and 8 to 10 mm) and hierarchical (F5-F1) follicles. Lepr gene expression was significantly higher in granulosa cells than in theca cells from follicles of 4 to 8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6- to 8-mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed that estradiol release was significantly stimulated, whereas progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr, Srebp1, Cyp51, StAR, and Cyp19a1 were induced by the addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17β-hsd, Cyp17, and 3β-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptin's effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor. PMID:25016410

  6. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells. PMID:24141419

  7. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  8. Human growth hormone binding and stimulation of insulin biosynthesis in cloned rat insulinoma cells

    DEFF Research Database (Denmark)

    Billestrup, Nils

    1985-01-01

    Binding of 125I labelled human growth hormone to cloned insulin producing RIN-5AH cells is described. Binding was specific for somatotropic hormones since both human and rat growth hormone could compete for binding sites, whereas much higher concentrations of lactogenic hormones were needed to in...

  9. Enzymology and Molecular Biology of Cell Wall Biosynthesis. Final Technical Report

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Peter M. Ray

    2000-04-01

    The following aspects of enzymology of cell wall synthesis were pursued under this cited grant: (1) Isolation of plasma membrane-localized glucan synthase II (GS-II) of pea; (2) Cloning of genes for possible plant GS-II components; (3) Golgi glucan synthase-I (GS-I); and (4) Golgi reversibly glycosylated protein 1 (RGP1).

  10. Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

    Science.gov (United States)

    Gaid, Mariam M; Sircar, Debabrata; Müller, Andreas; Beuerle, Till; Liu, Benye; Ernst, Ludger; Hänsch, Robert; Beerhues, Ludger

    2012-11-01

    Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

  11. Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

    International Nuclear Information System (INIS)

    A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S

  12. Towards an Understanding of How Protein Hydrolysates Stimulate More Efficient Biosynthesis in Cultured Cells

    Science.gov (United States)

    Siemensma, André; Babcock, James; Wilcox, Chris; Huttinga, Hans

    In the light of the growing demand for high quality plant-derived hydrolysates (i.e., HyPep™ and UltraPep™ series), Sheffield Bio-Science has developed a new hydrolysate platform that addresses the need for animal-free cell culture medium supplements while also minimizing variability concerns. The platform is based upon a novel approach to enzymatic digestion and more refined processing. At the heart of the platform is a rationally designed animal component-free (ACF) enzyme cocktail that includes both proteases and non-proteolytic enzymes (hydrolases) whose activities can also liberate primary components of the polymerized non-protein portion of the raw material. This enzyme system is added during a highly optimized process step that targets specific enzyme-substrate reactions to expand the range of beneficial nutritional factors made available to cells in culture. Such factors are fundamental to improving the bio-performance of the culture system, as they provide not merely growth-promoting peptides and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that improve both rate and quality of protein expression, and serve to improve culture life due to osmo-protectant and anti-apoptotic properties. Also of significant note is that, compared to typical hydrolysates, the production process is greatly reduced and requires fewer steps, intrinsically yielding a better-controlled and therefore more reproducible product. Finally, the more sophisticated approach to enzymatic digestion renders hydrolysates more amenable to sterile filtration, allowing hydrolysate end users to experience streamlined media preparation and bioreactor supplementation activities. Current and future development activities will evolve from a better understanding of the complex interactions within a handful of key biochemical pathways that impact the growth and productivity of industrially relevant organisms. Presented in this chapter are some examples of the efforts that

  13. Biosynthesis of intestinal microvillar proteins. Effect of castanospermine on cell-free synthesis of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Olsen, Jørgen

    1988-01-01

    the blocked removal of glucose residues. In contrast to its reduced expression in a mucosal explant system [(1986) Biochem. J. 240, 777-782], this molecular form of aminopeptidase N was at least as abundant in cell-free translation as its normal high mannose-glycosylated counterpart, ruling out degradation...... taking place in the rough endoplasmic reticulum. Degradation of newly produced, malprocessed enzyme must therefore occur at a later stage during intracellular transport, presumably in the sarcoplasmic reticulum or in transitional elements between this organelle and the Golgi complex....

  14. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

    Science.gov (United States)

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M.; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B.; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J.; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B.; Wiederhold, Nathan P.; Fothergill, Annette W.; Kirkpatrick, William R.; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L.; Luberto, Chiara; Nimrichter, Leonardo

    2015-01-01

    ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. PMID:26106079

  15. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    Science.gov (United States)

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  16. Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Genton, C.; Kruithof, E.K.; Schleuning, W.D.

    1987-03-01

    The tumor-promoting phorbol ester PMA induces changes in the histiocytic human lymphoma cell line U-937 akin to cellular differentiation and concomitantly stimulates the biosynthesis of plasminogen activator inhibitor 2 (PAI 2) and of urokinase-type plasminogen activator (u-PA). PAI 2 is found in a nonglycosylated intracellular and a glycosylated secreted form. The former appears to be identical to PAI 2 previously purified from placental extracts and large-scale U-937 cell cultures. The sixfold increase of PAI 2 antigen measured 24 h after PMA treatment in cell extracts and conditioned media is accompanied by an equal increase of active PAI 2 mRNA, whereas the 6 to 13-fold increase of u-PA antigen in the same samples is associated with only a 1.5-fold mRNA increase. The increase of PAI 2, but not of u-PA, biosynthesis requires transcription. A 50-fold molar excess of PAI 2 over u-PA is found in both extracts and conditioned media of PMA-treated cells. PAI 2 represents at least 0.3% of total de novo synthesized protein 24 h after induction with PMA. Thus, PAI 2, but not u-PA, is an abundant product of this precursor analogue of the mononuclear phagocyte lineage, and might represent a new marker for monocyte/macrophage differentiation.

  17. Occurrence of bioactive sphingolipids in meat and fish products

    DEFF Research Database (Denmark)

    Hellgren, Lars

    2001-01-01

    . Therefore we investigated the contents of sphingomyelin and neutral glycosphingolipids in commonly consumed meat and fish products. Sphingomyelin and glycosphingolipids were found in all foodstuffs studied. The total amount varied between 118 +/- 17 nmol/g (cod) to 589 +/- 39 nmol/g (chicken leg). Generally......, lower amounts of sphingolipids were determined in fish meat than in red meat and poultry, while poultry was the richest source of this class of lipids. However, fish meat contained a relatively high content of neutral glycolipids compared with other types of meat. Thus, in fish the ratio sphingomyelin....../neutral glycolipids varied from 1 to 2.9, while in poultry this ratio varied between 5.2 to 19.2 and in red meat it varied from 1.6 to 8.3. The fatty acid composition of sphingomyelin in fish was dominated by C24:1 (Delta (9)) or C22:1 (Delta (9)), while C16:0 and C18:0 were the dominating sphingomyelin species...

  18. [Role of a purified Sertoli cell protein (CMB-21) in the biosynthesis of Leydig cell testosterone in the immature rat].

    Science.gov (United States)

    Boujrad, N; Papadopoulos, V; Drosdowsky, M A; Carreau, S

    1989-09-01

    The existence of Sertoli cell factors which modulate the rat Leydig cell function prompted us to study the biological activity of selected proteins called CMB proteins and produced by immature rat Sertoli cells. Percoll purified Leydig cells (10(5)) from 20 days-old rats have been incubated 5 h at 32 degrees C in 1 ml Ham F12/DME medium with increasing concentrations of partially purified CMB proteins (0-1,000 ng/ml) either in presence or absence of oLH (25 ng/ml). Among the CMB proteins tested, only CMB-21 produces a dose related increase of testosterone production: from 2 to 500 pg/ml of CMB-21, testosterone output is unchanged (51 pg/10(5) cells) but 1 to 1,000 ng/ml of this protein produces a linear increase of testosterone productions (86 to 870 pg). In the presence of oLH which induces a 10-fold increase of testosterone production (499 pg), increasing doses of CMB-21 further stimulate testosterone output (775 to 2.272 pg/10(5) cells). Whatever the concentration of oLH used (0 to 50 ng/ml), CMB-21 (500 ng/ml) leads to a further 2 fold augmentation of testosterone synthesis; similarly, in the presence of dbcAMP (1 mM), CMB 21 increases the testosterone production but no effect is observed when Leydig cells are incubated in the presence of 22R-hydroxycholesterol (30 microM). The cAMP levels which are increased more than 4 fold by oLH, remain unchanged in the presence of CMB-21 either alone or with oLH, as observed when Sertoli cell culture medium is used.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

    Science.gov (United States)

    Hu, Minglue; Zhang, Xuehong; Wang, Zhilong

    2016-11-01

    Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.

  20. The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner.

    Science.gov (United States)

    Bojsen, Rasmus; Torbensen, Rasmus; Larsen, Camilla Eggert; Folkesson, Anders; Regenberg, Birgitte

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.

  1. The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane integrity in a sphingolipid dependent manner.

    Directory of Open Access Journals (Sweden)

    Rasmus Bojsen

    Full Text Available The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2. The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC and mannosyl di-(inositol phosphoryl ceramide (M(IP2C suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.

  2. Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids.

    Science.gov (United States)

    Cacas, Jean-Luc; Buré, Corinne; Grosjean, Kevin; Gerbeau-Pissot, Patricia; Lherminier, Jeannine; Rombouts, Yoann; Maes, Emmanuel; Bossard, Claire; Gronnier, Julien; Furt, Fabienne; Fouillen, Laetitia; Germain, Véronique; Bayer, Emmanuelle; Cluzet, Stéphanie; Robert, Franck; Schmitter, Jean-Marie; Deleu, Magali; Lins, Laurence; Simon-Plas, Françoise; Mongrand, Sébastien

    2016-01-01

    The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.

  3. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters.

    Science.gov (United States)

    Eich, Christina; Manzo, Carlo; de Keijzer, Sandra; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  4. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space

    Directory of Open Access Journals (Sweden)

    Zhen Yang

    2015-10-01

    Full Text Available Microcystins (MCs are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m2 not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine, MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space.

  5. Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae

    Directory of Open Access Journals (Sweden)

    Santamaria Anna

    2010-04-01

    Full Text Available Abstract Background Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT. At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production. Results The pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected. Conclusions The finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors.

  6. Polymethoxy flavones do not exert an inducing effect on the biosynthesis and secretion of insulin by pancreatic β-cells

    OpenAIRE

    Dong, Hao; Chen, Hong-Dong; ZHAO, YI-JUN; Li, Hong-Min

    2014-01-01

    Polymethoxylated flavones (PMFs), which are compounds characteristic of citrus plants, possess a wide range of biological properties, particularly affecting glucose and lipid metabolism. However, the mechanism underlying the antidiabetic activity of PMFs has not been fully elucidated. In this study, we aimed to investigate the potential effect of PMFs on the biosynthesis and secretion of insulin, which are crucial in diabetes. We investigated whether PMFs are able to induce insulin secretion ...

  7. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -chloroalanine

    Energy Technology Data Exchange (ETDEWEB)

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-09-06

    The effects of ..beta..-chloroalanine (..beta..-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with ..beta..-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with ..beta..-Cl-alanine and this treatment inhibited (/sup 14/C)serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of ..beta..-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, (/sup 14/C)serine uptake was not blocked, total lipid biosynthesis from (/sup 14/C)acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and (/sup 3/H)thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of (/sup 3/H)leucine, which was only decreased by 14%. Although ..beta..-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that ..beta..-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

  8. Inducing Effect of Dihydroartemisinic Acid in the Biosynthesis of Artemisinins with Cultured Cells of Artemisia annua by Enhancing the Expression of Genes

    Directory of Open Access Journals (Sweden)

    Jianhua Zhu

    2014-01-01

    Full Text Available Artemisinin has been used in the production of “artemisinin combination therapies” for the treatment of malaria. Feeding of precursors has been proven to be one of the most effective methods to enhance artemisinin production in plant cultured cells. At the current paper, the biosynthesis of artemisinin (ART and its four analogs from dihydroartemisinic acid (DHAA in suspension-cultured cells of Artemisia annua were investigated. ARTs were detected by HPLC/GC-MS and isolated by various chromatography methods. The structures of four DHAA metabolites, namely, dihydro-epi-deoxyarteannuin B, arteannuin I, arteannuin K, and 3-β-hydroxy-dihydro-epi-deoxyarteannuin B, were elucidated by physicochemical and spectroscopic analyses. The correlation between gene expression and ART content was investigated. The results of RT-PCR showed that DHAA could up-regulate expression of amorpha-4,11-diene synthase gene (ADS, amorpha-4,11-diene C-12 oxidase gene (CYP71AV1, and farnesyl diphosphate synthase gene (FPS (3.19-, 7.21-, and 2.04-fold higher than those of control group, resp., which indicated that biosynthesis processes from DHAA to ART were enzyme-mediated.

  9. Sphingosine Kinase 2 and Ceramide Transport as Key Targets of the Natural Flavonoid Luteolin to Induce Apoptosis in Colon Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Loubna Abdel Hadi

    Full Text Available The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC. Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P, two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

  10. Changes of sphingolipids profiles after ischemia-reperfusion injury in the rat liver

    Institute of Scientific and Technical Information of China (English)

    ZHAI Shu-ting; LIANG Ting-bo; LIU Guang-yi; XUE Fei; SUN Gong-ping; LIANG Liang; CHEN Wei; XU Guo-dong; LI Jun-jian; YANG Jun

    2009-01-01

    Background Hepatic ischemia-reperfusion (I/R) injury occurs in many clinical procedures. The molecular mechanisms responsible for hepatic I/R injury however remain unknown. Sphingolipids, in particular ceramide, play a role in stress and death receptor-induced hepatocellular death, contributing to the progression of several liver diseases including liver I/R injury. In order to further define the role of sphingolipids in hepatic I/R, systemic analysis of sphingolipids after reperfusion is necessary.Methods We investigated the lipidomic changes of sphingolipids in a rat model of warm hepatic I/R injury, by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). Results The total amounts of ceramide and sphingomyelin and the intensity of most kinds of sphingolipids, mainly sphingomyelin, significantly increased at 1 hour after reperfusion (P <0.05) and reached peaks at 6 hours after reperfusion (P<0.01) compared to controls. Six new forms of ceramide and sphingomyelins appeared 6 hours after reperfusion, they were (m/z) 537.8, 555.7, 567.7, 583.8, 683.5 and 731.4 respectively. A ceramide-monohexoside (m/z) 804.4 (CMH(d18:1C22:1+Na)~+) also increased after reperfusion and correlated with extent of liver injury after reperfursion.Conclusions Three main forms of sphingolipids, ceramide, sphingomyelin and ceramide-monohexoside, are related to hepatic I/R injury and provide a new perspective in understanding the mechanisms responsible for hepatic I/R injury.

  11. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    Directory of Open Access Journals (Sweden)

    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  12. Melatonin enhancement of the radiosensitivity of human breast cancer cells is associated with the modulation of proteins involved in estrogen biosynthesis.

    Science.gov (United States)

    Alonso-González, Carolina; González, Alicia; Martínez-Campa, Carlos; Menéndez-Menéndez, Javier; Gómez-Arozamena, José; García-Vidal, Angela; Cos, Samuel

    2016-01-01

    Enhancing the radiosensitivity of cancer cells is one of the most important tasks in clinical radiobiology. Endocrine therapy and radiotherapy are two cancer treatment modalities which are often given together in patients with locally-advanced breast cancer and positive hormone-receptor status. Oncostatic actions of melatonin are relevant on estrogen-dependent mammary tumors. In the present study, we wanted to evaluate the effects of the combination of ionizing radiation and melatonin on proteins involved in estrogen biosynthesis in breast cancer cells. We demonstrated a role of melatonin in mediating the sensitization of human breast cancer cells to the ionizing radiation by decreasing around 50% the activity and expression of proteins involved in the synthesis of estrogens in these cells. Thus, melatonin pretreatment before radiation reduces the amount of active estrogens at cancer cell level. Melatonin 1 nM induced a 2-fold change in p53 expression as compared to radiation alone. The regulatory action of melatonin on p53 could be a link between melatonin and its modulatory action on the sensitivity of breast cancer cells to ionizing radiation. These findings may have implications for designing clinical trials using melatonin and radiotherapy. PMID:26497762

  13. In vitro culture of the tropical sponge Axinella corrugata (Demospongiae): effect of food cell concentration on growth, clearance rate, and biosynthesis of stevensine.

    Science.gov (United States)

    Duckworth, Alan R; Samples, Gail A; Wright, Amy E; Pomponi, Shirley A

    2003-01-01

    In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 micro m in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h(-1) g DW(-1).Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.

  14. Sphingolipids in congenital diaphragmatic hernia; results from an international multicenter study

    NARCIS (Netherlands)

    K.G. Snoek (Kitty); I.K.M. Reiss (Irwin); J. Tibboel (Jeroen); J.M. van Rosmalen (Joost); I. Capolupo (Irma); A.F.J. van Heijst (Arno); T. Schaible (Thomas); M.R. Post (Martin); D. Tibboel (Dick)

    2016-01-01

    textabstractBackground: Congenital diaphragmatic hernia is a severe congenital anomaly with significant mortality and morbidity, for instance chronic lung disease. Sphingolipids have shown to be involved in lung injury, but their role in the pathophysiology of chronic lung disease has not been explo

  15. Low serum sphingolipids in children with attention deficit-hyperactivity disorder

    Directory of Open Access Journals (Sweden)

    Marcela Patricia Henríquez-Henríquez

    2015-08-01

    Full Text Available Background: Attention deficit-hyperactivity disorder (ADHD is the most prevalent neuropsychiatric condition in childhood. ADHD is a multifactorial trait with a strong genetic component. One neurodevelopmental hypothesis is that ADHD is associated with a lag in brain maturation. Sphingolipids are essential for brain development and neuronal functioning, but their role in ADHD pathogenesis is unexplored. We hypothesized that serum sphingolipid levels distinguish ADHD patients from unaffected subjects. Methods: We characterized serum sphingolipid profiles of ADHD patients and two control groups: non-affected relatives and non-affected subjects without a family history of ADHD. Sphingolipids were measured by LC-MS/MS in 77 participants (28 ADHD patients, 28 related controls and 21 unrelated controls. ADHD diagnosis was based on the Diagnostic and Statistical Manual of Mental Disorders (DSM IV-TR. Diagnostic criteria were assessed by 2 independent observers. Groups were compared by parametrical statistics. Results: Serum sphingomyelins C16:0, C18:0, C18:1, C24:1, ceramide C24:0 and deoxy-ceramide C24:1 were significantly decreased in ADHD patients at 20-30% relative reductions. In our sample, decreased serum sphingomyelin levels distinguished ADHD patients with 79% sensitivity and 78% specificity. Conclusions: Our results showed lower levels of all major serum sphingomyelins in ADHD. These findings may reflect brain maturation and affect neuro-functional pathways characteristic for ADHD.

  16. Antihypertensive Treatment Differentially Affects Vascular Sphingolipid Biology in Spontaneously Hypertensive Rats

    NARCIS (Netherlands)

    L.J.A. Spijkers; B.J.A. Janssen; J. Nelissen; M.J.P.M.T. Meens; D. Wijesinghe; C.E. Chalfant; J.G.R. de Mey; A.E. Alewijnse; S.L.M. Peters

    2011-01-01

    Background: We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vas

  17. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

    NARCIS (Netherlands)

    C. Eich (Christina); C. Manzo (Carlo); S. de Keijzer (Sandra); G.J. Bakker (Gert-Jan); I. Reinieren-Beeren (Inge); M.F. García-Parajo (Maria); A. Cambi (Alessandra)

    2016-01-01

    textabstractSphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how t

  18. Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism

    OpenAIRE

    Aljohani, Ayman J.; Edwards, Genea; Guerra, Yenifer; Dubovy, Sander; Miller, Darlene; Lee, Richard K; Bhattacharya, Sanjoy K.

    2014-01-01

    The mass spectrometry-identified sphingolipids and ceramides in the trabecular meshwork (TM) showed presence of nonmammalian sphingoid base Fumonisins. Taken together with Gomori methenamine silver staining and DNA amplification, this suggests widespread latent commensalism by Fusarium species in the human TM.

  19. Role of sphingolipid mediator ceramide in obesity and renal injury in mice fed a high-fat diet.

    Science.gov (United States)

    Boini, Krishna M; Zhang, Chun; Xia, Min; Poklis, Justin L; Li, Pin-Lan

    2010-09-01

    The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage. PMID:20543095

  20. Preservation of Bacillus firmus Strain 37 and Optimization of Cyclodextrin Biosynthesis by Cells Immobilized on Loofa Sponge

    Directory of Open Access Journals (Sweden)

    Cristiane Moriwaki

    2012-08-01

    Full Text Available The preservation of Bacillus firmus strain 37 cells by lyophilization was evaluated and response surface methodology (RSM was used to optimize the β-cyclodextrin (β-CD production by cells immobilized on loofa sponge. Interactions were studied with the variables temperature, pH and dextrin concentration using a central composite design (CCD. Immobilization time influence on β-CD production was also investigated. B. firmus strain 37 cells remained viable after one year of storage, showing that the lyophilization is a suitable method for preservation of the microorganism. From the three-dimensional diagrams and contour plots, the best conditions for β-CD production were determined: temperature 60 °C, pH 8, and 18% dextrin. Considering that the amount of dextrin was high, a new assay was carried out, in which dextrin concentrations of 10, 15, and 18% were tested and the temperature of 60 °C and pH 8 were maintained. The results achieved showed very small differences and therefore, for economic reasons, the use of 10% dextrin is suggested. Increasing the immobilization time of cells immobilized on synthetic sponge the β-CD production decreased and did not change for cells immobilized on loofa sponge. The results of this research are important for microorganism preservation and essential in the optimization of the biosynthesis of CD.

  1. Lignification: Flexibility, Biosynthesis and Regulation.

    Science.gov (United States)

    Zhao, Qiao

    2016-08-01

    Lignin is a complex phenolic polymer that is deposited in the secondary cell wall of all vascular plants. The evolution of lignin is considered to be a critical event during vascular plant development, because lignin provides mechanical strength, rigidity, and hydrophobicity to secondary cell walls to allow plants to grow tall and transport water and nutrients over a long distance. In recent years, great research efforts have been made to genetically alter lignin biosynthesis to improve biomass degradability for the production of second-generation biofuels. This global focus on lignin research has significantly advanced our understanding of the lignification process. Based on these advances, here I provide an overview of lignin composition, the biosynthesis pathway and its regulation. PMID:27131502

  2. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    DEFF Research Database (Denmark)

    Bojsen, Rasmus; Torbensen, Rasmus; Larsen, Camilla Eggert;

    2013-01-01

    The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicid...... observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids....... that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma...... membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX...

  3. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L.) cell suspension cultures

    International Nuclear Information System (INIS)

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway

  4. Antimicrobial Activity of Sphingolipids Isolated from the Stems of Cucumber (Cucumis sativus L.

    Directory of Open Access Journals (Sweden)

    Zhu Yu

    2010-12-01

    Full Text Available Three antimicrobial sphingolipids were separated by bioassay-guided isolation from the chloroform fraction of the crude methanol extract of cucumber (Cucumis sativus L. stems and identified as (2S,3S,4R,10E-2-[(2'R-2-hydroxytetra-cosanoylamino]-1,3,4-octadecanetriol-10-ene (1, 1-O-β-D-glucopyranosyl(2S,3S,4R,10E-2-[(2'R-2-hydroxy-tetracosanoylamino]-1,3,4-octadecanetriol-10-ene (2 and soya-cerebroside I (3 by their physicochemical properties and spectroscopic analysis. They were evaluated to show antifungal and antibacterial activity on test microorganisms including four fungal and three bacterial species. Among them, compound 1, a relatively low polarity aglycone,  exhibited stronger antimicrobial activity than its corresponding glycoside 2. The results indicated that sphingolipids could be the main antimicrobial compounds in the crude methanol extract of cucumber stems.

  5. Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

    OpenAIRE

    Fujibayashi, S; Kao, F T; Jones, C.; Morse, H; Law, M; Wenger, D A

    1985-01-01

    Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP...

  6. The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

    OpenAIRE

    Becker, Katrin Anne; Riethmüller, Joachim; Zhang, Yang; Gulbins, Erich

    2010-01-01

    Sphingolipids and in particular ceramide have been shown to be critically involved in the response to many receptor-mediated, but also receptor-independent, mainly stress stimuli. Recent studies demonstrate that ceramide plays an important role in the pathogenesis of cystic fibrosis, a hereditary metabolic disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator. Patients with cystic fibrosis suffer from chronic pulmonary inflammation and microbial lung infectio...

  7. Expression, Reinigung und biophysikalische Charakterisierung verschiedener Hydrolasen des Sphingolipid-Stoffwechsels

    OpenAIRE

    Ficht-Redmer, Susanne

    2015-01-01

    Sphingolipide sind eine wichtige Klasse von Lipiden, die nicht nur als Strukturmoleküle von Bedeutung sind sondern auch in Signaltransduktionsprozessen eine wichtige Rolle spielen. Insbesondere die Sphingolipidmetaboliten Ceramid, Sphingosin und Sphingosin-1-phosphat sind an zellulären Prozessen wie Differenzierung, Apoptose, Proliferation und Inflammation beteiligt. Sphingomyelinasen üben daher als katabole Enzyme des Sphingolipidstoffwechsels eine wichtige Funktion aus. Die vorliegende Ar...

  8. Sphingolipid metabolism correlates with cerebrospinal fluid Beta amyloid levels in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Alfred N Fonteh

    Full Text Available Sphingolipids are important in many brain functions but their role in Alzheimer's disease (AD is not completely defined. A major limit is availability of fresh brain tissue with defined AD pathology. The discovery that cerebrospinal fluid (CSF contains abundant nanoparticles that include synaptic vesicles and large dense core vesicles offer an accessible sample to study these organelles, while the supernatant fluid allows study of brain interstitial metabolism. Our objective was to characterize sphingolipids in nanoparticles representative of membrane vesicle metabolism, and in supernatant fluid representative of interstitial metabolism from study participants with varying levels of cognitive dysfunction. We recently described the recruitment, diagnosis, and CSF collection from cognitively normal or impaired study participants. Using liquid chromatography tandem mass spectrometry, we report that cognitively normal participants had measureable levels of sphingomyelin, ceramide, and dihydroceramide species, but that their distribution differed between nanoparticles and supernatant fluid, and further differed in those with cognitive impairment. In CSF from AD compared with cognitively normal participants: a total sphingomyelin levels were lower in nanoparticles and supernatant fluid; b levels of ceramide species were lower in nanoparticles and higher in supernatant fluid; c three sphingomyelin species were reduced in the nanoparticle fraction. Moreover, three sphingomyelin species in the nanoparticle fraction were lower in mild cognitive impairment compared with cognitively normal participants. The activity of acid, but not neutral sphingomyelinase was significantly reduced in the CSF from AD participants. The reduction in acid sphingomylinase in CSF from AD participants was independent of depression and psychotropic medications. Acid sphingomyelinase activity positively correlated with amyloid β42 concentration in CSF from cognitively normal but

  9. Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study

    Science.gov (United States)

    Chatterjee, Pratishtha; Lim, Wei L.F.; Shui, Guanghou; Gupta, Veer B.; James, Ian; Fagan, Anne M.; Xiong, Chengjie; Sohrabi, Hamid R.; Taddei, Kevin; Brown, Belinda M.; Benzinger, Tammie; Masters, Colin; Snowden, Stuart G.; Wenk, Marcus R.; Bateman, Randall J.; Morris, John C.; Martins, Ralph N.

    2016-01-01

    Background and Objective Aberrant lipid metabolism has been implicated in sporadic Alzheimer’s disease (AD). The current study investigated plasma phospholipid and sphingolipid profiles in individuals carrying PSEN1 mutations responsible for autosomal dominant AD (ADAD). Methods Study participants evaluated were from the Perth and Melbourne sites of the Dominantly Inherited Alzheimer Network (DIAN) study. Plasma phospholipid and sphingolipid profiles were measured using liquid chromatography coupled with mass spectrometry in 20 PSEN1 mutation carriers (MC; eight of whom were symptomatic and twelve asymptomatic, based on Clinical Dementia Rating scores) and compared with six non carriers (NC) using linear mixed models. Further, AD gold standard biomarker data obtained from the DIAN database were correlated with lipid species significantly altered between MC and NC, using Spearman’s correlation coefficient. Results One-hundred and thirty-nine plasma phospholipid and sphingolipid species were measured. Significantly altered species in MC compared to NC primarily belonged to choline and ethanolamine containing phospholipid classes and ceramides. Further phosphatidylcholine species (34:6, 36:5, 40:6) correlated with cerebrospinal fluid tau (p < 0.05), and plasmalogen ethanolamine species (34:2, 36:,4) correlated with both cerebrospinal fluid tau and brain amyloid load within the MC group (p < 0.05). Conclusion These findings indicate altered phospholipid and sphingolipid metabolism in ADAD and provide insight into the pathomolecular changes occurring with ADAD pathogenesis. Further, findings reported in this study allow comparison of lipid alterations in ADAD with those reported previously in sporadic AD. The findings observed in the current pilot study warrant validation in the larger DIAN cohort. PMID:26836186

  10. Sphingolipid metabolism enzymes as targets for anticancer therapy

    NARCIS (Netherlands)

    Kok, JW; Sietsma, H

    2004-01-01

    Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumour cells. Unfortunately, tumour cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ce

  11. Efficient biosynthesis of γ-decalactone in ionic liquids by immobilized whole cells of Yarrowia lipolytica G3-3.21 on attapulgite.

    Science.gov (United States)

    Zhao, Yuping; Xu, Yan; Jiang, Changxing

    2015-10-01

    In this study, the biosynthesis of γ-decalactone (GDL) was successfully conducted in an ionic liquid (IL)-containing cosolvent system using immobilized cells of Yarrowia lipolytica G3-3.21 on attapulgite (ATG). We found the immobilized Y. lipolytica G3-3.21 cells in N-butyl-pyridinium tetrafluoroborate ([BPy]BF4) solution gave the highest activity of C16-Acyl-CoA oxidase and the maximum yield of GDL. The optimum immobilization conditions for the highest yield of GDL were 20 g/L of ATG, 1.5 % of CaCl2 and 2 % of sodium alginate (NaAlg). The optimal [BPy]BF4 content, buffer pH, reaction temperature, shaking speed, castor oil and glucose contents were 7.5 %, 26 °C, 150 rpm, 100 g/L and 10 %, respectively. Under the optimized conditions, the GDL yield was up to 8.05 g/L. After ten times of reuse, the GDL yield was 7.51 g/L, corresponding to 93.3 % of that obtained in the first batch, suggesting a good reusability and potential for industrial applications. PMID:26091898

  12. Ceramide signaling in cancer and stem cells

    OpenAIRE

    Bieberich, Erhard

    2008-01-01

    Most of the previous work on the sphingolipid ceramide has been devoted to its function as an apoptosis inducer. Recent studies, however, have shown that in stem cells, ceramide has additional nonapoptotic functions. In this article, ceramide signaling will be reviewed in light of ‘systems interface biology’: as an interconnection of sphingolipid metabolism, membrane biophysics and cell signaling. The focus will be on the metabolic interconversion of ceramide and sphingomyelin or sphingosine-...

  13. PLP-dependent enzymes as entry and exit gates of sphingolipid metabolism

    Science.gov (United States)

    Bourquin, Florence; Capitani, Guido; Grütter, Markus Gerhard

    2011-01-01

    Sphingolipids are membrane constituents as well as signaling molecules involved in many essential cellular processes. Serine palmitoyltransferase (SPT) and sphingosine-1-phosphate lyase (SPL), both PLP (pyridoxal 5′-phosphate)-dependent enzymes, function as entry and exit gates of the sphingolipid metabolism. SPT catalyzes the condensation of serine and a fatty acid into 3-keto-dihydrosphingosine, whereas SPL degrades sphingosine-1-phosphate (S1P) into phosphoethanolamine and a long-chain aldehyde. The recently solved X-ray structures of prokaryotic homologs of SPT and SPL combined with functional studies provide insight into the structure–function relationship of the two enzymes. Despite carrying out different reactions, the two enzymes reveal striking similarities in the overall fold, topology, and residues crucial for activity. Unlike their eukaryotic counterparts, bacterial SPT and SPL lack a transmembrane helix, making them targets of choice for biochemical characterization because the use of detergents can be avoided. Both human enzymes are linked to severe diseases or disorders and might therefore serve as targets for the development of therapeutics aiming at the modulation of their activity. This review gives an overview of the sphingolipid metabolism and of the available biochemical studies of prokaryotic SPT and SPL, and discusses the major similarities and differences to the corresponding eukaryotic enzymes. PMID:21710479

  14. Effects of Chamomilla recutita flavonoids on age-related liver sphingolipid turnover in rats.

    Science.gov (United States)

    Babenko, Nataliya A; Shakhova, Elena G

    2006-01-01

    The increased sphingolipid turnover in the liver is associated with elevation of free radical production and state of chronic inflammation at old age. Plant polyphenols are reported to exhibit antioxidant and anti-inflammatory effects. In the present paper, the lipids contents and ceramide production in the liver and hepatocytes as well as the correction of sphingolipid metabolism at old age using the mixture of Chamomilla recutita flavonoids (chamiloflan) or apigenin-7-glucoside or luteolin-7-glucoside alone have been investigated. To study the sphingolipids turnover, the [14C]serine-pre-labeled hepatocytes and [14C-methyl]- or [14C]palmitate-pre-labeled sphingomyelin (SM) and ceramide were used. The ceramide content was higher in the liver and hepatocytes of 24- and 27-28-month-old animals as compared to adult 3-month-old Wistar rats. An addition of flavonoids to the culture medium did not influence significantly on the lipids contents and metabolism in the isolated hepatocytes. The administration of flavonoids to old rats decreased the elevated neutral and acid SMases activities and ceramide mass and did not affect both the lipid content in the liver of adult animals and ceramide conversion to the sphingosine or SM. These results suggest that the SMases play a key role in the flavonoid-induced decrease of ceramide levels in the liver of old rats. PMID:16183236

  15. Examination of the Effects of a Sphingolipid-Enriched Lipid Fraction from Wheat Gluten on the Incidence of Diabetes in BBdp Rats

    OpenAIRE

    Shi, Wenjuan

    2004-01-01

    This study was designed to examine if a sphingolipid-enriched lipid fraction from wheat gluten could affect the incidence of type I diabetes in BioBreeding diabetes prone (BBdp) rats. Wheat gluten was extracted with a chloroform-methanol (CM) mixture to isolate most of the lipids. Isolated lipids were subjected to silica gel column chromatography and saponification to remove most of neutral lipids and phospholipids, leaving behind a lipid fraction enriched in sphingolipids. This sphingolipid-...

  16. The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

    Institute of Scientific and Technical Information of China (English)

    Mara de Castro; Janice G Miller; Jose Luis Acebes; Antonio Encina; Penelope Garca-Angulo; Stephen C Fry

    2015-01-01

    Cell-suspension cultures (Zea mays L., Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile (DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to define the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose, and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H] xylose residues into (hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular trafficking of polymers, especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log-phase cultures, habituation increased the proportion of 3H-hemicelluloses ([3H]xylans and [3H]xyloglucan) sloughed into the medium. These findings could be related to the cell walls’ cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also reflect the habituated cells’ reduced capacity to integrate arabinox-ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.

  17. Leukocyte Adhesion Deficiency Type II is a generalized defect of de novo GDP-fucose biosynthesis. Endothelial cell fucosylation is not required for neutrophil rolling on human nonlymphoid endothelium.

    OpenAIRE

    Karsan, A.; Cornejo, C J; Winn, R K; Schwartz, B R; Way, W; Lannir, N; Gershoni-Baruch, R; Etzioni, A; Ochs, H. D.; Harlan, J. M.

    1998-01-01

    Leukocyte Adhesion Deficiency Type II (LAD II) is a recently described syndrome and the two patients with this defect lack fucosylated glycoconjugates. These glycoconjugates include the selectin ligand, sialyl LewisX, and various fucosylated blood group antigens. To date, the molecular anomaly in these patients has not been identified. We localized the defect in LAD II to the de novo pathway of GDP-fucose biosynthesis, by inducing cell-surface expression of fucosylated glycoconjugates after e...

  18. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2014-01-01

    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

  19. Effects of yeast extract and methyl jasmonate on the enhancement of solasodine biosynthesis in cell cultures of Solanum hainanense Hance

    Directory of Open Access Journals (Sweden)

    NGUYEN HOANG LOC

    2014-04-01

    Full Text Available In this work, the effects of the elicitors methyl jasmonate (MeJA and yeast extract (YE on the growth and solasodine production of Solanum hainanense cells were investigated. The results showed that various concentrations of MeJA (50-250 µM and YE (1-4 g/L have different eliciting influences. The increase of solasodine content induced by the elicitation of 3 g/L of YE and 50 µM of MeJA at the beginning of cell culture was about 1.9- and 1.3-fold, respectively, as compared with that of the non-elicitated cells. In general, YE (biotic elicitor was more effective in enhancing solasodine production than MeJA (abiotic elicitor.

  20. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    Science.gov (United States)

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

  1. Biosynthesis, Antibacterial Activity and Anticancer Effects Against Prostate Cancer (PC-3) Cells of Silver Nanoparticles Using Dimocarpus Longan Lour. Peel Extract.

    Science.gov (United States)

    He, Yan; Du, Zhiyun; Ma, Shijing; Cheng, Shupeng; Jiang, Sen; Liu, Yue; Li, Dongli; Huang, Huarong; Zhang, Kun; Zheng, Xi

    2016-12-01

    Metal nanoparticles, particularly silver nanoparticles (AgNPs), are developing more important roles as diagnostic and therapeutic agents for cancers with the improvement of eco-friendly synthesis methods. This study demonstrates the biosynthesis, antibacterial activity, and anticancer effects of silver nanoparticles using Dimocarpus Longan Lour. peel aqueous extract. The AgNPs were characterized by UV-vis absorption spectroscopy, X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscope (FTIR). The bactericidal properties of the synthesized AgNPs were observed via the agar dilution method and the growth inhibition test. The cytotoxicity effect was explored on human prostate cancer PC-3 cells in vitro by trypan blue assay. The expressions of phosphorylated stat 3, bcl-2, survivin, and caspase-3 were examined by Western blot analysis. The longan peel extract acted as a strong reducing and stabilizing agent during the synthesis. Water-soluble AgNPs of size 9-32 nm was gathered with a face-centered cubic structure. The AgNPs had potent bactericidal activities against gram-positive and gram-negative bacteria with a dose-related effect. AgNPs also showed dose-dependent cytotoxicity against PC-3 cells through a decrease of stat 3, bcl-2, and survivin, as well as an increase in caspase-3. These findings confirm the bactericidal properties and explored a potential anticancer application of AgNPs for prostate cancer therapy. Further research should be focused on the comprehensive study of molecular mechanism and in vivo effects on the prostate cancer. PMID:27316741

  2. Biosynthesis, Antibacterial Activity and Anticancer Effects Against Prostate Cancer (PC-3) Cells of Silver Nanoparticles Using Dimocarpus Longan Lour. Peel Extract

    Science.gov (United States)

    He, Yan; Du, Zhiyun; Ma, Shijing; Cheng, Shupeng; Jiang, Sen; Liu, Yue; Li, Dongli; Huang, Huarong; Zhang, Kun; Zheng, Xi

    2016-06-01

    Metal nanoparticles, particularly silver nanoparticles (AgNPs), are developing more important roles as diagnostic and therapeutic agents for cancers with the improvement of eco-friendly synthesis methods. This study demonstrates the biosynthesis, antibacterial activity, and anticancer effects of silver nanoparticles using Dimocarpus Longan Lour. peel aqueous extract. The AgNPs were characterized by UV-vis absorption spectroscopy, X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscope (FTIR). The bactericidal properties of the synthesized AgNPs were observed via the agar dilution method and the growth inhibition test. The cytotoxicity effect was explored on human prostate cancer PC-3 cells in vitro by trypan blue assay. The expressions of phosphorylated stat 3, bcl-2, survivin, and caspase-3 were examined by Western blot analysis. The longan peel extract acted as a strong reducing and stabilizing agent during the synthesis. Water-soluble AgNPs of size 9-32 nm was gathered with a face-centered cubic structure. The AgNPs had potent bactericidal activities against gram-positive and gram-negative bacteria with a dose-related effect. AgNPs also showed dose-dependent cytotoxicity against PC-3 cells through a decrease of stat 3, bcl-2, and survivin, as well as an increase in caspase-3. These findings confirm the bactericidal properties and explored a potential anticancer application of AgNPs for prostate cancer therapy. Further research should be focused on the comprehensive study of molecular mechanism and in vivo effects on the prostate cancer.

  3. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  4. Ceramide and S1P signaling in embryonic stem cell differentiation

    OpenAIRE

    Bieberich, Erhard

    2012-01-01

    Recent studies show that bioactive lipids are important regulators for stem cell survival and differentiation. The sphingolipid ceramide and its derivative, sphingosine-1-phosphate (S1P), can act synergistically on embryonic stem (ES) cell differentiation. We show here simple methods to analyze sphingolipids in differentiating ES cells and to use ceramide and S1P analogs for the guided differentiation of mouse ES cells toward neuronal and glial lineage.

  5. The mechanism of Acetobacter xylinum cellulose biosynthesis: direction of chain elongation and the role of lipid pyrophosphate intermediates in the cell membrane

    Energy Technology Data Exchange (ETDEWEB)

    Han, N.S.; Robyt, J.F. [Laboratory of Carbohydrate Chemistry and Enzymology, Iowa State University, Ames, IA (United States)

    1998-12-01

    The biosynthesis of Acetobacter xylinum ATCC 10821 cellulose has been studied with resting cells and a membrane preparation using {sup 14}C-pulse and chase reactions, with d-glucose and UDPGlc, respectively. Cellulose was biosynthesized from UDPGlc, and it was found to be tightly associated with both the cells and the membrane. The cellulose chains could be released from the cells and the membrane preparation by treating at pH 2, 100 C for 20 min. The cellulose chains that were released from the pulse and pulse-chase reactions were purified and separated from any low molecular weight substances by gel chromatography on Bio-Gel P4. They were then reduced with sodium borohydride and hydrolyzed with 4 M trifluoroacetic acid at 121 C for 2 h. Labeled products from the acid hydrolyzates were separated by paper chromatography and found to be d-glucose and d-glucitol. The amount of radioactivity in the products was determined by liquid scintillation counting. It was found that the pulsed products from the resting cells gave a ratio of d-[{sup 14}C]glucitol to d-[{sup 14}C]glucose of 1:11, and after chasing, the ratio decreased to 1:36. The pulsed products from the membrane gave a ratio of d-[{sup 14}C]glucitol to d-[{sup 14}C]glucose of 1:12, and after chasing for 5 min the ratio decreased to 1:43, and after 10 min, the ratio decreased to 1:66. These results show that the labeled d-glucitol obtained from the reducing end of the cellulose chain is chased into the interior of the cellulose chain during synthesis, showing that the cellulose chain is elongated from the reducing end. An insertion mechanism for the synthesis of cellulose from UDPGlc is proposed that involves lipid pyrophosphate glycosyl intermediates and three membrane enzymes: lipid phosphate:UDPGlc phosphotransferase, cellulose synthase, and lipid pyrophosphate phosphohydrolase. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  6. Biosynthesis of tylophora alkaloids

    International Nuclear Information System (INIS)

    Using labelled precursors, biosynthesis of the tylophora alkaloids, tylophorine, tylophorinidine and tylophorinide has been investigated in Tylophora asthmatica plants. The radioactive precursors, phenylalanine-2-14C, benzoic acid-1-14C, benzoic acid-ring 14C, acetate-2-14C, ornithine-5-14C, acetate-2-14C, ornithine-5-14C and cinnamic acid-2-14C were administered to the plants individually by wick technique. Tylophorine was isolated in each case and assayed for its radioactivity to find out the incorporation of the label into it. The results indicate that: (1) phenylalanine via cinnamic acid is an important precursor in the biosynthesis of tylophorine (2) orinithine participates in tylophorine biosynthesis via pyrroline and (3) tylophorinidine may be a direct precursor of tylophorine. (M.G.B.)

  7. The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover

    DEFF Research Database (Denmark)

    Luo, Guangzuo; Gruhler, Albrecht; Liu, Ying;

    2008-01-01

    eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating......Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1...... and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized...

  8. Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids

    Directory of Open Access Journals (Sweden)

    Michel Oelschlägel

    2015-09-01

    Full Text Available The styrene oxide isomerase (SOI represents a membrane-bound enzyme of the microbial styrene degradation pathway and has been discussed as promising biocatalyst. It catalyzes the isomerization of styrene oxide to phenylacetaldehyde. In this study a styC gene, which encodes the SOI of Rhodococcus opacus 1CP, was optimized for optimal expression in Escherichia coli BL21(DE3 pLysS. The expression of this synthetic styC was investigated and subsequently optimized. Highly active biomass was obtained yielding an SOI activity of 44.5 ± 8.7 U mg−1 after 10 h. This represents the highest SOI activity reported for crude cell extracts of SOI-containing bacterial strains. Remarkably, this biomass can be applied as whole cell biocatalyst for the production of phenylacetic acids from styrene oxides. In the case of non-substituted styrene oxide, nearly 730 mg l−1 phenylacetic acid (∼85% yield was formed over a period of 20 days.

  9. Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide.

    Directory of Open Access Journals (Sweden)

    Léon J A Spijkers

    Full Text Available BACKGROUND: Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. METHODS AND FINDINGS: In isolated carotid arteries from spontaneously hypertensive rats (SHR and normotensive Wistar-Kyoto (WKY rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10, that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05. Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA(2, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7, but not in WKY (-12±10%; n = 6. Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3-5 respectively, p<0.05. These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6-12, p<0.05. Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05. CONCLUSIONS: Hypertension is associated with marked alterations in vascular

  10. Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic.

    Science.gov (United States)

    Kester, Mark; Bassler, Jocelyn; Fox, Todd E; Carter, Carly J; Davidson, Jeff A; Parette, Mylisa R

    2015-06-01

    Despite the therapeutic potential of sphingolipids, the ability to develop this class of compounds as active pharmaceutical ingredients has been hampered by issues of solubility and delivery. Beyond these technical hurdles, significant challenges in completing the necessary preclinical studies to support regulatory review are necessary for commercialization. This review seeks to identify the obstacles and potential solutions in the translation of a novel liposomal technology from the academic bench to investigational new drug (IND) stage by discussing the preclinical development of the Ceramide NanoLiposome (CNL), which is currently being developed as an anticancer drug for the initial indication of hepatocellular carcinoma (HCC).

  11. Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Mitsui, T; Akazawa, T

    1986-12-01

    Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

  12. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    Directory of Open Access Journals (Sweden)

    Carla Busquets-Cortés

    2016-01-01

    Full Text Available Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA supplementation (1.14 g/day on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs from sportsmen. Subjects were assigned to an intervention (N=9 or placebo groups (N=7 in a randomized double-blind trial. Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in terms of circulating PBMCs, Mn-superoxide dismutase protein levels, and their capability to produce reactive oxygen species. The proteins related to mitochondrial dynamics were, in general, increased after an 8-week training and this increase was enhanced by DHA supplementation. The content in mitofusins Mtf-1 and Mtf-2, optic atrophy protein-1 (Opa-1, and mitochondrial transcription factor A (Tfam were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX-IV activity and uncoupling proteins UCP-2 and UCP-3 protein levels were increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response was modulated by DHA supplementation.

  13. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    Science.gov (United States)

    Busquets-Cortés, Carla; Capó, Xavier; Tur, Josep A.; Sureda, Antoni

    2016-01-01

    Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA) supplementation (1.14 g/day) on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs) from sportsmen. Subjects were assigned to an intervention (N = 9) or placebo groups (N = 7) in a randomized double-blind trial. Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in terms of circulating PBMCs, Mn-superoxide dismutase protein levels, and their capability to produce reactive oxygen species. The proteins related to mitochondrial dynamics were, in general, increased after an 8-week training and this increase was enhanced by DHA supplementation. The content in mitofusins Mtf-1 and Mtf-2, optic atrophy protein-1 (Opa-1), and mitochondrial transcription factor A (Tfam) were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX-IV) activity and uncoupling proteins UCP-2 and UCP-3 protein levels were increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response was modulated by DHA supplementation. PMID:27698953

  14. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    Science.gov (United States)

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  15. Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

    Science.gov (United States)

    Robitaille, Stephan; Mailloux, Ryan J; Chan, Hing Man

    2016-08-10

    Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity. PMID:27180086

  16. DNA damage response and sphingolipid signaling in liver diseases

    Science.gov (United States)

    Matsuda, Yasunobu; Moro, Kazuki; Tsuchida, Junko; Soma, Daiki; Hirose, Yuki; Kobayashi, Takashi; Kosugi, Shin-ichi; Takabe, Kazuaki; Komatsu, Masaaki; Wakai, Toshifumi

    2016-01-01

    Patients with unresectable hepatocellular carcinoma (HCC) cannot generally be cured by systemic chemotherapy or radiotherapy due to their poor response to conventional therapeutic agents. The development of novel and efficient targeted therapies to increase their treatment options depends on the elucidation of the molecular mechanisms that underlie the pathogenesis of HCC. The DNA damage response (DDR) is a network of cell-signaling events that are triggered by DNA damage. Its dysregulation is thought to be one of the key mechanisms underlying the generation of HCC. Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important signaling molecule that has been found to be involved in many cellular functions. In the liver, the alteration of S1P signaling potentially affects the DDR pathways. In this review, we explore the role of the DDR in hepatocarcinogenesis of various etiologies, including hepatitis B and C infection and non-alcoholic steatohepatitis. Furthermore, we discuss the metabolism and functions of S1P that may affect the hepatic DDR. The elucidation of the pathogenic role of S1P may create new avenues of research into therapeutic strategies for patients with HCC. PMID:26514817

  17. Role of Sphingolipids in Infant Gut Health and Immunity.

    Science.gov (United States)

    Nilsson, Åke

    2016-06-01

    Sphingomyelin (SM), glycosphingolipids, and gangliosides are important polar lipids in the milk fat globule membrane but are not found in standard milk replacement formulas. Because digestion and absorption of SM and glycosphingolipids generate the bioactive metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P), and because intact gangliosides may have beneficial effects in the gut, this may be important for gut integrity and immune maturation in the neonate. The brush border enzymes that hydrolyze milk SM, alkaline sphingomyelinase (nucleotide phosphodiesterase pyrophosphatase 7), and neutral ceramidase are expressed at birth in both term and preterm infants. Released sphingosine is absorbed, phosphorylated to S1P, and converted to palmitic acid via S1P-lyase in the gut mucosa. Hypothetically, S1P also may be released from absorptive cells and exert important paracrine actions favoring epithelial integrity and renewal, as well as immune function, including secretory IgA production and migration of T lymphocyte subpopulations. Gluco-, galacto-, and lactosylceramide are hydrolyzed to ceramide by lactase-phlorizin hydrolase, which also hydrolyzes lactose. Gangliosides may adhere to the brush border and is internalized, modified, and possibly transported into blood, and may exert protective functions by their interactions with bacteria, bacterial toxins, and the brush border. PMID:27234412

  18. Preparation of sphingolipid fatty acid methyl esters for determination by gas-liquid chromatography.

    Science.gov (United States)

    MacGee, J; Williams, M G

    1981-01-30

    Sphingolipid fatty acids are first converted to a mixture of free acids and their n-butyl esters by heating the specimen at 85 degree C in aqueous butanolic hydrogen chloride; the butyl esters are then saponified with methanolic potassium hydroxide. After acidification and extraction into hexane, the fatty acids are extracted into a very small volume of aqueous trimethyl(m-trifluorotolyl)ammonium hydroxide (TMTFTH), injection of an aliquot of the TMTFTH extract into the gas chromatograph yields the fatty acid methyl esters by pyrolytic methylation of the quaternary ammonium salts of the fatty acids. The preparation of a specimen ready for the gas--liquid chromatographic (GLC) analysis with quantitative recovery of the sphingolipid fatty acids can be accomplished in less than 2 h. By comparison, none of a number of well-accepted techniques for the release of sphingomyelin fatty acids by hydrolysis or methanolysis released the fatty acids quantitatively in less than 3 h, and all required additional manipulations before GLC analysis. PMID:7217267

  19. Ceramide in Stem Cell Differentiation and Embryo Development: Novel Functions of a Topological Cell-Signaling Lipid and the Concept of Ceramide Compartments

    Directory of Open Access Journals (Sweden)

    Erhard Bieberich

    2011-01-01

    Full Text Available In the last two decades, the view on the function of ceramide as a sole metabolic precursor for other sphingolipids has completely changed. A plethora of studies has shown that ceramide is an important lipid cell-signaling factor regulating apoptosis in a variety of cell types. With the advent of new stem cell technologies and knockout mice for specific steps in ceramide biosynthesis, this view is about to change again. Recent studies suggest that ceramide is a critical cell-signaling factor for stem cell differentiation and cell polarity, two processes at the core of embryo development. This paper discusses studies on ceramide using in vitro differentiated stem cells, embryo cultures, and knockout mice with the goal of linking specific developmental stages to exciting and novel functions of this lipid. Particular attention is devoted to the concept of ceramide as a topological cell-signaling lipid: a lipid that forms distinct structures (membrane domains and vesicles termed “sphingosome”, which confines ceramide-induced cell signaling pathways to localized and even polarized compartments.

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

    Science.gov (United States)

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

  1. Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19)

    International Nuclear Information System (INIS)

    Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds—trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC50: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers. - Highlights: • Two natural products inhibited estrogen biosynthesis in human ovarian granulosa cells. • They inhibited

  2. Two natural products, trans-phytol and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol, inhibit the biosynthesis of estrogen in human ovarian granulosa cells by aromatase (CYP19)

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jiajia [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Yuan, Yun [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang (China); Lu, Danfeng; Du, Baowen [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Xiong, Liang; Shi, Jiangong [State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Yang, Lijuan [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Liu, Wanli [MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Yuan, Xiaohong [School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang (China); Zhang, Guolin, E-mail: zhanggl@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu (China); Wang, Fei, E-mail: wangfei@cib.ac.cn [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu (China); Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu (China)

    2014-08-15

    Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds—trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC{sub 50}: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levels in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers. - Highlights: • Two natural products inhibited estrogen biosynthesis in human ovarian granulosa cells. • They

  3. Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

    Energy Technology Data Exchange (ETDEWEB)

    Perera, Rushika M.; Riley, Catherine; Isaac, Georgis; Hopf- Jannasch, Amber; Moore, Ronald J.; Weitz, Karl K.; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Adamec, Jiri; Kuhn, Richard J.

    2012-03-22

    Dengue virus causes {approx}50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

  4. A Hyphenated Technique based on High-Performance Thin Layer Chromatography for Determining Neutral Sphingolipids: A Proof of Concept

    Directory of Open Access Journals (Sweden)

    Andrés Domínguez

    2015-04-01

    Full Text Available Hyphenated HPTLC has been used to analyze several neutral sphingolipids acting as lysosomal storage disease (LSD biomarkers. Automated multiple development (AMD provides separation of lipid peaks, which are detected and quantified using fluorescence detection by intensity changes (FDIC after primuline post-impregnation. A final online transfer to a mass spectrometer by means of an elution-based interface allows their identification using electrospray ionization (ESI and atmospheric pressure chemical ionization (APCI.Given that the increases in fluorescent emission detected by FDIC are produced by non-specific, electrostatic interactions between the primuline and hydrocarbon chains in the ceramide backbones of sphingolipids, it is a non-destructive detection technique, allowing the precise location and transfer of biomarker peaks to a mass spectrometer using an elution interface. By using primuline as a fluorophore, the technique is also compatible with ESI-APCI and does not interfere with the MS of sphingolipids. APCI provides useful and complementary structural information to the ESI for sphingolipid identification. Moreover, FDIC emission can be used for quantitative purposes. Results include the determination of sphingomyelin (SM in human-plasma samples (RSD < 6% by means of a standard addition method with non-linear calibration, and the identification of globotriaosylceramide (Gb3 in the plasma of a Fabry patient. Only one HPTLC plate is needed to perform the analysis.

  5. On the influence of ionizing radiation on polyamine biosynthesis and content in animal cells and on the possibility of involvement of polyamines in the formation and recovery from radiation damage

    International Nuclear Information System (INIS)

    The initial section of this monograph provides a review of the present data on distribution, biosynthesis, catabolism and biological function of polyamines, putrescine, spermidine, and spermine in animal cells. The conclusion is drawn that there is a possibility of participation of these compounds in the formation and recovery from radiation damage. In the investigations presented in the experimental section, it was established that ionizing radiation can induce changes of the polyamine content and activity of the enzymes of polyamine metabolism (ornithine decarboxylase, S-adenosylmethionine decarboxylase, diamine oxidase, and polyamine oxidase) in animal cells. The results were also obtained which indicate that a close relationship exists between the post-irradiation synthesis and accumulation of polyamines and such recovery processes from radiation insult as restorative cell proliferation and repair of chromosome damage. Moreover, it was found that products of enzymatic and radiolytic oxidative deamination of spermidine and spermine can cause inhibition of cell proliferation and induction of chromosome aberrations. (author)

  6. Acid ceramidase (AC)--a key enzyme of sphingolipid metabolism--correlates with better prognosis in epithelial ovarian cancer.

    Science.gov (United States)

    Hanker, Lars Christian; Karn, Thomas; Holtrich, Uwe; Gätje, Regine; Rody, Achim; Heinrich, Tomas; Ruckhäberle, Eugen; Engels, Knut

    2013-05-01

    Acid ceramidase (AC), a key enzyme of sphingolipid metabolism, seems to play an important role in cancer progression. The objective of this study was to explore the expression of AC in ovarian cancer and its impact on prognosis. Expression analysis of AC in n=112 ovarian cancer patients was performed by immunohistochemical analysis of primary paraffin-embedded tumor samples. The results were scored on the basis of the staining intensity and percentage of positive tumor cells, resulting in an immunoreactive score from 0 to 12. These results were correlated to clinical and pathologic characteristics and survival. AC expression correlated significantly only with FIGO stage (0.047). In serous carcinoma, low level of AC was independently associated with reduced progression-free survival and overall survival of 12.0 mo [95% confidence interval (CI), 5.78-18.23] versus 18.1 mo (95% CI, 11.61-24.59; P=0.008) and 35.7 mo (95% CI, 22.24-47.16) versus 58.7 mo (95% CI, 36.48-80.91; P=0.032), respectively. In multivariate analysis, AC presents as an independent prognostic factor for progression-free survival (hazard ratio 1.88; 95% CI, 1.13-3.11; P=0.015). AC is a prognostic factor in epithelial ovarian cancer. Low AC expression can be associated with tumor progression in carcinoma of the ovaries. These results are in contrast to the concept of AC as a promoter for cancer progression. Nevertheless, they are supported by the lately discovered tumor-suppressing function of sphingosine, the enzymatic product of AC.

  7. BIOSYNTHESIS OF YEAST CAROTENOIDS

    Science.gov (United States)

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  8. Mechanistic Insight into the Biosynthesis and Detoxification of Fumonisin Mycotoxins.

    Science.gov (United States)

    Burgess, Kevin M N; Renaud, Justin B; McDowell, Tim; Sumarah, Mark W

    2016-09-16

    Fumonisins, notably FB1, FB2, FB3, and FB4, are economically important mycotoxins produced by a number Fusarium sp. that occur on corn, rice, and sorghum as well as by Aspergillus sp. on grapes. The fumonisin scaffold is comprised of a C18 polyketide backbone functionalized with two tricarballylic esters and an alanine derived amine. These functional groups contribute to fumonisin's ability to inhibit sphingolipid biosynthesis in animals, plants, and yeasts. We report for the first time the isolation and structure elucidation of two classes of nonaminated fumonisins (FPy and FLa) produced by Aspergillus welwitschiae. Using a Lemna minor (duckweed) bioassay, these new compounds were significantly less toxic in comparison to the fumonisin B mycotoxins, providing new insight into the mechanism of fumonisin toxicity. Time course fermentations monitoring the production of FB4, FPy4, and FLa4, as well as (13)C and (15)N stable isotope incorporation, suggest a novel postbiosynthetic oxidative deamination process for fumonisins. This pathway was further supported by a feeding study with FB1, a fumonisin not produced by Aspergillus sp., which resulted in its transformation to FPy1. This study demonstrates that Aspergillus have the ability to produce enzymes that could be used for fumonisin detoxification. PMID:27444057

  9. Functional specialization in proline biosynthesis of melanoma.

    Directory of Open Access Journals (Sweden)

    Jessica De Ingeniis

    Full Text Available Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C, and then to proline via pyrroline-5-carboxylate reductases (PYCRs. Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

  10. Functional Specialization in Proline Biosynthesis of Melanoma

    Science.gov (United States)

    Richardson, Adam D.; Scott, David A.; Aza-Blanc, Pedro; De, Surya K.; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L.; Smith, Jeffrey W.

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis. PMID:23024808

  11. Antihypertensive treatment differentially affects vascular sphingolipid biology in spontaneously hypertensive rats.

    Directory of Open Access Journals (Sweden)

    Léon J A Spijkers

    Full Text Available BACKGROUND: We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR, is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A(2, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A(2. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR. METHODS AND FINDINGS: For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg. The blood pressure lowering was associated with a 20-25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A(2. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction. CONCLUSION: In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.

  12. A profile of sphingolipids and related compounds tentatively identified in yak milk.

    Science.gov (United States)

    Qu, S; Barrett-Wilt, G; Fonseca, L M; Rankin, S A

    2016-07-01

    This work characterized a fraction of constituents in yak milk within the realm of approximately 1,000 to 3,000 Da using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Eleven samples of yak milk powder from the Sichuan province of China were received by the Department of Food Science, University of Wisconsin-Madison, and stored at room temperature until analysis. Sample preparation involved delipidation and deproteinization of yak milk samples and cold ethanol precipitation. Subsequently, MALDI time-of-flight mass spectrometry was performed in positive ion, reflector mode (AB Sciex TOF/TOF 4800 MALDI; AB Sciex, Foster City, CA). The instrument was first calibrated with the manufacturer's 6-peptide mixture, and each spectrum was internally calibrated using the accurate mass of ACTH Fragment 18-39 standard peptide (protonated mass at m/z 2464.199) present in each sample. Laser power was adjusted for the calibration standards and for each sample so that the signal obtained for the most-abundant ion in each spectrum could be maximized, or kept below ~2×10(4) to preserve spectral quality. Structure and name based on mass were matched using the Metlin metabolite database (https://metlin.scripps.edu/index.php). Results of the current work for yak milk powder showed a large variety of sphingolipid structures with clusters around 1,200, 1,600, and 2,000 Da. The profiling matched several glycosphingolipids, such as gangliosides GA1, GD1a, GD1b, GD3, GM1, GM2, GM3, and GT2 and several other unique moieties, including deaminated neuraminic acid (KDN) oligosaccharides, and fucose containing gangliosides. Matrix preparation and MALDI time-of-flight parameters were important factors established in this work to allow high resolution profiling of complex sphingolipids in yak powder milk. PMID:27085416

  13. Levels of Blood Organophosphorus Flame Retardants and Association with Changes in Human Sphingolipid Homeostasis.

    Science.gov (United States)

    Zhao, Fanrong; Wan, Yi; Zhao, Haoqi; Hu, Wenxin; Mu, Di; Webster, Thomas F; Hu, Jianying

    2016-08-16

    While a recent toxicological study has shown that organophosphorus flame retardants (OPFRs) may disrupt sphingolipid homeostasis, epidemiologic evidence is currently lacking. In this study, a total of 257 participants were recruited from Shenzhen, China. Eleven OPFRs were for the first time simultaneously determined in the human blood samples by ultraperformance liquid chromatography and tandem mass spectrometry. Six OPFRs, tributyl phosphate (TNBP), 2-ethylhexyl diphenyl phosphate (EHDPP), tris(2-chloroisopropyl) phosphate (TCIPP), tris(2-butoxyethyl) phosphate (TBOEP), triethyl phosphate (TEP), and TPHP, were detectable in at least 90% of participants, with median concentrations of 37.8, 1.22, 0.71, 0.54, 0.49, and 0.43 ng/mL, respectively. Sphingomyelin (SM) levels in the highest quartile of EHDPP, TPHP, TNBP, TBOEP, TEP, and TCIPP were 45.3% [95% confidence interval; 38.1%, 53.0%], 51.9% (45.5%, 58.6%), 153.6% (145.1%, 162.3%), 20.6% (14.5%, 27.0%), 59.0% (52.1%, 66.2%), and 62.8% (55.2%, 70.6%) higher than those in the lowest quartile, respectively, after adjusting for covariates. Sphingosine 1-phosphate (S1P) levels in the highest quartile of EHDPP, TPHP, and TNBP were 36% (-39%, -33%), 16% (-19%, -14%), and 36% (-38%, -33%) lower than those in the lowest quartile, respectively. A similar pattern emerged when exposures were modeled continuously. We for the first time found the associations between OPFRs and changes in human sphingolipid homeostasis. PMID:27434659

  14. The tRNA-Dependent Biosynthesis of Modified Cyclic Dipeptides

    OpenAIRE

    Giessen, Tobias W.; Mohamed A. Marahiel

    2014-01-01

    In recent years it has become apparent that aminoacyl-tRNAs are not only crucial components involved in protein biosynthesis, but are also used as substrates and amino acid donors in a variety of other important cellular processes, ranging from bacterial cell wall biosynthesis and lipid modification to protein turnover and secondary metabolite assembly. In this review, we focus on tRNA-dependent biosynthetic pathways that generate modified cyclic dipeptides (CDPs). The essential peptide bond...

  15. Biosynthesis of fibronectin by human embryo fibroblasts

    International Nuclear Information System (INIS)

    The biosynthesis of fibronectin by human embryo fibroblasts, transformed by SV-40 virus, was investigated in intact cells and in a cell-free protein-synthesizing system on free and membrane-bound polyribosomes, isolated from the same cells. It was shown that fibronectin production in the culture medium by transformed fibroblasts is reduced by a factor of 4.5, while its percent content in the medium is decreased by a factor of 2. The content of fibronectin material precipitable by antibodies during the immunoprecipitation reaction proved to be somewhat greater in transformed cells than in normal cells, although the percentage was decreased by a factor of 1.5. However, the content of the fibronectin monomer with molecular weight 220 kilodaltons was 1.6 times as great in the cellular material of normal fibroblasts. In an investigation of fibronectin biosynthesis in a cell-free system it was established that in transformed cells 45% of the fibronectin is synthesized on free polyribosomes; in normal fibroblasts only 13% of the fibronectin is synthesized on free polyribosomes. It is suggested that one of the consequences of the transformation of human fibroblasts by SV-40 virus, leading to a decrease in fibronectin production, is the spatial uncoupling of the polyribosomes and membrane structures responsible for protein transport out of the cell, as a result of which a substantial portion of the fibronectin synthesized by transformed fibroblasts is subjected to intracellular degradation

  16. Role of Sphingolipid Mediator Ceramide in Obesity and Renal Injury in Mice Fed a High-Fat DietS⃞

    OpenAIRE

    Boini, Krishna M.; Zhang, Chun; Xia, Min; Poklis, Justin L.; Li, Pin-Lan

    2010-01-01

    The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD)...

  17. Glycosyltransferases Involved in Xylan Biosynthesis in Plant Cell Walls%参与植物细胞壁半纤维素木聚糖合成的糖基转移酶

    Institute of Scientific and Technical Information of China (English)

    秦丽霞; 张德静; 李龙; 李学宝; 许文亮

    2011-01-01

    Xylans are the major hemicelluloses in secondary cell walls of dicots and are critical for normal plant growth and development. Xylan-containing lignocellulosic secondary cell walls are the most abundant repository of biomass on earth and are widely used for energy, pulping, paper-making and textiles. However, the pen-tose composition of xylans makes them difficult to be used efficiently. Thus, understanding the detailed mechanism of xylan biosynthesis may lead to new strategies to manipulate the xylan composition in cell walls and to modify their structures. To date the characterization of various xylan-deficient Arabidopsis mutants has identified many genes encoding members of glycosyltransferase family GT43, GT8 and GT47 that are involved in biosynthesis of xylan backbone, reducing end sequence and side chains. In this review, we summarize the recent progress on glycosyltransferases involved in xylan biosynthesis.%木聚糖是双子叶植物次生细胞壁中最主要的半纤维素,合有木聚糖的次生壁是最丰富的植物生物质,广泛应用于能源、制浆、造纸和纺织业中,但其主要组分戊糖对细胞壁生物质利用具有较大影响.揭示木聚糖合成的分子机制,为遗传修饰细胞壁组成,更好地利用细胞壁生物质提供新的策略.近年来对模式植物拟南芥中多个木聚糖合成有缺陷的突变体的分析表明:GT43家族的IRX9、IRX9-L、IRX14、IRX14-L,GT47家族的FRA8、F8H、IRX10、IRX10-L,GT8家族的IRX8、PARVUS、QUA1、GUX1、GUX2等参与了木聚糖主链、还原末端序列和侧链的合成.本文主要对这些研究进展做一综述,并讨论了木聚糖合成的机制及亟待解决的问题,展望了其发展趋势.

  18. LC-MS Based Sphingolipidomic Study on A2780 Human Ovarian Cancer Cell Line and its Taxol-resistant Strain

    Science.gov (United States)

    Huang, Hao; Tong, Tian-Tian; Yau, Lee-Fong; Chen, Cheng-Yu; Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    Drug resistance elicited by cancer cells continue to cause huge problems world-wide, for example, tens of thousands of patients are suffering from taxol-resistant human ovarian cancer. However, its biochemical mechanisms remain unclear. Sphingolipid metabolic dysregulation has been increasingly regarded as one of the drug-resistant mechanisms for various cancers, which in turn provides potential targets for overcoming the resistance. In the current study, a well-established LC-MS based sphingolipidomic approach was applied to investigate the sphingolipid metabolism of A2780 and taxol-resistant A2780 (A2780T) human ovarian cancer cell lines. 102 sphingolipids (SPLs) were identified based on accurate mass and characteristic fragment ions, among which 12 species have not been reported previously. 89 were further quantitatively analyzed by using multiple reaction monitoring technique. Multivariate analysis revealed that the levels of 52 sphingolipids significantly altered in A2780T cells comparing to those of A2780 cells. These alterations revealed an overall increase of sphingomyelin levels and significant decrease of ceramides, hexosylceramides and lactosylceramides, which concomitantly indicated a deviated SPL metabolism in A2780T. This is the most comprehensive sphingolipidomic analysis of A2780 and A2780T, which investigated significantly changed sphingolipid profile in taxol-resistant cancer cells. The aberrant sphingolipid metabolism in A2780T could be one of the mechanisms of taxol-resistance. PMID:27703266

  19. Serum sphingolipids reflect the severity of chronic HBV infection and predict the mortality of HBV-acute-on-chronic liver failure.

    Directory of Open Access Journals (Sweden)

    Feng Qu

    Full Text Available Patients with HBV-acute-on-chronic liver failure (HBV-ACLF have high mortality and frequently require liver transplantation; few reliable prognostic markers are available. As a class of functional lipids, sphingolipids are extensively involved in the process of HBV infection. However, their role in chronic HBV infection remains unknown. The aim of this study was to determine the serum sphingolipid profile in a population of patients with chronic HBV infection, paying special attention to exploring novel prognostic markers in HBV-ACLF. High performance liquid chromatography tandem mass spectrometry was used to examine the levels of 41 sphingolipids in 156 serum samples prospectively collected from two independent cohorts. The training and validation cohorts comprised 20 and 28 healthy controls (CTRL, 29 and 23 patients with chronic hepatitis B (CHB, and 30 and 26 patients with HBV-ACLF, respectively. Biometric analysis was used to evaluate the association between sphingolipid levels and disease stages. Multivariate analysis revealed difference of sphingolipid profiles between CHB and HBV-ACLF was more drastic than that between CTRL and CHB, which indicated that serum sphingolipid levels were more likely to associate with the progression HBV-ACLF rather than CHB. Furthermore, a 3-month mortality evaluation of HBV-ACLF patients showed that dhCer(d18 : 0/24 : 0 was significantly higher in survivors than in non-survivors (including deceased patients and those undergoing liver transplantation, p < 0.05, and showed a prognostic performance similar to that of the MELD score. The serum sphingolipid composition varies between CTRL and chronic HBV infection patients. In addition, dhCer(d18 : 0/24 : 0 may be a useful prognostic indicator for the early prediction of HBV-ACLF.

  20. Uptake of long chain fatty acids is regulated by dynamic interaction of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts

    Directory of Open Access Journals (Sweden)

    Herrmann Thomas

    2008-08-01

    Full Text Available Abstract Background Mechanisms of long chain fatty acid uptake across the plasma membrane are important targets in treatment of many human diseases like obesity or hepatic steatosis. Long chain fatty acid translocation is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but certain membrane proteins can also accelerate the transport. However, we now can provide further evidence that not only proteins but also lipid microdomains play an important part in the regulation of the facilitated uptake process. Methods Dynamic association of FAT/CD36 a candidate fatty acid transporter with lipid rafts was analysed by isolation of detergent resistant membranes (DRMs and by clustering of lipid rafts with antibodies on living cells. Lipid raft integrity was modulated by cholesterol depletion using methyl-β-cyclodextrin and sphingolipid depletion using myriocin and sphingomyelinase. Functional analyses were performed using an [3H]-oleate uptake assay. Results Overexpression of FAT/CD36 and FATP4 increased long chain fatty acid uptake. The uptake of long chain fatty acids was cholesterol and sphingolipid dependent. Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains. FAT/CD36 co-localized with the lipid raft marker PLAP in antibody-clustered domains at the plasma membrane and segregated away from the non-raft marker GFP-TMD. Antibody cross-linking increased DRM association of FAT/CD36 and accelerated the overall fatty acid uptake in a cholesterol dependent manner. Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane. Conclusion Our observations suggest the existence of two pools of FAT/CD36 within cellular membranes. As increased raft association of FAT/CD36 leads to an increased fatty acid uptake, dynamic association of FAT/CD36 with lipid rafts might regulate the process. There is no

  1. Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

    Science.gov (United States)

    Libisch, B; Michaelson, L V; Lewis, M J; Shewry, P R; Napier, J A

    2000-12-29

    The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets. PMID:11162428

  2. Plasma lipidomics reveal profound perturbation of glycerophospholipids, fatty acids, and sphingolipids in diet-induced hyperlipidemia.

    Science.gov (United States)

    Miao, Hua; Chen, Hua; Pei, Songwen; Bai, Xu; Vaziri, Nosratola D; Zhao, Ying-Yong

    2015-02-25

    Hyperlipidemia is a major risk factor for coronary heart disease and has emerged as an important public health problem. Lipidomics is a powerful technology for assessment of global lipid metabolites in a biological system and for biomarker discovery. In the present study, hyperlipidemia was induced by feeding rats a high fat diet. A sensitive ultra-performance liquid chromatography coupled with quadrupole time-of-flight synapt high-definition mass spectrometry method was used for the analysis of plasma lipids. Orthogonal partial least squares-discriminant analysis, correlation analysis and heatmap analysis were performed to investigate the metabolic changes in rats with diet-induced hyperlipidemia. Potential biomarkers were detected using S-plot and were identified by accurate mass data, isotopic pattern and MS(E) fragments information. Significantly increased total cholesterol, triglycerides and low-density lipoprotein cholesterol as well as decreased high-density lipoprotein cholesterol were observed in diet-induced hyperlipidemic rats. Combined with standard serum biochemical results, significant differences in plasma lipid compounds including eleven glycerophospholipids, six fatty acids, two sphingolipids, one eicosanoid, one sterol lipid and one glycerolipid were observed, highlighting the perturbation of lipid metabolism in diet-induced hyperlipidemia. These findings provide further insights into the lipid profile across a wide range of biochemical pathways in diet-induced hyperlipidemia.

  3. Imaging MALDI mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system.

    Science.gov (United States)

    Chen, Yanfeng; Liu, Ying; Allegood, Jeremy; Wang, Elaine; Cachón-González, Begoña; Cox, Timothy M; Merrill, Alfred H; Sullards, M Cameron

    2010-01-01

    Matrix deposition is a critical step in tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It greatly affects the quality of MALDI imaging, especially for the analytes (such as lipids) that may easily dissolve in the solvent used for the matrix application. This chapter describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This protocol allows visualization of many different lipid species and, of particular interest, sphingolipids in tissue slices of Tay-Sachs/Sandhoff disease by imaging MALDI-MS. The structures of these lipids were identified by analysis of tissue extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS and MS(3)). These results illustrate the usefulness of tissue imaging MALDI-MS with matrix deposition by OCN for the molecular analysis in normal physiology and pathology. In addition, the observation of numerous lipid subclasses with distinct localizations in the brain slices demonstrates that imaging MALDI-MS could be effectively used for "lipidomic" studies. PMID:20680588

  4. Diverse inhibitors of aflatoxin biosynthesis.

    Science.gov (United States)

    Holmes, Robert A; Boston, Rebecca S; Payne, Gary A

    2008-03-01

    Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

  5. Serine biosynthesis and transport defects.

    Science.gov (United States)

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. PMID:27161889

  6. Involved in the secondary cell wall biosynthesis in popar%杨树中与次生细胞壁生物合成相关的糖基转移酶研究进展

    Institute of Scientific and Technical Information of China (English)

    杨少辉; 王洁华; 宋英今; 胡荣峰

    2013-01-01

    随着杨树基因组测序计划的完成,加之生物信息学、生物化学、分子生物学和遗传学等研究方法的不断完善,杨树功能基因组学研究近年来被大大推进.目前,有关杨树材性的研究主要集中于MYBs、NACs、bZIP、HB、WRKY、MADS等转录因子的调控机制以及木质素的合成途径.与之相比,有关次生细胞壁中关键碳水化合物结构的合成及代谢仍然不清楚.糖基转移酶参与木本植物次生细胞壁中碳水化合物的合成和架构,直接影响木质部的发育过程.着重介绍了木本模式植物——杨树中参与次生细胞壁生物合成的糖基转移酶基因家族及其生物学功能,并对其在利用基因工程手段改良杨树材性和选育新品种方面进行了展望.%With the completion of genome sequencing of populus trichacarpa,together with the methods of bioinformatics,biochemistry,molecular biology and genetics,the functional genomics of poplar were greatly promoted.So far,the wood-related reseaches in poplar were mainly concentrated on the transcripional factors including MYBs,NACs,bZIP,HB,WRKY,MADS and on the lignin biosynthesis pathway.In comparison,the metabolic pathways of the key structural carbohydrates still remain unclear.Glycosyltransferases are involved in the biosynthesis and remodeling of the carbahydrates in poplar xylem,thus directly affecting xylem developmental process.In this review,an emphasis on the glycosyltransferase gene family and their biological functions involved in secondary cell wall biosynthesis in the woody model system-poplar weere summarized,and the future perspectives on the modification of wood properties in poplar by genetic engineering and new variety breeding were given.

  7. Glycolipid biosynthesis in cyanobacteria

    International Nuclear Information System (INIS)

    The biosynthesis of monogalactosyldiacyl-glycerol (MGDG) was studied in five different cyanobacteria. Previous work has shown Anabaena variabilis to synthesize both MGDG and monoglucosyl-diacylglycerol (MG1cDG) with MG1cDG being the precursor of MGDG. They have examined four other cyanobacteria to determine if a similar relationship exists. The cyanobacteria studied were Anabaena variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis nidulans, and Anacystis marina. Each were grown in liquid culture and lipids were labeled with 14C]CO2 for 20 min., 1.0 hr, 1.0 hr + 10 hr chase. Glycolipids were analyzed by initial separation of MGDG and MG1cDG by TLC followed by further analysis by HPLC. Complete separation of molecular species was obtained isocratically on an ODS column. All of the cyanobacteria labeled 16-C and 18-C fatty acids except for A. marina which labeled only 14-C and 16-C fatty acids. Desaturation of the fatty acids could be observed in the 1.0 hr and chase experiments. All were capable of labeling both MG1cDG and MGDG with the precursor-product relationship being observed. There does not appear to be a direct relationship between the epimerization of the sugar moiety and fatty acid desaturation

  8. The yeast PH domain proteins Slm1 and Slm2 are targets of sphingolipid signaling during the response to heat stress.

    Science.gov (United States)

    Daquinag, Alexes; Fadri, Maria; Jung, Sung Yun; Qin, Jun; Kunz, Jeannette

    2007-01-01

    The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P(2), TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization. PMID:17101780

  9. Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its γ-aminobutyric acid biosynthesis ability in milk.

    Science.gov (United States)

    Wu, Qinglong; Law, Yee-Song; Shah, Nagendra P

    2015-08-06

    Most high γ-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.

  10. Phosphoregulation of the ceramide transport protein CERT at serine 315 in the interaction with VAMP-associated protein (VAP) for inter-organelle trafficking of ceramide in mammalian cells.

    Science.gov (United States)

    Kumagai, Keigo; Kawano-Kawada, Miyuki; Hanada, Kentaro

    2014-04-11

    The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.

  11. Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells*

    Science.gov (United States)

    Kumagai, Keigo; Kawano-Kawada, Miyuki; Hanada, Kentaro

    2014-01-01

    The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids. PMID:24569996

  12. The Yeast PH Domain Proteins Slm1 and Slm2 Are Targets of Sphingolipid Signaling during the Response to Heat Stress▿ †

    OpenAIRE

    Daquinag, Alexes; Fadri, Maria; Jung, Sung Yun; Qin, Jun; Kunz, Jeannette

    2006-01-01

    The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P2) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosph...

  13. Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

    Science.gov (United States)

    Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

    2015-02-01

    Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis. PMID:25104168

  14. Biosynthesis of silver nanoparticle and its application in cell wall disruption to release carbohydrate and lipid from C. vulgaris for biofuel production

    Directory of Open Access Journals (Sweden)

    Sirajunnisa Abdul Razack

    2016-09-01

    Full Text Available Microalgae are the fledging feedstocks yielding raw materials for the production of third generation biofuel. Assorted and conventional cell wall disruption techniques were helpful in extracting lipids and carbohydrates, nevertheless the disadvantages have led the biotechnologists to explore new process to lyse cell wall in a faster and an economical manner. Silver nanoparticles have the ability to break the cell wall of microalgae and release biomolecules effectively. Green synthesis of silver nanoparticles was performed using a novel bacterial isolate of Bacillus subtilis. Characterisation of nanosilver and its effect on cell wall lysis of microalgae were extensively analysed. Cell wall damage was confirmed by lactate dehydrogenase assay and visually by SEM analysis. This first piece of research work on direct use of nanoparticles for cell wall lysis would potentially be advantageous over its conventional approaches and a greener, cost effective and non laborious method for the production of biodiesel.

  15. Alternate biosynthesis of valerenadiene and related sesquiterpenes.

    Science.gov (United States)

    Paknikar, Shashikumar K; Kadam, Shahuraj H; Ehrlich, April L; Bates, Robert B

    2013-09-01

    It is proposed that the biosynthesis of the sesquiterpene valerenadiene, a key intermediate in the biosynthesis of a sedative in valerian, involves cyclopropane and not cyclobutane intermediates and includes as a key step a cyclopropylcarbinylcation-cyclopropylcarbinylcation rearrangement analogous to the one observed in the conversion of presqualene to squalene in triterpene and steroid biosynthesis. Similar mechanisms are proposed for the biosynthesis of the related sesquiterpenes pacifigorgiol, tamariscene and (+)-pacifigorgia-1,10-diene. PMID:24273843

  16. Plasma membrane targeting by short chain sphingolipids inserted in liposomes improves anti-tumor activity of mitoxantrone in an orthotopic breast carcinoma xenograft model.

    Science.gov (United States)

    Cordeiro Pedrosa, Lília R; van Tellingen, Olaf; Soullié, Thomas; Seynhaeve, Ann L; Eggermont, Alexander M M; Ten Hagen, Timo L M; Verheij, Marcel; Koning, Gerben A

    2015-08-01

    Mitoxantrone (MTO) is clinically used for treatment of various types of cancers providing an alternative for similarly active, but more toxic chemotherapeutic drugs such as anthracyclines. To further decrease its toxicity MTO was encapsulated into liposomes. Although liposomal drugs can accumulate in target tumor tissue, they still face the plasma membrane barrier for effective intracellular delivery. Aiming to improve MTO tumor cell availability, we used short chain lipids to target and modulate the tumor cell membrane, promoting MTO plasma membrane traversal. MTO was encapsulated in liposomes containing the short chain sphingolipid (SCS), C8-Glucosylceramide (C8-GluCer) or C8-Galactosylceramide (C8-GalCer) in their bilayer. These new SCS-liposomes containing MTO (SCS-MTOL) were tested in vivo for tolerability, pharmacokinetics, biodistribution, tumor drug delivery by intravital microscopy and efficacy, and compared to standard MTO liposomes (MTOL) and free MTO. Liposomal encapsulation decreased MTO toxicity and allowed administration of higher drug doses. SCS-MTOL displayed increased clearance and lower skin accumulation compared to standard MTOL. Intratumoral liposomal drug delivery was heterogeneous and rather limited in hypoxic tumor areas, yet SCS-MTOL improved intracellular drug uptake in comparison with MTOL. The increased MTO availability correlated well with the improved antitumor activity of SCS-MTOL in a MDAMB-231 breast carcinoma model. Multiple dosing of liposomal MTO strongly delayed tumor growth compared to free MTO and prolonged mouse survival, whereas among the liposomal MTO treatments, C8-GluCer-MTOL was most effective. Targeting plasma membranes with SCS improved MTO tumor availability and thereby therapeutic activity and represents a promising approach to improve MTO-based chemotherapy.

  17. In vitro biosynthesis of complement protein D

    Energy Technology Data Exchange (ETDEWEB)

    Barnum, S.R.

    1985-01-01

    The aim of this study was twofold: to determine site(s) of complement protein D biosynthesis and to examine D biosynthesis with respect to the kinetics of D secretion, the post-translational modification of D and the tissue-specific differences in D secretion and processing. Antigenic D was detected in the culture supernatants of two cell lines, U937 and HepG2, and adherent blood monocytes by a solid-phase radioimmunoassay. D secreted by U937 cells was hemolytically active with a specific activity comparable to D in serum. De novo synthesis of D by U937 cells was demonstrated with the use of cycloheximide. Biosynthetic labeling using /sup 35/S labeled methionine or cysteine, followed by immunoprecipitation demonstrated a single d band intra- and extra-cellularly in all three cell types as analyzed by SDS-PAGE and auto-radiography. Elevated serum D levels in individuals with IgA nephropathy led to studies on the D levels in serum and urine of individuals with chronic renal failure and an individual with Fanconi's syndrome. The former group had elevated serum D levels, compared to normals, and insignificant levels of D in their urine while the patient with Fanconi's syndrome had normal serum D levels but markedly elevated urinary D levels. These studies demonstrate that the monocyte and hepatocyte are both sites of D synthesis and that there are no apparent differences in the secretion rates and processing of D produced by these cell types. The results also suggest that D is not synthesized or secreted as a precursor molecule. Additionally, these studies suggest that the kidney is a major site of D catabolism.

  18. Xyloglucan and Its Biosynthesis

    OpenAIRE

    Zabotina, Olga A.

    2012-01-01

    The hemicellulosic polysaccharide xyloglucan (XyG), found in the primary cell walls of most plant tissues, is important for structural organization of the cell wall and regulation of growth and development. Significant recent progress in structural characterization of XyGs from different plant species has shed light on the diversification of XyG during plant evolution. Also, identification of XyG biosynthetic enzymes and examination of their interactions suggests the involvement of a multipro...

  19. Xyloglucan and its biosynthesis

    OpenAIRE

    Zabotina, Olga A.

    2012-01-01

    The hemicellulosic polysaccharide xyloglucan (XyG), found in the primary cell walls of most plant tissues, is important for structural organization of the cell wall and regulation of growth and development. Significant recent progress in structural characterization of XyGs from different plant species has shed light on the diversification of XyG during plant evolution. Also, identification of XyG biosynthetic enzymes and examination of their interactions suggests the involvement of a multipro...

  20. The biosynthesis of phytoalexins in Dianthus caryophyllus L. cell cultures: induction of benzoyl-CoA:anthranilate N-benzoyltransferase activity.

    Science.gov (United States)

    Reinhard, K; Matern, U

    1989-11-15

    It has been shown that cell cultures of Dianthus caryophyllus L. c.v. Eleganz accumulate N-benzoyl-4-methoxyanthranilic acid, previously identified as the phytoalexin methoxydianthramide B, in response to treatment either with a crude elicitor isolated from the cell walls of Phytophthora megasperma f.sp. glycinea or with a commercial yeast extract. Cell-free extracts from the induced cells efficiently catalyzed the N-benzoylation of anthranilate in the presence of benzoyl-CoA. The partially purified transferase was shown to be specific for anthranilate with almost no activity toward 4-hydroxyanthranilate, whereas acyl donors other than benzoyl-CoA such as salicyloyl-, cinnamoyl-, or 4-coumaroyl-CoA were also accepted. Elicitor treatment of the cells additionally induced an S-adenosyl-L-methionine:N-benzoyl-4-hydroxyanthranilate 4-O-methyltransferase activity. We propose, therefore, that methoxydianthramide B is derived from N-benzoylanthranilic acid via N-benzoyl-4-hydroxyanthranilic acid. Dark-grown cells contained little N-benzoyltransferase activity (approx 8 mu kat/kg), which increased roughly ninefold within 6 h following the addition of the elicitor. In addition, phenylalanine ammonia-lyase activity of the cells increased about twofold under these conditions to a maximum (approx 40 mu kat/kg) at 5 h. The rapid induction of both enzyme activities suggests that the shikimate pathway is of crucial importance in the disease resistance response of carnation cells.

  1. LOCALIZATION OF THE PATHWAY OF THE PENICILLIN BIOSYNTHESIS IN PENICILLIUM-CHRYSOGENUM

    NARCIS (Netherlands)

    MULLER, WH; VANDERKRIFT, TP; KROUWER, AJJ; WOSTEN, HAB; VANDERVOORT, LHM; SMAAL, EB; VERKLEIJ, AJ

    1991-01-01

    The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locati

  2. The Evolution of Aflatoxin Biosynthesis

    Science.gov (United States)

    The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and O-methylsterigmatocysin (OMST), the respective penultimate and ultimate precursors of AF. Although ST, OMST, and ...

  3. Biosynthesis and transport of terpenes

    NARCIS (Netherlands)

    Ting, H.M.

    2014-01-01

    Terpenoids are the largest class of natural product that are produced by plants, with functions that range from a role in plant development to direct defence against pathogens and indirect defence against insects through the attraction of natural enemies. While terpene biosynthesis genes have been w

  4. Breeding grasses for capacity to biofuel production or silage feeding value: an updated list of genes involved in maize secondary cell wall biosynthesis and assembly

    OpenAIRE

    Courtial, Audrey; Soler, Marçal; Chateigner-Boutin, Anne Laure; Reymond, Matthieu; Mechin, Valerie; WANG Hua; Grima-Pettenati, Jacqueline

    2013-01-01

    In the near future, maize, sorghum, or switchgrass stovers and cereal straws will be a significant source of carbohydratesfor sustainable biofuel production, in addition to the current use of grass silage in cattle feeding. However, cell wall properties, including the enzymatic degradability of structural polysaccharides in industrial fermenters or animal rumen, is greatly influenced by the embedding of cell wall carbohydrates in lignin matrix, and the linkages between lignins, p-hydroxycinna...

  5. (-)-Menthol biosynthesis and molecular genetics

    Science.gov (United States)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  6. Effective pretreatment of sugarcane bagasse with combination pretreatment and its hydrolyzates as reaction media for the biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by whole cells of E. coli CCZU-K14.

    Science.gov (United States)

    He, Yu-Cai; Zhang, Dan-Ping; Di, Jun-Hua; Wu, Yin-Qi; Tao, Zhi-Cheng; Liu, Feng; Zhang, Zhi-Jun; Chong, Gang-Gang; Ding, Yun; Ma, Cui-Luan

    2016-07-01

    In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Compared with glucose, arabinose and cellobiose couldn't promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of (S)-CHBE (∼500g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of β-CD (0.4mol β-CD/mol COBE), l-glutamine (200mM) and glycine (500mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate (S)-CHBE. PMID:27060248

  7. Final Report on Regulation of Guaiacyl and Syringyl Monolignol Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Vincent L. Chiang

    2006-03-09

    The focus of this research is to understand syringyl monolignol biosynthesis that leads to the formation of syringyl lignin, a type of lignin that can be easily removed during biomass conversion. We have achieved the three originally proposed goals for this project. (1) SAD and CAD genes (enzyme catalytic and kinetic properties) and their functional relevance to CAld5H/AldOMT pathway, (2) spatiotemporal expression patterns of Cald5H, AldOMT, SAD and CAD genes, and (3) functions of CAld5H, AldOMT, and SAD genes in vivo using transgenic aspen. Furthermore, we also found that microRNA might be involved in the upstream regulatory network of lignin biosynthesis and wood formation. The achievements are as below. (1) Based on biochemical and molecular studies, we discovered a novel syringyl-specific alcohol dehydrogenase (SAD) involved in monolignol biosynthesis in angiosperm trees. Through CAld5H/OMT/SAD mediation, syringyl monolignol biosynthesis branches out from guaiacyl pathway at coniferaldehyde; (2) The function of CAld5H gene in this syringyl monolignol biosynthesis pathway also was confirmed in vivo in transgenic Populus; (3) The proposed major monolignol biosynthesis pathways were further supported by the involving biochemical functions of CCR based on a detailed kinetic study; (4) Gene promoter activity analysis also supported the cell-type specific expression of SAD and CAD genes in xylem tissue, consistent with the cell-specific locations of SAD and CAD proteins and with the proposed pathways; (5) We have developed a novel small interfering RNA (siRNA)-mediated stable gene-silencing system in transgenic plants; (6) Using the siRNA and P. trichocarpa transformation/regeneration systems we are currently producing transgenic P. trichocarpa to investigate the interactive functions of CAD and SAD in regulating guaiacyl and syringyl lignin biosynthesis; (7) We have cloned for the first time from a tree species, P. trichocarpa, small regulatory RNAs termed micro

  8. Biosynthesis and biological action of pineal allopregnanolone

    Directory of Open Access Journals (Sweden)

    Kazuyoshi eTsutsui

    2014-05-01

    Full Text Available The pineal gland transduces photoperiodic changes to the neuroendocrine system by rhythmic secretion of melatonin. We recently provided new evidence that the pineal gland is a major neurosteroidogenic organ and actively produces a variety of neurosteroids de novo from cholesterol in birds. Notably, allopregnanolone is a major pineal neurosteroid that is far more actively produced in the pineal gland than the brain and secreted by the pineal gland in juvenile birds. Subsequently, we have demonstrated the biological action of pineal allopregnanolone on Purkinje cells in the cerebellum during development in juvenile birds. Pinealectomy (Px induces apoptosis of Purkinje cells, whereas allopregnanolone administration to Px chicks prevents cell death. Furthermore, Px increases the number of Purkinje cells that express active caspase-3, a crucial mediator of apoptosis, and allopregnanolone administration to Px chicks decreases the number of Purkinje cells expressing active caspase-3. It thus appears that pineal allopregnanolone prevents cell death of Purkinje cells by suppressing the activity of caspase-3 during development. This paper highlights new aspects of the biosynthesis and biological action of pineal allopregnanolone.

  9. Essences in Metabolic Engineering of Lignan Biosynthesis

    Directory of Open Access Journals (Sweden)

    Honoo Satake

    2015-05-01

    Full Text Available Lignans are structurally and functionally diverse phytochemicals biosynthesized in diverse plant species and have received wide attentions as leading compounds of novel drugs for tumor treatment and healthy diets to reduce of the risks of lifestyle-related non-communicable diseases. However, the lineage-specific distribution and the low-amount of production in natural plants, some of which are endangered species, hinder the efficient and stable production of beneficial lignans. Accordingly, the development of new procedures for lignan production is of keen interest. Recent marked advances in the molecular and functional characterization of lignan biosynthetic enzymes and endogenous and exogenous factors for lignan biosynthesis have suggested new methods for the metabolic engineering of lignan biosynthesis cascades leading to the efficient, sustainable, and stable lignan production in plants, including plant cell/organ cultures. Optimization of light conditions, utilization of a wide range of elicitor treatments, and construction of transiently gene-transfected or transgenic lignan-biosynthesizing plants are mainly being attempted. This review will present the basic and latest knowledge regarding metabolic engineering of lignans based on their biosynthetic pathways and biological activities, and the perspectives in lignan production via metabolic engineering.

  10. Biosynthesis of pectin

    DEFF Research Database (Denmark)

    Scheller, Henrik Vibe; Jensen, Jacob Krüger; Sørensen, Susanne Oxenbøll;

    2007-01-01

    for a putative glucuronosyltransferase (GUT1) involved in making RGII, a putative arabinosyltransferase (ARAD1) involved in making arabinan, and a putative xylosyltransferase (XGD1) involved in making XGA. In several other cases, enzymes have been identified as involved in making pectin but because of ambiguity......). These polysaccharides appear to be present in all cells but their relative abundance and structural details differ between cell types and species. Pectin is synthesized in the Golgi vesicles and its complexity dictates that a large number of enzymes must be involved in the process. The biosynthetic enzymes required...... are glycosyltransferases and decorating enzymes including methyltransferases, acetyltransferases and feruloyltransferases. Biochemical methods successfully led to the recent identification of a pectin biosynthetic galacturonosyltransferase (GAUT1), and recent functional genomics and mutant studies have allowed...

  11. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity

    NARCIS (Netherlands)

    Wiedemann, [No Value; Breukink, E; van Kraaij, C; Kuipers, OP; Bierbaum, G; de Kruijff, B; Sahl, HA

    2001-01-01

    Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the Lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin

  12. Biosynthesis of 10 kDa and 7.5 kDa insulin-like growth factor II in a human rhabdomyosarcoma cell line

    DEFF Research Database (Denmark)

    Nielsen, F C; Haselbacher, G; Christiansen, Jan;

    1993-01-01

    In the present study we have analysed the expression of insulin-like growth factor II (IGF-II) in the human rhabdomyosarcoma cell line IN157.IN157 cells express high levels of three IGF-II mRNAs of 6.0 kb, 4.8 kb and 4.2 kb. In contrast, normal skeletal muscle expresses a negligible amount of IGF......-II mRNA. Two forms of IGF-II with molecular masses of 7.5 kDa and 10 kDa, corresponding to the mature IGF-II and IGF-II with a C-terminal extension of 21 amino acids (IGF-IIE21), were secreted into the culture medium at amounts of 17 ng/ml (2.3 nM) and 15 ng/ml (1.5 nM), respectively. IN157 cells also......-II and IGF-IIE21 with Kd values of 0.5 nM and 2 nM, respectively, and IGF-I with about 500 times lower affinity. IGF-II and IGF-IIE21 stimulated DNA synthesis via the IGF-I receptor, whereas the IGF-II/Man 6-P receptor mediated their rapid internalization and inactivation. During culture of IN157 cells about...

  13. The antimalarial drug quinine interferes with serotonin biosynthesis and action.

    Science.gov (United States)

    Islahudin, Farida; Tindall, Sarah M; Mellor, Ian R; Swift, Karen; Christensen, Hans E M; Fone, Kevin C F; Pleass, Richard J; Ting, Kang-Nee; Avery, Simon V

    2014-01-01

    The major antimalarial drug quinine perturbs uptake of the essential amino acid tryptophan, and patients with low plasma tryptophan are predisposed to adverse quinine reactions; symptoms of which are similar to indications of tryptophan depletion. As tryptophan is a precursor of the neurotransmitter serotonin (5-HT), here we test the hypothesis that quinine disrupts serotonin function. Quinine inhibited serotonin-induced proliferation of yeast as well as human (SHSY5Y) cells. One possible cause of this effect is through inhibition of 5-HT receptor activation by quinine, as we observed here. Furthermore, cells exhibited marked decreases in serotonin production during incubation with quinine. By assaying activity and kinetics of the rate-limiting enzyme for serotonin biosynthesis, tryptophan hydroxylase (TPH2), we showed that quinine competitively inhibits TPH2 in the presence of the substrate tryptophan. The study shows that quinine disrupts both serotonin biosynthesis and function, giving important new insight to the action of quinine on mammalian cells.

  14. ORMDL3 contributes to the risk of atherosclerosis in Chinese Han population and mediates oxidized low-density lipoprotein-induced autophagy in endothelial cells.

    Science.gov (United States)

    Ma, Xiaochun; Qiu, Rongfang; Dang, Jie; Li, Jiangxia; Hu, Qin; Shan, Shan; Xin, Qian; Pan, Wenying; Bian, Xianli; Yuan, Qianqian; Long, Feng; Liu, Na; Li, Yan; Gao, Fei; Zou, Chengwei; Gong, Yaoqin; Liu, Qiji

    2015-11-25

    ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) is a universally confirmed susceptibility gene for asthma and has recently emerged as a crucial modulator in lipid metabolism, inflammation and endoplasmic reticulum (ER) stress-the mechanisms also closely involved in atherosclerosis (AS). Here we first presented the evidence of two single nucleotide polymorphisms regulating ORMDL3 expression (rs7216389 and rs9303277) significantly associated with AS risk and the evidence of increased ORMDL3 expression in AS cases compared to controls, in Chinese Han population. Following the detection of its statistical correlation with AS, we further explored the functional relevance of ORMDL3 and hypothesized a potential role mediating autophagy as autophagy is activated upon modified lipid, inflammation and ER stress. Our results demonstrated that in endothelial cells oxidized low-density lipoprotein (ox-LDL) up-regulated ORMDL3 expression and knockdown of ORMDL3 alleviated not only ox-LDL-induced but also basal autophagy. BECN1 is essential for autophagy initiation and silencing of ORMDL3 suppressed ox-LDL-induced as well as basal BECN1 expression. In addition, deletion of ORMDL3 resulted in greater sensitivity to ox-LDL-induced cell death. Taken together, ORMDL3 might represent a causal gene mediating autophagy in endothelial cells in the pathogenesis of AS.

  15. Biosynthesis of enediyne antitumor antibiotics.

    Science.gov (United States)

    Van Lanen, Steven G; Shen, Ben

    2008-01-01

    The enediyne polyketides are secondary metabolites isolated from a variety of Actinomycetes. All members share very potent anticancer and antibiotic activity, and prospects for the clinical application of the enediynes has been validated with the recent marketing of two enediyne derivatives as anticancer agents. The biosynthesis of these compounds is of interest because of the numerous structural features that are unique to the enediyne family. The gene cluster for five enediynes has now been cloned and sequenced, providing the foundation to understand natures' means to biosynthesize such complex, exotic molecules. Presented here is a review of the current progress in delineating the biosynthesis of the enediynes with an emphasis on the model enediyne, C-1027. PMID:18397168

  16. Biosynthesis of Enediyne Antitumor Antibiotics

    OpenAIRE

    Van Lanen, Steven G.; Shen, Ben

    2008-01-01

    The enediyne polyketides are secondary metabolites isolated from a variety of Actinomycetes. All members share very potent anticancer and antibiotic activity, and prospects for the clinical application of the enediynes has been validated with the recent marketing of two enediyne derivatives as anticancer agents. The biosynthesis of these compounds is of interest because of the numerous structural features that are unique to the enediyne family. The gene cluster for five enediynes has now been...

  17. Fatty acid biosynthesis in actinomycetes

    OpenAIRE

    Gago, Gabriela; Diacovich, Lautaro; Arabolaza, Ana; Tsai, Shiou-Chuan; Gramajo, Hugo

    2011-01-01

    All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation fo...

  18. Taxol biosynthesis and molecular genetics

    OpenAIRE

    Croteau, Rodney; Ketchum, Raymond E.B.; Long, Robert M.; Kaspera, Rüdiger; Wildung, Mark R.

    2006-01-01

    Biosynthesis of the anticancer drug Taxol in Taxus (yew) species involves 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methyl erythritol phosphate pathway for isoprenoid precursor supply. Following the committed cyclization to the taxane skeleton, eight cytochrome P450-mediated oxygenations, three CoA-dependent acyl/aroyl transfers, an oxidation at C9, and oxetane (D-ring) formation yield the intermediate baccatin III, to which the fu...

  19. Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

    Science.gov (United States)

    Krijt, J; van Holsteijn, I; Hassing, I; Vokurka, M; Blaauboer, B J

    1993-01-01

    The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides. PMID:8517781

  20. The translation elongation factor eEF-1Bβ1 is involved in cell wall biosynthesis and plant development in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Zakir Hossain

    Full Text Available The eukaryotic translation elongation factor eEF-1Bβ1 (EF1Bβ is a guanine nucleotide exchange factor that plays an important role in translation elongation. In this study, we show that the EF1Bβ protein is localized in the plasma membrane and cytoplasm, and that the transcripts should be expressed in most tissue types in seedlings. Sectioning of the inflorescence stem revealed that EF1Bβ predominantly localizes to the xylem vessels and in the interfascicular cambium. EF1Bβ gene silencing in efβ caused a dwarf phenotype with 38% and 20% reduction in total lignin and crystalline cellulose, respectively. This loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type plants, but no changes were detected in a gain-of-function mutant transformed with the EF1Bβ gene. Histochemical analysis showed a reduced vascular apparatus, including smaller xylem vessels in the inflorescence stem of the loss-of-function mutant. Over-expression of EF1Bβ in an eli1 mutant background restored a WT phenotype and abolished ectopic lignin deposition as well as cell expansion defects in the mutant. Taken together, these data strongly suggest a role for EF1Bβ in plant development and cell wall formation in Arabidopsis.

  1. Polyamine biosynthesis is critical for growth and differentiation of the pancreas.

    Science.gov (United States)

    Mastracci, Teresa L; Robertson, Morgan A; Mirmira, Raghavendra G; Anderson, Ryan M

    2015-08-24

    The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation.

  2. Identification of Protein-Protein Interactions Involved in Pectin Biosynthesis in the golgi Apparatus

    DEFF Research Database (Denmark)

    Lund, Christian Have

    for instance as food additives, nutraceutical, for paper and energy production. Pectin is a cell wall glycan that crucial for every plant growing on land. Pectin is said to be one of the most complex glycans on earth and it is hypothesized that at least 67 enzymatic reactions are involved in its biosynthesis....... To date, only seven glycosyltransferase (GT) genes have been identified and characterized comprising only four biosynthetic activities within pectin biosynthesis. Therefore, increased knowledge about pectin biosynthesis is of great importance if we in the future wants to fully manipulate and exploit...... the diverse pectin structures for industrial, agronomic and biomedical uses. Increasing evidence suggests that complex formation is important in governing functional coordination of proteins involved in cell wall biosynthesis. In Arabidopsis thaliana, a homogalacturonan (HG) synthase core complex between...

  3. Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

    Institute of Scientific and Technical Information of China (English)

    Kerry H. Caffall; Sivakumar Pattathil; Sarah E. Phillips; Michael G. Hahn; Debra Mohnen

    2009-01-01

    Galacturonosyltransferase 1 (GAUT1) is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1(GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution com-parable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as dem-onstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mu-tant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.

  4. Lignin biosynthesis and its molecular regulation

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Lignin biosynthesis has become increasingly highlighted because it plays an important role in the growth and development of plant, in the systematic evolution of plant and in the human life. Due to the progress in the field of lignin studies in recent years, the lignin biosynthesis pathway has been 修订日期:. Here we discuss some genetic engineering approaches on lignin biosynthesis, and conceive strategy to regulate lignin biosynthesis in order to use lignin resource more efficiently in agricultural and industrial productions.

  5. A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

    Science.gov (United States)

    Ferrieri, Abigail P; Arce, Carla C M; Machado, Ricardo A R; Meza-Canales, Ivan D; Lima, Eraldo; Baldwin, Ian T; Erb, Matthias

    2015-10-01

    Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory. PMID:26017581

  6. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    OpenAIRE

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthet...

  7. Biosynthesis and Function of Extracellular Glycans in Cyanobacteria

    OpenAIRE

    Jan-Christoph Kehr; Elke Dittmann

    2015-01-01

    The cell surface of cyanobacteria is covered with glycans that confer versatility and adaptability to a multitude of environmental factors. The complex carbohydrates act as barriers against different types of stress and play a role in intra- as well as inter-species interactions. In this review, we summarize the current knowledge of the chemical composition, biosynthesis and biological function of exo- and lipo-polysaccharides from cyanobacteria and give an overview of sugar-binding lectins c...

  8. Improving penicillin biosynthesis in Penicillium chrysogenum by glyoxalase overproduction

    OpenAIRE

    Scheckhuber, Christian Q.; Veenhuis, Marten; van der Klei, Ida J

    2013-01-01

    Genetic engineering of fungal cell factories mainly focuses on manipulating enzymes of the product pathway or primary metabolism. However, despite the use of strong promoters or strains containing the genes of interest in multiple copies, the desired strongly enhanced enzyme levels are often not obtained. Here we present a novel strategy to improve penicillin biosynthesis by Penicillium chrysogenum by reducing reactive and toxic metabolic by-products, 2-oxoaldehydes. This was achieved by over...

  9. Sulfur deficiency–induced repressor proteins optimize glucosinolate biosynthesis in plants

    Science.gov (United States)

    Aarabi, Fayezeh; Kusajima, Miyuki; Tohge, Takayuki; Konishi, Tomokazu; Gigolashvili, Tamara; Takamune, Makiko; Sasazaki, Yoko; Watanabe, Mutsumi; Nakashita, Hideo; Fernie, Alisdair R.; Saito, Kazuki; Takahashi, Hideki; Hubberten, Hans-Michael; Hoefgen, Rainer; Maruyama-Nakashita, Akiko

    2016-01-01

    Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (−S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links –S to GSL biosynthesis has remained understudied. We report here the identification of the –S marker genes sulfur deficiency induced 1 (SDI1) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under –S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of –S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions.

  10. Oleic acid biosynthesis in cyanobacteria

    International Nuclear Information System (INIS)

    The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with 14CO2. None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating 14CO2 into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria

  11. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  12. Biosynthesis and Function of Extracellular Glycans in Cyanobacteria

    Directory of Open Access Journals (Sweden)

    Jan-Christoph Kehr

    2015-01-01

    Full Text Available The cell surface of cyanobacteria is covered with glycans that confer versatility and adaptability to a multitude of environmental factors. The complex carbohydrates act as barriers against different types of stress and play a role in intra- as well as inter-species interactions. In this review, we summarize the current knowledge of the chemical composition, biosynthesis and biological function of exo- and lipo-polysaccharides from cyanobacteria and give an overview of sugar-binding lectins characterized from cyanobacteria. We discuss similarities with well-studied enterobacterial systems and highlight the unique features of cyanobacteria. We pay special attention to colony formation and EPS biosynthesis in the bloom-forming cyanobacterium, Microcystis aeruginosa.

  13. Biosynthesis of collagen by fibroblasts kept in culture

    International Nuclear Information System (INIS)

    The sinthesis of collagen is studied in fibroblasts of different origins with the purpose of obtaining an appropriate system for the study of its biosynthesis and processing. The percentage of collagen synthesis vary according to the fibroblast origin. Experiences are performed with fibroblasts kept in culture from: chicken - and guinea pig embryos, carragheenin - induced granulomas in adult guinea pig and from human skin. The collagen pattern synthesized after acetic acid - or saline extractions in the presence of inhibitors is also determined. This pattern is then assayed by poliacrilamide - 5% - SDS gel electrophoresis accompanied by fluorography. The importance of the cell culture system in the elucidation of collagen biosynthesis is pointed out. (M.A.)

  14. Steroid biosynthesis in adipose tissue.

    Science.gov (United States)

    Li, Jiehan; Papadopoulos, Vassilios; Vihma, Veera

    2015-11-01

    Tissue-specific expression of steroidogenic enzymes allows the modulation of active steroid levels in a local manner. Thus, the measurement of local steroid concentrations, rather than the circulating levels, has been recognized as a more accurate indicator of the steroid action within a specific tissue. Adipose tissue, one of the largest endocrine tissues in the human body, has been established as an important site for steroid storage and metabolism. Locally produced steroids, through the enzymatic conversion from steroid precursors delivered to adipose tissue, have been proven to either functionally regulate adipose tissue metabolism, or quantitatively contribute to the whole body's steroid levels. Most recently, it has been suggested that adipose tissue may contain the steroidogenic machinery necessary for the initiation of steroid biosynthesis de novo from cholesterol. This review summarizes the evidence indicating the presence of the entire steroidogenic apparatus in adipose tissue and discusses the potential roles of local steroid products in modulating adipose tissue activity and other metabolic parameters.

  15. Acylphloroglucinol Biosynthesis in Strawberry Fruit.

    Science.gov (United States)

    Song, Chuankui; Ring, Ludwig; Hoffmann, Thomas; Huang, Fong-Chin; Slovin, Janet; Schwab, Wilfried

    2015-11-01

    Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis.

  16. Role of acid sphingomyelinase in the age-dependent dysregulation of sphingolipids turnover in the tissues of rats.

    Science.gov (United States)

    Babenko, Nataliya A; Garkavenko, Vladimir V; Storozhenko, Galina V; Timofiychuk, Olga A

    2016-04-01

    Old age-associated pathologies usually coincide with altered sphingolipid metabolism. In the present article, the role of acid sphingomyelinase (ASMase) in the age-dependent changes of sphingomyelin (SM) and ceramide contents in the tissues has been investigated by means of ASMase inhibitors, imipramine and zoledronic acid. It has been determined that ceramide content and ceramide/SM ratio increased, while SM level decreased in the heart, liver, blood serum and skeletal muscles of 24-month old rats in contrast to 3-month old animals. Injections of imipramine or zoledronic acid to 24-month old rats resulted in significant downregulation of ASMase in the liver and skeletal and heart muscles. The both inhibitors decreased the ceramide content and ceramide/SM ratio and increased the SM content in all tissues studied, except the heart, of old rats to the levels close to those observed in the young animals. Long-term treatment of rats by inhibitors, which have different mechanisms of action on ASMase, exerts the similar, but not equal effects on enzyme activity and SM turnover. In summary, the data above strongly suggest that the age-dependent up-regulation of ASMase plays an important role in the modulation of ceramide and SM contents in rat tissues and that imipramine and zoledronic acid are useful tools for SM turnover manipulation at old age. PMID:26830134

  17. Phosphoglycerate Mutase 1 Coordinates Glycolysis and Biosynthesis to Promote Tumor Growth

    Energy Technology Data Exchange (ETDEWEB)

    Hitosugi, Taro [Emory Univ. School of Medicine, Atlanta, GA (United States); Zhou, Lu [Univ. of Chicago, IL (United States); Elf, Shannon [Emory Univ. School of Medicine, Atlanta, GA (United States); Fan, Jun [Emory Univ. School of Medicine, Atlanta, GA (United States); Kang, Hee-Bum [Emory Univ. School of Medicine, Atlanta, GA (United States); Seo, Jae Ho [Emory Univ. School of Medicine, Atlanta, GA (United States); Shan, Changliang [Emory Univ. School of Medicine, Atlanta, GA (United States); Dai, Qing [Univ. of Chicago, IL (United States); Zhang, Liang [Univ. of Chicago, IL (United States); Xie, Jianxin [Cell Signaling Technology, Inc., Danvers, MA (United States); Gu, Ting-Lei [Cell Signaling Technology, Inc., Danvers, MA (United States); Jin, Peng [Emory Univ. School of Medicine, Atlanta, GA (United States); Alečković, Masa [Princeton Univ., NJ (United States); LeRoy, Gary [Princeton Univ., NJ (United States); Kang, Yibin [Princeton Univ., NJ (United States); Sudderth, Jessica A. [UT Southwestern Medical Center, Dallas, TX (United States); DeBerardinis, Ralph J. [UT Southwestern Medical Center, Dallas, TX (United States); Luan, Chi-Hao [Northwestern Univ., Evanston, IL (United States); Chen, Georgia Z. [Emory Univ. School of Medicine, Atlanta, GA (United States); Muller, Susan [Emory Univ. School of Medicine, Atlanta, GA (United States); Shin, Dong M. [Emory Univ. School of Medicine, Atlanta, GA (United States); Owonikoko, Taofeek K. [Emory Univ. School of Medicine, Atlanta, GA (United States); Lonial, Sagar [Emory Univ. School of Medicine, Atlanta, GA (United States); Arellano, Martha L. [Emory Univ. School of Medicine, Atlanta, GA (United States); Khoury, Hanna J. [Emory Univ. School of Medicine, Atlanta, GA (United States); Khuri, Fadlo R. [Emory Univ. School of Medicine, Atlanta, GA (United States); Lee, Benjamin H. [Novartis Inst. for BioMedical Research, Cambridge, MA (United States); Ye, Keqiang [Emory Univ. School of Medicine, Atlanta, GA (United States); Boggon, Titus J. [Yale Univ. School of Medicine, New Haven, CT (United States); Kang, Sumin [Emory Univ. School of Medicine, Atlanta, GA (United States); He, Chuan [Univ. of Chicago, IL (United States); Chen, Jing [Emory Univ. School of Medicine, Atlanta, GA (United States)

    2012-11-12

    It is unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation partially by controlling intracellular levels of its substrate, 3-phosphoglycerate (3-PG), and product, 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.

  18. Evolution of catalase activity during nystatin biosynthesis

    Directory of Open Access Journals (Sweden)

    Cristina Bota

    2009-03-01

    Full Text Available The research studies focused on the dynamics of catalase during nystatin biosynthesis by Streptomyces noursei. The catalase activity was determined by growing a pure culture of Streptomyces noursei from the strain collection owned by the company S.C. Antibiotice Iasi on biosynthesis medium. The test was performed on two experimental models of biosynthesis, one using sunflower oil, while the other soybean oil as basic nutrients. Special attention was paid to the connection between the evolution of the biomass and the level of catalase activity.

  19. Effect of Methyl Jasmonic Acid on Baccatin Ⅲ Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jianfeng; GUO Zhigang

    2006-01-01

    As baccatin Ⅲ is an immediate diterpenoid precursor of taxol, the increase of baccatin Ⅲ is beneficial to the biosynthesis of taxol. Addition of methyl jasmonic acid (M J) enhances the activity of 10- deaceyle baccatin (DAB) Ⅲ acetyl transferase which catalyzes the bioconversion from 10-DAB Ⅲ to beccatin Ⅲ. In this paper, the baccatin Ⅲ content was increased by 174% by the addition of 100 μmol/L MJ in suspension cultures of Taxus cuspidate. Induction by MJ also increased the expression of a 49.0-kDa protein. This paper describes the cell free acetylation of 10-DAB Ⅲ in crude extracts of enzyme from the suspension cultures of Taxus cuspidate. The reaction product was confirmed by high performance liquid chromatograph (HPLC). About 25.0% of the 10-DAB Ⅲ was acetylized into baccatin Ⅲ on the 4th day with 100 μmol/L MJ.The 10-DAB Ⅲ acetyl transferase activity reached a peak on the 2nd day with 100 μmol/L M J, with 54.7% of the 10-DAB Ⅲ transformed into baccatin Ⅲ. The baccatin Ⅲ content increased with the increase of 10-DAB Ⅲ acetyl transferase activity, although the biosynthesis was delayed by more than 24 h. The remarkable induction of MJ on baccatin Ⅲ biosynthesis shows a promising way to increase the production of taxol.

  20. Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi.

    Science.gov (United States)

    Freitag, Johannes; Ast, Julia; Linne, Uwe; Stehlik, Thorsten; Martorana, Domenica; Bölker, Michael; Sandrock, Björn

    2014-07-01

    Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell. PMID:24835306

  1. The tRNA-Dependent Biosynthesis of Modified Cyclic Dipeptides

    Directory of Open Access Journals (Sweden)

    Tobias W. Giessen

    2014-08-01

    Full Text Available In recent years it has become apparent that aminoacyl-tRNAs are not only crucial components involved in protein biosynthesis, but are also used as substrates and amino acid donors in a variety of other important cellular processes, ranging from bacterial cell wall biosynthesis and lipid modification to protein turnover and secondary metabolite assembly. In this review, we focus on tRNA-dependent biosynthetic pathways that generate modified cyclic dipeptides (CDPs. The essential peptide bond-forming catalysts responsible for the initial generation of a CDP-scaffold are referred to as cyclodipeptide synthases (CDPSs and use loaded tRNAs as their substrates. After initially discussing the phylogenetic distribution and organization of CDPS gene clusters, we will focus on structural and catalytic properties of CDPSs before turning to two recently characterized CDPS-dependent pathways that assemble modified CDPs. Finally, possible applications of CDPSs in the rational design of structural diversity using combinatorial biosynthesis will be discussed before concluding with a short outlook.

  2. The tRNA-dependent biosynthesis of modified cyclic dipeptides.

    Science.gov (United States)

    Giessen, Tobias W; Marahiel, Mohamed A

    2014-01-01

    In recent years it has become apparent that aminoacyl-tRNAs are not only crucial components involved in protein biosynthesis, but are also used as substrates and amino acid donors in a variety of other important cellular processes, ranging from bacterial cell wall biosynthesis and lipid modification to protein turnover and secondary metabolite assembly. In this review, we focus on tRNA-dependent biosynthetic pathways that generate modified cyclic dipeptides (CDPs). The essential peptide bond-forming catalysts responsible for the initial generation of a CDP-scaffold are referred to as cyclodipeptide synthases (CDPSs) and use loaded tRNAs as their substrates. After initially discussing the phylogenetic distribution and organization of CDPS gene clusters, we will focus on structural and catalytic properties of CDPSs before turning to two recently characterized CDPS-dependent pathways that assemble modified CDPs. Finally, possible applications of CDPSs in the rational design of structural diversity using combinatorial biosynthesis will be discussed before concluding with a short outlook. PMID:25196600

  3. Effect of intestinal resection on hydrogen sulfide biosynthesis and the damage of Cajal interstitial cells%肠道手术对肠道硫化氢生成及Cajal间质细胞破坏的影响

    Institute of Scientific and Technical Information of China (English)

    黄颖; 邵宇; 叶道雄; 黄胜辉; 徐宗斌; 池畔

    2015-01-01

    目的:探讨小鼠肠切除术对肠道内源性硫化氢(H2S)生成与Cajal间质细胞(ICC)破坏的影响。方法制作肠切除术模型小鼠,检测术后吻合口近端过氧化物酶(MPO)活性,敏感硫电极法测定H2S含量,反转录PCR检测肠组织H2S生成酶[胱硫醚β合成酶(CBS)和胱硫醚γ裂解酶(CSE)] mRNA表达,免疫荧光检测肠组织c-kit表达以了解ICC的分布。结果小鼠小肠组织表达CSE mRNA,但未检测到CBS mRNA表达。小鼠肠切除术后,吻合口近端3 cm处CSE mRNA表达在术后1 h开始增加(P<0.05),术后6 h达峰值,而后逐渐下降,至术后24 h与对照组的差异无统计学意义(P>0.05)。术前腹腔注射CSE拮抗剂propargylglycine(PPG)后,小鼠吻合口近端3 cm处肠组织CSE mRNA表达下降[(1.16±0.18)比(1.63±0.13),P<0.05],MPO活性降低[(0.54±0.07) U/g比(0.83±0.09) U/g,P<0.05],H2S含量减少[(36.1±6.1) nmol/mg比(45.3±5.6) nmol/mg,P<0.05],肌间丛ICC阳性面积百分比增加[(2.26±0.19)%比(1.65±0.24)%, P<0.01]。结论小鼠肠切除诱发的肌层炎性反应,可增加吻合口近端小肠组织CSE表达,产生过量的H2S,破坏ICC,造成肠动力紊乱。术前抑制内源性H2S的产生可对ICC具有保护作用。%Objective To investigate the effect of intestinal resection on hydrogen sulfide (H2S) biosynthesis and interstitial cells of Cajal (ICC) in mice. Methods After intestinal resection mouse model was established , the activity of MPO in the proximal anastomosis intestinal tissue were detected. Sensitive sulphur electrode assay was applied to measure the H2S level. RT-PCR technique was employed to investigate the mRNA expression of the endogenous H2S biosynthesis enzymes , cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE). Immunofluorescence staining was used to detecte

  4. Very long-chain fatty acid-containing lipids rather than sphingolipids per se are required for raft association and stable surface transport of newly synthesized plasma membrane ATPase in yeast.

    Science.gov (United States)

    Gaigg, Barbara; Toulmay, Alexandre; Schneiter, Roger

    2006-11-10

    The proton-pumping H+-ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid "rafts") already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide.

  5. The regulation and biosynthesis of antimycins

    Directory of Open Access Journals (Sweden)

    Ryan F. Seipke

    2013-11-01

    Full Text Available Antimycins (>40 members were discovered nearly 65 years ago but the discovery of the gene cluster encoding antimycin biosynthesis in 2011 has facilitated rapid progress in understanding the unusual biosynthetic pathway. Antimycin A is widely used as a piscicide in the catfish farming industry and also has potent killing activity against insects, nematodes and fungi. The mode of action of antimycins is to inhibit cytochrome c reductase in the electron transport chain and halt respiration. However, more recently, antimycin A has attracted attention as a potent and selective inhibitor of the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL. Remarkably, this inhibition is independent of the main mode of action of antimycins such that an artificial derivative named 2-methoxyantimycin A inhibits Bcl-xL but does not inhibit respiration. The Bcl-2/Bcl-xL family of proteins are over-produced in cancer cells that are resistant to apoptosis-inducing chemotherapy agents, so antimycins have great potential as anticancer drugs used in combination with existing chemotherapeutics. Here we review what is known about antimycins, the regulation of the ant gene cluster and the unusual biosynthetic pathway.

  6. Estrogen biosynthesis in human uterine adenomyosis

    Energy Technology Data Exchange (ETDEWEB)

    Urabe, Mamoru; Yamamoto, Takara; Kitawaki, Jo; Honjo, Hideo; Okada, Hiroji (Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto (Japan))

    1989-01-01

    Estrogen biosynthesis (aromatiase activity) was investigated in human adenomyosis tissue and compared with that of the normal myometrium, endometrium, and endometrical cancer tissues. Homogenates were incubated with (1,2,6,7-{sup 3}H)androstenedione and NADPH at 37 deg. C for 1 h. After stopping the enzymatic reaction with ethyl acetate, (4-{sup 14}C)estrone and (4-{sup 14}C)estradiol-17{beta} were added to the incubated sample. Estrone and estradiol were purified and identified by Bio-Rad AG1-X2 column chromatography, thin-layer chromatography and co-crystallization. Estrogen formed in the incubated sample was calculated from the {sup 3}H/{sup 14}C ratio of the final crystal. The value for estrone formed from androstenedione was 52-132 fmol{sup .}h{sup -1.}g{sup -1} wet weight. Aromatase activity in the adenomyosis tissues was higher than that in normal endometrial or myometrial tissues, but lower than that found in myometrial or endometrial tumour tissue. Furthermore, we investigated the effect of danazol, progresterone, and medroxyprogesterone acetate on adenomyosis cells in primary cultures. Aromatase activity in adenomyosis was blocked by danazol, but stimulated by progesterone and MPA. These results indicate that aromatase activity in adenomyosis may contribute to the growth of the ectopic endometrial tissue which occurs in this disease. (author).

  7. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  8. Sterols of the fungi - Distribution and biosynthesis

    Science.gov (United States)

    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.

  9. Biosynthesis of monoterpene scent compounds in roses

    OpenAIRE

    Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frederic; Nicolè, Florence; Raymond, Olivier; Huguet, Stephanie; Baltenweck-Guyot, Raymonde; Meyer, Sophie; Claudel, Patricia

    2015-01-01

    The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribut...

  10. Antibacterial Targets in Fatty Acid Biosynthesis

    OpenAIRE

    Wright, H. Tonie; Reynolds, Kevin A.

    2007-01-01

    The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for development of new anti-bacterial agents. The extended use of the anti-tuberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for anti-bacterial development. Differences in subcellular organization of the bacterial and eukaryotic multi-enzyme fatty acid synthase systems offer the prospect of inhibitors with host vs...

  11. Carotenoid Biosynthesis in Daucus carota.

    Science.gov (United States)

    Simpson, Kevin; Cerda, Ariel; Stange, Claudia

    2016-01-01

    Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota. PMID:27485223

  12. Effect of apolipoprotein a-I complex with tetrahydrocortisone on protein biosynthesis and glucose absorption by rat hepatocytes.

    Science.gov (United States)

    Sumenkova, D V; Knyazev, R A; Guschya, R S; Polyakov, L M; Panin, L E

    2009-08-01

    We studied the effect of apolipoprotein A-I-tetrahydrocortisone complex on (14)C glucose absorption and lactate accumulation and on the rate of protein biosynthesis in isolated rat hepatocytes. The presence of apolipoprotein A-I-tetrahydrocortisone complex in the incubation medium increased absorption of labeled glucose by hepatocytes by 52%, while lactate content in the conditioning medium increased 4-fold. The rate of protein biosynthesis increased by 80% in comparison with control cells. It is hypothesized that the increase in protein biosynthesis rate in hepatocytes under the effect of apolipoprotein A-I-tetrahydrocortisone complex is due to stimulation of energy metabolism, specifically, of its glycolytic component. PMID:20027330

  13. Nfasc155H and MAG are specifically susceptible to detergent extraction in the absence of the myelin sphingolipid sulfatide.

    Science.gov (United States)

    Pomicter, A D; Deloyht, J M; Hackett, A R; Purdie, N; Sato-Bigbee, C; Henderson, S C; Dupree, J L

    2013-12-01

    Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

  14. Animal source food intake and association with blood cholesterol, glycerophospholipids and sphingolipids in a northern Swedish population

    Directory of Open Access Journals (Sweden)

    Wilmar Igl

    2013-08-01

    Full Text Available Background . The high intake of game meat in populations with a subsistence-based diet may affect their blood lipids and health status. Objective . To examine the association between diet and circulating levels of blood lipid levels in a northern Swedish population. Study design . We compared a group with traditional lifestyle (TLS based on reindeer herding (TLS group with those from the same area with a non-traditional lifestyle (NTLS typical of more industrialized regions of Sweden (NTLS group. The analysis was based on self-reported intake of animal source food (i.e. non-game meat, game meat, fish, dairy products and eggs and the serum blood level of a number of lipids [total cholesterol (TC, low-density lipoprotein cholesterol (LDL, high-density lipoprotein cholesterol (HDL, triglycerides (TG, glycerophospholipids and sphingolipids]. Results . The TLS group had higher cholesterol, LDL and HDL levels than the reference group. Of the TLS group, 65% had cholesterol levels above the threshold for increased risk of coronary heart disease (≥240 mg/dl, as compared to 38% of the NTLS group. Self-reported consumption of game meat was positively associated with TC and LDL. Conclusions . The high game meat consumption of the TLS group is associated with increased cholesterol levels. High intake of animal protein and fat and low fibre is known to increase the risk of cardiovascular disease, but other studies of the TLS in northern Sweden have shown comparable incidences of cardiovascular disease to the reference (NTLS group from the same geographical area. This indicates that factors other than TC influence disease risk. One such possible factor is dietary phospholipids, which are also found in high amounts specifically in game meat and have been shown to inhibit cholesterol absorption.

  15. Ceramide formation is involved in Lactobacillus acidophilus-induced IFN-beta response in dendritic cells

    DEFF Research Database (Denmark)

    Fuglsang, Eva; Henningsen, Louise; Frøkiær, Hanne

    The sphingolipid ceramide is known to play a role in lipid raft fusion and receptor clustering in the plasma membrane (PM). Upon bacterial encounter, dendritic cells (DCs) endocytose the bacteria and initiate a bacteria-specific downstream signaling event. We hypothesized that conversion...

  16. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase

    OpenAIRE

    Austin, Michael B; Saito, Tamao; Bowman, Marianne E.; Haydock, Stephen; Kato, Atsushi; Moore, Bradley S.; Kay, Robert R.; Noel, Joseph P.

    2006-01-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two ~3,000-residue D. discoideum proteins, termed ‘steely’, as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid syntha...

  17. Biosynthesis of secondary metabolites in sugarcane

    Directory of Open Access Journals (Sweden)

    S.C. França

    2001-12-01

    Full Text Available A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC; chalcone synthase (CHS, the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS, involved in plant defense and root nodulation; isoflavone reductase (IFR, a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.Este trabalho foi realizado com os objetivos de gerar uma coleção de genes

  18. Biosynthesis of Triacylglycerols (TAGs in plants and algae

    Directory of Open Access Journals (Sweden)

    Alexandro Cagliari

    2011-12-01

    Full Text Available Triacylglycerols (TAGs, which consist of three fatty acids bound to a glycerol backbone, are major storage lipids that accumulate in developing seeds, flower petals, pollen grains, and fruits of innumerous plant species. These storage lipids are of great nutritional and nutraceutical value and, thus, are a common source of edible oils for human consumption and industrial purposes. Two metabolic pathways for the production of TAGs have been clarified: an acyl¬ CoA-dependent pathway and an acyl-CoA-independent pathway. Lipid metabolism, specially the pathways to fatty acids and TAG biosynthesis, is relatively well understood in plants, but poorly known in algae. It is generally accepted that the basic pathways of fatty acid and TAG biosynthesis in algae are analogous to those of higher plants. However, unlike higher plants where individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, the complete pathway, from carbon dioxide fixation to TAG synthesis and sequestration, takes place within a single algal cell. Another distinguishing feature of some algae is the large amounts of very long-chain polyunsaturated fatty acids (VLC- PUFAs as major fatty acid components. Nowadays, the focus of attention in biotechnology is the isolation of novel fatty acid metabolizing genes, especially elongases and desaturases that are responsible for PUFAs synthesis, from different species of algae, and its transfer to plants. The aim is to boost the seed oil content and to generate desirable fatty acids in oilseed crops through genetic engineering approaches. This paper presents the current knowledge of the neutral storage lipids in plants and algae from fatty acid biosynthesis to TAG accumulation.

  19. Biosynthesis of gold nanoparticles: A green approach.

    Science.gov (United States)

    Ahmed, Shakeel; Annu; Ikram, Saiqa; Yudha S, Salprima

    2016-08-01

    Nanotechnology is an immensely developing field due to its extensive range of applications in different areas of technology and science. Different types of methods are employed for synthesis of nanoparticles due to their wide applications. The conventional chemical methods have certain limitations with them either in the form of chemical contaminations during their syntheses procedures or in later applications and use of higher energy. During the last decade research have been focussed on developing simple, clean, non-toxic, cost effective and eco-friendly protocols for synthesis of nanoparticles. In order to get this objective, biosynthesis methods have been developed in order to fill this gap. The biosynthesis of nanoparticles is simple, single step, eco-friendly and a green approach. The biochemical processes in biological agents reduce the dissolved metal ions into nano metals. The various biological agents like plant tissues, fungi, bacteria, etc. are used for biosynthesis for metal nanoparticles. In this review article, we summarised recent literature on biosynthesis of gold nanoparticles which have revolutionised technique of synthesis for their applications in different fields. Due to biocompatibility of gold nanoparticles, it has find its applications in biomedical applications. The protocol and mechanism of biosynthesis of gold nanoparticles along with various applications have also been discussed. PMID:27236049

  20. Mechanisms for Recycling and Biosynthesis of Endogenous Cannabinoids Anandamide and 2-Arachidonylglycerol

    OpenAIRE

    Placzek, Ekaterina A.; Okamoto, Yasuo; Ueda, Natsuo; Barker, Eric L.

    2008-01-01

    The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid...

  1. High-temperature injury and auxin biosynthesis in microsporogenesis.

    Directory of Open Access Journals (Sweden)

    Atsushi eHigashitani

    2013-03-01

    Full Text Available Plant reproductive development is more sensitive than vegetative growth to many environmental stresses. With global warming, in particular, plant high temperature injury is becoming an increasingly serious problem. In wheat, barley, and various other commercially important crops, the early phase of anther development is especially susceptible to high temperatures. We recently demonstrated that high temperature causes cell-proliferation arrest and represses auxin signaling in a tissue-specific manner of the anther cells of barley and Arabidopsis. These phenomena were accompanied by comprehensive alterations in transcription including repression of cell-proliferation related genes and YUCCA auxin biosynthesis genes. Moreover, application of auxin completely improved the transcriptional alterations, the production of normal pollen grains, and seed setting rate under increasing temperatures. These denote that auxin, which has been used widely as potent and selective herbicides, is useful for the promotion of plant fertility and maintenance of crop yields under the global warming conditions.

  2. Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

    NARCIS (Netherlands)

    Boer, L. de; Vrijbloed, J.W.; Grobben, G.; Dijkhuizen, L.

    1989-01-01

    The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenyl

  3. Crystallization and preliminary crystallographic analysis of the cellulose biosynthesis-related protein CMCax from Acetobacter xylinum

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Shin; Yasutake, Yoshiaki; Tajima, Kenji; Satoh, Yasuharu; Yao, Min, E-mail: yao@castor.sci.hokudai.ac.jp; Tanaka, Isao; Munekata, Masanobu

    2005-02-01

    The cellulose biosynthesis-related protein CMCax from A. xylinum has been purified and crystallized. The crystals of CMCax belong to the primitive hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 89.1, c = 94.2 Å.

  4. Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

    Science.gov (United States)

    St-Pierre, B; Vazquez-Flota, FA; De Luca V

    1999-01-01

    In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed. PMID:10330473

  5. The Terpenoid Biosynthesis Toolkit of Trichoderma.

    Science.gov (United States)

    Bansal, Ravindra; Mukherjee, Prasun Kumar

    2016-04-01

    The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters. PMID:27396184

  6. The Terpenoid Biosynthesis Toolkit of Trichoderma.

    Science.gov (United States)

    Bansal, Ravindra; Mukherjee, Prasun Kumar

    2016-04-01

    The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters.

  7. Flavonoids: Biosynthesis, Biological functions and Biotechnological applications

    Directory of Open Access Journals (Sweden)

    Maria Lorena eFalcone Ferreyra

    2012-09-01

    Full Text Available Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, bHLH and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.

  8. Feeding a Modified Fish Diet to Bottlenose Dolphins Leads to an Increase in Serum Adiponectin and Sphingolipids.

    Directory of Open Access Journals (Sweden)

    Philip Michael Sobolesky

    2016-04-01

    Full Text Available Feeding a modified fish diet has been suggested to improve insulin sensitivity in bottlenose dolphins; however insulin sensitivity was not directly measured. Since demonstrating an improvement in insulin sensitivity is technically difficult in dolphins, we postulated that directional changes in the hormone axis: fibroblast growth factor 21 (FGF21/Adiponectin/Ceramide (Cer, could provide further support to this hypothesis. We measured two hour post-prandial serum FGF21, total adiponectin, % unmodified adiponectin, ceramide, and sphingosine levels from dolphins fed a diet rich in heptadecanoic acid (C17:0 over 24 weeks. Serum FGF21 was quantified by ELISA with an observed range of 129-1599 pg/ml, but did not significantly change over the 24 week study period. Total adiponectin levels (mean ± SD significantly increased from 776 ± 400 pmol/ml at week 0 to 1196 ± 467 pmol/ml at week 24. The % unmodified adiponectin levels (mean ± SD decreased from 23.8 ± 6.0% at week 0 to 15.2 ± 5.2% at week 24. Interestingly, although FGF21 levels did not change, there was a good correlation between FGF21 and total adiponectin (ρ = 0.788, P < 0.001. We quantified the abundances of serum ceramides and sphingosines because adiponectin has a defined role in sphingolipid metabolism through adiponectin receptor mediated activation of ceramidases. The most abundant ceramide in dolphin sera was Cer 24:1 comprising 49% of the ceramides measured. Significant reductions were observed in the unsaturated Cer 18:1, Cer 20:1, and Cer 24:1, whereas significant increases were observed in saturated Cer 22:0, Cer 24:0, Cer 26:0. However, total serum ceramides did not change. Significant elevations were detected for total sphingosine, dihydrosphingosine, sphingosine-1-phosphate, and dihydrosphingosine-1-phosphate. Proteomic analysis of the serum proteins revealed few changes in serum proteins over the study period. In conclusion, shifting the dolphin diet to fishes rich in odd

  9. Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles.

    Science.gov (United States)

    Alboresi, Alessandro; Perin, Giorgio; Vitulo, Nicola; Diretto, Gianfranco; Block, Maryse; Jouhet, Juliette; Meneghesso, Andrea; Valle, Giorgio; Giuliano, Giovanni; Maréchal, Eric; Morosinotto, Tomas

    2016-08-01

    The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level. PMID:27325666

  10. MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells

    NARCIS (Netherlands)

    Klappe, K; Hinrichs, JWJ; Kroesen, BJ; Sietsma, H; Kok, JW

    2004-01-01

    Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipean

  11. Triterpenoid biosynthesis in Euphorbia lathyris latex

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, D.R.

    1987-11-01

    The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I/sub 50/ concentration of 3.2 ..mu..M. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I/sub 50/ of 4 ..mu..M. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4-/sup 3/H-mevalonic acid and incubating latex with a mixture of this and /sup 14/C-mevalonic acid. From the /sup 3/H//sup 14/C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs.

  12. Triterpenoid biosynthesis in Euphorbia lathyris latex

    International Nuclear Information System (INIS)

    The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I50 concentration of 3.2 μM. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I50 of 4 μM. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4-3H-mevalonic acid and incubating latex with a mixture of this and 14C-mevalonic acid. From the 3H/14C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs

  13. Fermentation kinetics makeover in poly-ε-lysine biosynthesis by Streptomyces noursei NRRL 5126

    Directory of Open Access Journals (Sweden)

    Sandip Balasaheb Bankar

    2012-04-01

    Full Text Available An unstructured model has been used to predict microbial growth based on glycerol consumption and poly-ε-lysine (ε-PL biosynthesis by Streptomyces noursei NRRL 5126. The logistic and Luedeking-Piret equations have been proposed to describe the time course of ε-PL formation, substrate consumption and cell growth. The shake flask level data from kinetic studies was illustrated and compared with fermenter studies. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of Streptomyces noursei during efficient ε-PL production from glycerol. Optimized oxygen supply into the fermenter shifted mixed growth associated biosynthesis of ε-PL from shake flask level to growth associated biosynthesis

  14. The structural biology of phenazine biosynthesis.

    Science.gov (United States)

    Blankenfeldt, Wulf; Parsons, James F

    2014-12-01

    The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis. PMID:25215885

  15. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Barwal Indu

    2011-12-01

    Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

  16. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    Directory of Open Access Journals (Sweden)

    Fernando D Villarreal

    Full Text Available Myo-inositol (Ins is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus. Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS and inositol monophosphatase (IMPase, by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1 were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P, mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

  17. Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry

    DEFF Research Database (Denmark)

    Ejsing, Christer S.; Moehring, Thomas; Bahr, Ute;

    2006-01-01

    and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends...

  18. Inhibition of honokiol on the proliferation and melanin biosynthesis on B16 melanoma cells in vitro%和厚朴酚对小鼠黑色素瘤B16细胞增殖以及黑色素合成的影响

    Institute of Scientific and Technical Information of China (English)

    喻丽红; 张超; 谭茵

    2012-01-01

    Objective To investigate the inhibition of honokiol on proliferation and melanin biosynthesis in B16 melanoma cells in vitro. Methods B16 cells were cultured in vitro. MTT assay, DAPI and NaOH lysis test were applied for assessment of cell proliferation, cellular morphologic observation and cellular melanin content, respectively. Results B16 cells were treated with honokiol with different concentrations ( 5,10,20,40 and 80 μmol/L ) and durations ( 12,24 and 48 h ). The IC50 of honokiol on proliferation of B16 cells were 23. 4, 13. 1 and 11.4 μmol/L, respectively, with durations of 12,24 and 48 h. Apoptosis of B16 cells were induced with honokiol in DAPI staining. Furthermore, the melanin biosynthesis was inhibited with honokiol with concentration over 20 (xmol/L. Conclusion Honokiol effectively inhibits the proliferation of B16 in dose - and time - depend manners. Apoptotic bodies were observed in B16 cells treated with honokiol. Although melanin biosynthesis is also inhibited by honokiol, this effect is insignificant.%目的 探讨和厚朴酚对小鼠黑色素瘤B16细胞增殖以及细胞内黑色素合成的影响.方法 体外培养小鼠B16细胞,MTT法检测和厚朴酚对B16细胞增殖的影响;DAPI染色法观察和厚朴酚对B16细胞细胞形态的影响;NaOH裂解法检测和厚朴酚对黑色素含量的影响.结果 和厚朴酚浓度为5、10、20、40、80 μmol/L 作用B16细胞,在不同的用药时间12、24和48 h,和厚朴酚对B16细胞增殖的IC50分别为23.4、13.1和11.4 μmol/L;同时和厚朴酚作用B16细胞12 h后,经DAPI染色,B16细胞呈现典型的凋亡形态;另外采用20、40、80 μmol/L的和厚朴酚分别作用B16细胞,B16细胞内黑色素合成量也呈现降低的趋势.结论 和厚朴酚能有效地抑制B16细胞增殖,且其抑制作用具有时间和浓度依赖性;药物作用后的B16细胞呈现凋亡形态并出现凋亡小体;和厚朴酚对B16细胞内黑色素合成也呈现抑制作用但不明显.

  19. Outlining eicosanoid biosynthesis in the crustacean Daphnia

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    Timmermans Martijn JTN

    2008-07-01

    Full Text Available Abstract Background Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX pathway; the lipoxygenase (LOX pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Results Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. Conclusion We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

  20. Combinatorial biosynthesis of medicinal plant secondary metabolites

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Koulman, Albert; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2006-01-01

    Combinatorial biosynthesis is a new tool in the generation of novel natural products and for the production of rare and expensive natural products. The basic concept is combining metabolic pathways in different organisms on a genetic level. As a consequence heterologous organisms provide precursors

  1. Bile acid biosynthesis and its regulation

    Directory of Open Access Journals (Sweden)

    Areta Hebanowska

    2010-10-01

    Full Text Available Bile acid biosynthesis is the main pathway of cholesterol catabolism. Bile acids are more soluble than cholesterol so are easier to excrete. As amphipathic molecules they participate in lipid digestion and absorption in the intestine and they help to excrete free cholesterol with bile. They are also ligands for nuclear receptors regulating the expression of genes involved in cholesterol metabolism. Interconversion of cholesterol into bile acids is an important point of its homeostasis. Seventeen enzymes are engaged in this process and many of them are cytochromes P450. Bile acid synthesis initiation may proceed with the “classical” pathway (starting with cholesterol hydroxylation at the C7α position or the “alternative” pathway (starting with cholesterol hydroxylation at the C27 position. Two additional pathways are possible, though their quantitative significance is small (initiated with cholesterol hydroxylations of C24 and C25 positions. Oxysterols produced are not only intermediates of bile acid biosynthesis but also important regulators of metabolism. Bile acid biosynthesis takes place in the liver, but some enzymes are also present in other organs, where they participate in regulation of cholesterol metabolism. Those enzymes are potential targets for new drugs against cholesterol metabolism disturbances. This article is a brief description of the bile acid biosynthesis pathway and participating enzymes.

  2. Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

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    Nabin Malla

    Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

  3. Sphingolipids in Major Depression

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    Peter L. Jernigan

    2015-12-01

    Full Text Available Major depression is one of the most common and severe diseases affecting the world's population. However, the pathogenesis of the disease remains inadequately defined. Previously, a lack of monoaminergic neurotransmitters was the focus of pathophysiological concepts; however, recent concepts focus on a alteration of neurogenesis in the hippocampus. This concept suggests that neurogenesis is decreased in major depression with a rarefication of neuronal networks and a lack of new, immature neurons in the hippocampus, events that may result in the clinical symptoms of major depression. However, molecular targets involved in the pathogenesis of major depression and, in particular, a reduction of neurogenesis, are largely unknown. We have recently discovered that an inhibition of the acid sphingomyelinase/ceramide system mediates the effects of tri- and tetracyclic antidepressants. Moreover, an accumulation of ceramide in the hippocampus results in depression-like symptoms. This suggests the acid sphingomyelinase/ceramide system is very important in the pathogenesis of major depression.

  4. Synthesis of N-acetyl Glucosamine Analogs as Inhibitors for Hyaluronan Biosynthesis

    OpenAIRE

    Wasonga, Gilbert; Tatara, Yota; Kakizaki, Ikuko; Huang, Xuefei

    2013-01-01

    Elevated hyaluronan expression is a hallmark of many types of cancer. Therefore, inhibition of hyaluronan biosynthesis can potentially slow the growth of tumor cells. Herein, we explore a chain termination strategy to reduce hyaluronan synthesis by tumor cells. Several analogs of glucosamine were prepared, which contained modifications at the C-3 positions. These analogs can possibly cap the nonreducing end of a growing hyaluronan chain, thus lowering the amount of hyaluronan synthesized. Upo...

  5. Methionine salvage pathway in relation to ethylene biosynthesis

    International Nuclear Information System (INIS)

    The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H3CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, [14C]MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of [ribose-U-14C]MTR with avocado extract resulted in the production of [14C]formate, with little evolution of other 14C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR

  6. Cellulose biosynthesis in Acetobacter xylinum

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.C.

    1988-01-01

    Time-lapse video microscopy has shown periodic reversals during the synthesis of cellulose. In the presence of Congo Red, Acetobacter produces a band of fine fibrils. The direction of cell movement is perpendicular to the longitudinal axis of cell, and the rate of movement was decreased. A linear row of particles, presumably the cellulose synthesizing complexes, was found on the outer membrane by freeze-fracture technique. During the cell cycle, the increase of particles in linear row, the differentiation to four linear rows and the separation of the linear rows have been observed. A digitonin-solubilized cellulose synthase was prepared from A. xylinum, and incubated under conditions known to lead to active in vitro synthesis of 1,4-{beta}-D-glucan polymer. Electron microscopy revealed that clusters of fibrils were assembled within minutes. Individual fibrils are 17 {plus minus} 2 angstroms in diameter. Evidence for the cellulosic composition of newly synthesized fibrils was based on incorporation of tritium from UDP-({sup 3}H) glucose binding of gold-labeled cellobiohydrolase, and an electron diffraction pattern identified as cellulose II polymorph instead of cellulose I.

  7. How peroxisomes affect aflatoxin biosynthesis in Aspergillus flavus.

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    Massimo Reverberi

    Full Text Available In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived, an hyper-oxidant cell environment and of hormone-like signals (oxylipins enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.

  8. Creatine biosynthesis and transport in health and disease.

    Science.gov (United States)

    Joncquel-Chevalier Curt, Marie; Voicu, Pia-Manuela; Fontaine, Monique; Dessein, Anne-Frédérique; Porchet, Nicole; Mention-Mulliez, Karine; Dobbelaere, Dries; Soto-Ares, Gustavo; Cheillan, David; Vamecq, Joseph

    2015-12-01

    Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of

  9. Composition of Cassia fistula oil and its antifungal activity by disrupting ergosterol biosynthesis.

    Science.gov (United States)

    Irshad, Md; Ahmad, Aijaz; Zafaryab, Md; Ahmad, Farah; Manzoor, Nikhat; Singh, Man; Rizvi, M Moshahid A

    2013-02-01

    Cassia fistula oil was investigated for antifungal activities against standard and clinical isolates of Candida species. Gas chromatography coupled with mass spectrometric (GC-MS) analysis of C. fistula oil revealed the presence of antimicrobial compounds like beta-sitosterol, stigmasterol, ergosterol, betulinic acid, lupeol, fucosterol, alpha-amyrin and friedelin. The minimum inhibitory concentration (MIC) of the pulp and seed oils ranged between 250-300 and 350-500 microg/mL respectively. Both oils also inhibited by > or = 63.8% ergosterol bio-synthesis in Candida cell wall {fluconazole (standard) > or = 89.1%)}. The MICs were significantly correlated with the ergosterol content decrease in the cell wall (Student's t test p Cassia fistula oil that primarily target ergosterol biosynthesis in Candida cell wall.

  10. Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis.

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    Courtney Nosak

    Full Text Available The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.

  11. Carotenoid Metabolism: Biosynthesis, Regulation,and Beyond

    Institute of Scientific and Technical Information of China (English)

    Shan Lu; Li Li

    2008-01-01

    Carotenoids are Indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied.Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis,regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.

  12. Microbial Exopolysaccharides: Biosynthesis and Potential Applications

    Directory of Open Access Journals (Sweden)

    K. V. Madhuri

    2014-09-01

    Full Text Available Many bacteria synthesize extracellular polysaccharides (EPSs with commercially significant physiological and therapeutic activities. Microbial polysaccharides have also been reported to have potential therapeutic applications. Recently, much attention has been devoted to the microbial exopolysaccharides (EPSs due to their numerous health benefits.EPSs from lactic acid bacteria are reported to possess antitumor effects, immunostimulatory activity, and the ability to lower blood cholesterol. EPSs also offer an alternative class of biothickeners that are widely used in the food and dairy industries and have been proven to provide strong emulsifying activity, which is important in many food formulations. It is also important to understand the mechanism of microbial biosynthesis of EPSs in order to enhance their production by genetic alterations. The potential applications and the mode of microbial biosynthesis of the EPSs have been presented in this article.

  13. Biosynthesis and toxicological effects of patulin.

    Science.gov (United States)

    Puel, Olivier; Galtier, Pierre; Oswald, Isabelle P

    2010-04-01

    Patulin is a toxic chemical contaminant produced by several species of mold, especially within Aspergillus, Penicillium and Byssochlamys. It is the most common mycotoxin found in apples and apple-derived products such as juice, cider, compotes and other food intended for young children. Exposure to this mycotoxin is associated with immunological, neurological and gastrointestinal outcomes. Assessment of the health risks due to patulin consumption by humans has led many countries to regulate the quantity in food. A full understanding of the molecular genetics of patulin biosynthesis is incomplete, unlike other regulated mycotoxins (aflatoxins, trichothecenes and fumonisins), although the chemical structures of patulin precursors are now known. The biosynthetic pathway consists of approximately 10 steps, as suggested by biochemical studies. Recently, a cluster of 15 genes involved in patulin biosynthesis was reported, containing characterized enzymes, a regulation factor and transporter genes. This review includes information on the current understanding of the mechanisms of patulin toxinogenesis and summarizes its toxicological effects.

  14. Structural basis for phosphatidylinositol-phosphate biosynthesis

    Science.gov (United States)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  15. Plant Terpenoids: Biosynthesis and Ecological Functions

    Institute of Scientific and Technical Information of China (English)

    Ai-Xia Cheng; Yong-Gen Lou; Ying-Bo Mao; Shan Lu; Ling-Jian Wang; Xiao-Ya Chen

    2007-01-01

    Among plant secondary metabolites terpenoids are a structurally most diverse group; they function as phytoalexins in plant direct defense, or as signals in indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the investigation of the ecological role of plant terpenoids. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes include the synthesis of C5 precursor isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases (TPSs) play a key role in volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to increase terpenoid production. This review mainly summarizes the recent research progress in elucidating the ecological role of terpenoids and characterization of the enzymes involved in the terpenoid biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.

  16. Efficiency of Lignin Biosynthesis: a Quantitative Analysis

    OpenAIRE

    Amthor, Jeffrey S.

    2003-01-01

    Lignin is derived mainly from three alcohol monomers: p‐coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Biochemical reactions probably responsible for synthesizing these three monomers from sucrose, and then polymerizing the monomers into lignin, were analysed to estimate the amount of sucrose required to produce a unit of lignin. Included in the calculations were amounts of respiration required to provide NADPH (from NADP+) and ATP (from ADP) for lignin biosynthesis. Two pathways in...

  17. Structural basis for phosphatidylinositol-phosphate biosynthesis

    OpenAIRE

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinosito...

  18. The structural biology of phenazine biosynthesis

    OpenAIRE

    Blankenfeldt, Wulf; Parsons, James F.

    2014-01-01

    The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in ...

  19. A mitochondrial pathway for biosynthesis of lipid mediators

    Science.gov (United States)

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

  20. Leukotriene C4 biosynthesis in isolated August rat peritoneal leukocytes

    Directory of Open Access Journals (Sweden)

    J. M. Huebner

    1996-01-01

    Full Text Available The mixed leukocyte population obtained from the peritoneum of the August rat is a potentially important experimental model of inherent eosinophilia that has not been well characterized. In the present study, isolated cell preparations generated a concentration-dependent release of leukotriene (LT C4 when exposed to the Ca2+ ionophore A23187, reaching maximal stimulation at 5.0 μM. This response was inhibited by the 5-lipoxygenase activating protein antagonist MK-886 (0.1 μM, nominally Ca2+ and Mg2+-free incubation media and by activation of protein kinase C via phorbol 12-myristate 13-acetate (50 nM. These findings establish a model system for investigating LTC4 profiles contingent with innate peritoneal eosinophilia and are consistent with the hypothesis that cellular LTC4 biosynthesis is phosphoregulated.

  1. Oxidative stress induces the biosynthesis of citrinin by Penicillium verrucosum at the expense of ochratoxin.

    Science.gov (United States)

    Schmidt-Heydt, Markus; Stoll, Dominic; Schütz, Peter; Geisen, Rolf

    2015-01-01

    Penicillium verrucosum is a fungus that can produce ochratoxin A and citrinin, two structurally related nephrotoxic mycotoxins. P. verrucosum usually occurs on wheat but can occasionally also be found in NaCl rich habitats such as salted cheeses or olives, indicating that this fungus can adapt to different environments. The ratio of ochratoxin A to citrinin produced by P. verrucosum is shifted to one of either mycotoxin at the expense of the other dependent on the environmental conditions. High NaCl concentrations shift secondary metabolite biosynthesis towards ochratoxin A production. P. verrucosum copes with NaCl stress by increased ochratoxin A biosynthesis, ensuring chloride homeostasis. Ochratoxin A carries chlorine in its molecule and can excrete chlorine from the cell. It was further shown that the regulation of ochratoxin A by high NaCl conditions is mediated by the HOG MAP kinase signal transduction pathway. Here it is shown that high oxidative stress conditions, evoked for example by increasing concentrations of Cu(2+) cations in the growth medium, shift secondary metabolite biosynthesis of P. verrucosum from ochratoxin A to citrinin. The production of citrinin normalizes the oxidative status of the fungal cell under oxidative stress conditions leading to an adaptation to these environmental conditions and protects against increased oxidative stress caused by increased Cu(2+) concentrations. Moreover citrinin also protects against light of short wavelength, which may also increase the oxidative status of the environment. The biosynthesis of citrinin is apparently regulated by a cAMP/PKA signaling pathway, because increasing amounts of external cAMP reduce citrinin biosynthesis in a concentration dependent manner. These conditions lead to the cross-regulation of the ochratoxin A/citrinin secondary metabolite pair and support the adaptation of P. verrucosum to different environments. PMID:25279858

  2. A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize.

    Science.gov (United States)

    Tanaka, Shigeyuki; Brefort, Thomas; Neidig, Nina; Djamei, Armin; Kahnt, Jörg; Vermerris, Wilfred; Koenig, Stefanie; Feussner, Kirstin; Feussner, Ivo; Kahmann, Regine

    2014-01-01

    The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin-proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses. DOI: http://dx.doi.org/10.7554/eLife.01355.001.

  3. MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems.

    Science.gov (United States)

    Öhman, David; Demedts, Brecht; Kumar, Manoj; Gerber, Lorenz; Gorzsás, András; Goeminne, Geert; Hedenström, Mattias; Ellis, Brian; Boerjan, Wout; Sundberg, Björn

    2013-01-01

    The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103. PMID:22967312

  4. Effects of brassinazole, an inhibitor of brassinosteroid biosynthesis, on light- and dark-grown Chlorella vulgaris.

    Science.gov (United States)

    Bajguz, Andrzej; Asami, Tadao

    2004-03-01

    Treatment of cultured Chlorella vulgaris Beijerinck cells with 0.1-10 microM brassinazole (Brz2001), an inhibitor of brassinosteroid (BR) biosynthesis, inhibits their growth during the first 48 h of cultivation in the light. This inhibition is prevented by the co-application of BR. This result suggests that the presence of endogenous BRs during the initial steps of the C. vulgaris cell cycle is indispensable for their normal growth in the light. In darkness, a treatment with 10 nM brassinolide (BL) promotes growth through the first 24 h of culture, but during the following 24 h the cells undergo complete stagnation. Treatment of dark-grown cells with either Brz2001 alone, or a mixture of 10 nM BL and 0.1/10 microM Brz2001, also stimulates their growth. The effects of treatment with 10 nM BL mixed with 0.1-10 microM of a mevalonate-pathway inhibitor (mevinolin), or a non-mevalonate-pathway inhibitor (clomazone), were also investigated. Mevinolin at these concentrations did not inhibit growth of C. vulgaris; however, clomazone did. Addition of BL overcame the inhibition. These results suggest that the mevalonate pathway does not function in C. vulgaris, and that the non-mevalonate pathway for isopentenyl diphosphate biosynthesis is responsible for the synthesis of one of the primary precursors in BR biosynthesis.

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

    Directory of Open Access Journals (Sweden)

    Wanderley de Souza

    2009-01-01

    Full Text Available 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

  6. Production of α-galactosylceramide by a prominent member of the human gut microbiota.

    Directory of Open Access Journals (Sweden)

    Laura C Wieland Brown

    2013-07-01

    Full Text Available While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf, which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000 that is the prototypical agonist of CD1d-restricted natural killer T (iNKT cells. We demonstrate that α-GalCer(Bf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

  7. 细胞外信号激酶蛋白5在卵泡刺激素介导的卵泡颗粒细胞甾体激素生成中的作用%Role of Extracellular Signal-regulated Protein Kinase 5 in the Biosynthesis of Follicle-stimulating Hormone-stimulated Progesterone in Primary Granulosa Cells

    Institute of Scientific and Technical Information of China (English)

    高小博; 姚楠; 马旭; 陆彩玲; 杨伯清; 彭小忠

    2011-01-01

    Objective To study the role of extracellular signal-regulated protein kinase 5 ( ERK5 ) during the biosynthesis of follicle-stimulating hormone ( FSH ) -mediated progesterone in primary granulosa cells. Methods The expressions of phophorylated and general forms of ERK5 in primary granulosa cells afterthe treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors. Results ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caM-EK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production. Conclusion ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.%目的 研究细胞外信号激酶蛋白5(ERK5)在卵巢颗粒细胞甾体激素生成中的作用.方法 采用Western blot检测在卵泡刺激素(FSH)刺激原代颗粒细胞甾体激素生成过程中ERK5的表达变化和激活情况,荧光共聚焦技术分析ERK5的亚细胞定位,腺病毒技术观察活化型ERK5和显性负性ERK5对颗粒细胞甾体激素生成的影响.结果 Western blot检测结果表明,FSH处理颗粒细胞后ERK5的表达随处理时间延长而下调,而ERK5活性形式的表达随处理时间延长而逐渐上升.荧光共聚焦技术分析结果显示,FSH的处理并没有明显影响ERK5在颗粒细胞中的定位.腺病毒技术观察结果发现,在细胞中联合感染活化型MEK5

  8. The Effect of Sodium Nitrite and Arginine on the Biosynthesis of Nitric Oxide and Triterpenoid in Birch (Betula platyphylla Suk) Suspension Cells%亚硝酸钠和精氨酸对白桦悬浮细胞中NO和三萜合成的影响

    Institute of Scientific and Technical Information of China (English)

    许道琦; 张杰琨; 王晓东; 詹亚光; 范桂枝

    2012-01-01

    The aim was to analyze the relation of nitric oxide (NO) and triterpenoid production. Sodium nitrite and arginine were added in the eight-day-old birch suspension cell culture, the change of triterpene content and NO content were analyzed by chemical colorimetry and fluorescence microscopy. NO and triterpenoid content were increased by 500 μg/L and 100 μg/L sodium nitrite and arginine, and the higher increasing was induced by 500 μg/L sodium nitrite and arginine. The content of NO and triterpenoid in birch cells induced by 500 μg/L sodium nitrite was higher than that of 500 μg/L arginine, the increasing rate of triterpenoid was 1.5 times. Inhibitor of NaN3 and L-NAME decreased the effect of sodium nitrite and arginine on the biosynthesis of NO and triterpenoid, the content of NO and triterpenoid were near to the level of control. These results preliminary indicated that NR and NOS were involved in triterpenoids biosynthesis in birch suspension culture, and NR might be play an important role in triterpenoids biosynthesis.%旨在分析亚硝酸钠和精氨酸处理12h后白桦悬浮细胞中一氧化氮(NO)和三萜含量的变化.在白桦悬浮细胞的生长末期添加硝酸还原酶(NR)和一氧化氮合成酶(NOS)的底物亚硝酸钠和精氨酸,采用比色法和荧光显微镜方法分析白桦悬浮体系中三萜含量和NO含量的变化.500 μg/L和100 μg/L的亚硝酸钠和精氨酸均促进了白桦悬浮细胞中NO和三萜的合成,其中500μg/L的亚硝酸钠和精氨酸促进作用最强.500μg/L亚硝酸钠处理下白桦细胞中的NO荧光强度和三萜含量均高于500μg/L精氨酸处理,其中三萜含量增加了1.5倍.将NR和NOS的抑制剂NaN3和L-NAME分别添加到亚硝酸钠和精氨酸处理的白桦悬浮体系中,发现亚硝酸钠和精氨酸对NO和三萜合成的促进作用被抑制了.NR和NOS来源途径的NO参与了白桦三萜的合成,可能NR来源途径的贡献大于NOS途径.

  9. Relationship between carbon catabolite repression and the biosynthesis regulation of the prolidase PepQ from Lactobacillus delbrueckii ssp. bulgaricus CNRZ 397

    OpenAIRE

    Lamarque, Mauld; Morel, Fabienne; Bissardon, Isabelle; Galinier, Anne; Portalier, Raymond; Atlan, Danièle

    2001-01-01

    International audience Lactobacillus delbrueckii ssp. bulgaricus CNRZ 397 (L. bulgaricus) displays several enzymes specific of proline-containing peptides. We focused on the prolidase PepQ which specifically cleaves X-Pro dipeptides. PepQ biosynthesis was previously shown to be independent of the peptide concentration of the culture medium in contrast to the cell surface proteinase PrtB and several aminopeptidases. Regulation of PepQ biosynthesis can be explained by the genetic organizatio...

  10. Elucidation and Chemical Modulation of Sulfolipid-1 Biosynthesis in Mycobacterium tuberculosis *

    OpenAIRE

    Seeliger, Jessica C.; Holsclaw, Cynthia M.; Schelle, Michael W.; Botyanszki, Zsofia; Gilmore, Sarah A.; Tully, Sarah E.; Niederweis, Michael; Cravatt, Benjamin F.; Leary, Julie A.; Bertozzi, Carolyn R.

    2011-01-01

    Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The m...

  11. In Vitro Biosynthesis of Ether-Type Glycolipids in the Methanoarchaeon Methanothermobacter thermautotrophicus▿

    OpenAIRE

    Morii, Hiroyuki; Eguchi, Tadashi; Koga, Yosuke

    2007-01-01

    The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the β-d-glucosyl-(1→6)-d-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose β-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose β-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarch...

  12. Fermentation kinetics makeover in poly-ε-lysine biosynthesis by Streptomyces noursei NRRL 5126

    OpenAIRE

    Sandip Balasaheb Bankar; Singhal, Rekha S.

    2012-01-01

    An unstructured model has been used to predict microbial growth based on glycerol consumption and poly-ε-lysine (ε-PL) biosynthesis by Streptomyces noursei NRRL 5126. The logistic and Luedeking-Piret equations have been proposed to describe the time course of ε-PL formation, substrate consumption and cell growth. The shake flask level data from kinetic studies was illustrated and compared with fermenter studies. In all cases, the model simulation matched well with the ...

  13. Characterization of the Fucosylation Pathway in the Biosynthesis of Glycopeptidolipids from Mycobacterium avium Complex▿

    OpenAIRE

    Miyamoto, Yuji; Mukai, Tetsu; Maeda, Yumi; Nakata, Noboru; Kai, Masanori; Naka, Takashi; Yano, Ikuya; Makino, Masahiko

    2007-01-01

    The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal regio...

  14. Identification and characterization of an operon in Salmonella typhimurium involved in thiamine biosynthesis.

    OpenAIRE

    Petersen, L A; Downs, D M

    1997-01-01

    Thiamine pyrophosphate (TPP) is synthesized de novo in Salmonella typhimurium and is a required cofactor for many enzymes in the cell. Five kinase activities have been implicated in TPP synthesis, which involves joining a 4-methyl-5-(beta-hydroxyethyl)thiazole (THZ) moiety and a 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety. We report here identification of a 2-gene operon involved in thiamine biosynthesis and present evidence that the genes in this operon, thiMD, encode two previou...

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

    OpenAIRE

    Fabris, M; Matthijs, M.; Carbonelle, S.; Moses, T.; Pollier, J.; Dasseville, R.; Baart, G.J.E.; Vyverman, W.; Goossens, A

    2014-01-01

    Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the path...

  16. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

    OpenAIRE

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou; Fichant, Gwennaele; Cam, Kaymeuang

    2015-01-01

    Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on...

  17. Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

    OpenAIRE

    de Boer, L; Vrijbloed, J W; Grobben, G.; Dijkhuizen, L.

    1989-01-01

    The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhib...

  18. In vitro biosynthesis of the C-glycosidic bond in aloin.

    Science.gov (United States)

    Grün, M; Franz, G

    1981-10-01

    Biosynthetic studies with cell-free extracts from Aloe arborescens Mill. demonstrate the transfer of the glucose moiety from UDP-glucose to aloe emodin anthrone, forming the C-glycosidic linkage in the anthracene derivative aloin. The pH-dependence and the specificity of UDP-glucose and aloe emodin anthrone for the biosynthesis of the C-glycosidic bond in aloin are shown. PMID:24301161

  19. Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Bing Lü; Feng Chen; Zhong-Hua Gong; Hong Xie; Jian-Sheng Liang

    2007-01-01

    We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid (ABA) in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction.Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser (GRGDS), which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-like proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.

  20. Plasma membrane rafts engaged in T cell signalling: new developments in an old concept

    Directory of Open Access Journals (Sweden)

    Sangani Dhaval

    2009-09-01

    Full Text Available Abstract Considerable controversy arose over the concept that cholesterol/sphingolipid-rich rafts in the T cell plasma membrane serve as a platform for TCR signalling reactions. This controversy was founded on the initial definition of rafts as detergent resistant membranes which later turned out to misrepresent many features of cell membrane organisation under physiological conditions. Raft-organisation was subsequently studied using a number of detergent-free experimental approaches. The results led to a refined perception of membrane rafts which resolves the controversies. Here we review new biophysical and biochemical data which provide an updated picture of the highly dynamic nanometer-sized cholesterol/sphingolipid-rich raft domains stabilised by protein-networks to form TCR signalling platforms in the T cell plasma membrane.

  1. Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase.

    Directory of Open Access Journals (Sweden)

    Tarun Chopra

    2008-07-01

    Full Text Available In recent years, remarkable versatility of polyketide synthases (PKSs has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative or by using these catalytic domains only once (modular during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM. The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of

  2. Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

    NARCIS (Netherlands)

    Lokman, Christien; Franken, A.C.; Ram, A.F.; Punt, P.J.; Hondel, C.A. van den; Weert, S. de

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  3. Heme biosynthesis and its regulation: Towards understanding and improvement of heme biosynthesis in filamentous fungi

    NARCIS (Netherlands)

    Franken, A.C.W.; Lokman, B.C.; Ram, A.F.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Weert, S. de

    2011-01-01

    Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

  4. Evolution of the Kdo2-lipid A Biosynthesis in Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    S Opiyo; R Pardy; H Moriyama; E Moriyama

    2011-12-31

    BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

  5. Evolution of the Kdo2-lipid A biosynthesis in bacteria

    Directory of Open Access Journals (Sweden)

    Moriyama Hideaki

    2010-11-01

    Full Text Available Abstract Background Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS. It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. Results In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB. Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. Conclusions The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

  6. Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation

    Science.gov (United States)

    Christiansen, Guntram; Fastner, Jutta; Erhard, Marcel; Börner, Thomas; Dittmann, Elke

    2003-01-01

    Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases. PMID:12511503

  7. Blakeslea trispora Genes for Carotene Biosynthesis

    OpenAIRE

    Rodríguez-Sáiz, M.; Paz, B.; de la Fuente, J L; López-Nieto, M J; Cabri, W.; Barredo, J L

    2004-01-01

    We cloned the carB and carRA genes involved in β-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and c...

  8. Biosynthesis of Iron-Sulfur Clusters

    OpenAIRE

    Yuvaniyama, Pramvadee

    1999-01-01

    It is not known whether biosynthesis of [Fe-S] clusters occurs through a spontaneous self-assembly process or an enzymatic process. However, in the Azotobacter vinelandii nitrogenase system, it has been proposed that NifS and NifU are involved in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfur for the in vitro catalytic formation of [Fe-S] cl...

  9. Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana

    NARCIS (Netherlands)

    Birke, Hannah; Haas, Florian H.; De Kok, Luit J.; Balk, Janneke; Wirtz, Markus; Hell, Ruediger

    2012-01-01

    In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oa

  10. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters

    NARCIS (Netherlands)

    Kleerebezem, M.; Bongers, R.; Rutten, G.; Vos, de W.M.; Kuipers, O.P.

    2004-01-01

    The production of the type 1 antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editor

  11. Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis

    NARCIS (Netherlands)

    Boels, I.C.; Beerthuyzen, M.M.; Kosters, M.H.; Kaauwen, van M.P.W.; Kleerebezem, M.; Vos, de W.M.

    2004-01-01

    dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the

  12. Genetics, Biosynthesis, Structure, and Mode of Action of Lantibiotics

    Science.gov (United States)

    Kuipers, Anneke; Rink, Rick; Moll, Gert N.

    Lantibiotics are lanthionine-containing peptide antibiotics. They are characterized by having meso-lanthionine(s) and/or β-methyllanthionine(s) or both. These intramolecular monosulfide cross-links render the peptide resistant against breakdown by peptidases. Moreover, in several cases, the (methyl)lanthionines are essential for interaction with the so-called docking molecule lipid II. The best known lantibiotic, nisin, highly effectively inhibits growth of target cells via two mechanisms: (1) abduction of the cell wall precursor lipid II from the septum and (2) formation of pores composed of lipid II and nisin. (Methyl)lanthionines result from two enzyme-catalyzed posttranslational modifications: dehydration of serines/threonines and coupling of the resulting dehydro amino acids to cysteines. Besides the localization of the thioether bridges and dehydro amino acids in the lantibiotics, also the three-dimensional structure of some lantibiotics has been resolved by NMR. Genes encoding proteins involved in the biosynthesis of lantibiotics are present in clusters and may comprise combinations of the following genes in varying order: a structural gene that encodes a leader peptide and the lantibiotic propeptide, modification enzyme(s), a transporter responsible for the export of the lantibiotic and in some cases for cleavage of the leader peptide, a leader peptidase, a so-called immunity protein involved in self-protection of the host cell, components of a transporter also involved in self-protection, and two components of an autoinduction system.

  13. Calcium ionophore (A-23187) induced peritoneal eicosanoid biosynthesis: a rapid method to evaluate inhibitors of arachidonic acid metabolism in vivo

    OpenAIRE

    Rao, T S; Currie, J. L.; A. F. Shaffer; Isakson, P C

    1993-01-01

    The present investigation characterizes calcium ionophore (A-23187) induced peritoneal eicosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat) stimulated marked biosynthesis of 6-keto-PGF1α (6-KPA), TxB2, LTC4 and LTB4, with no detectable changes on levels of PGE2. Levels of all eicosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased r...

  14. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis

    International Nuclear Information System (INIS)

    Inhibin, a gonadal hormone capable of preferential suppression of pituitary follicle-stimulating hormone (FSH) secretion, has recently been purified. The major form of this protein is an αβ heterodimer encoded by two separate genes. In contrast to the FSH-suppressing action of the αβ heterodimer, the ββ homodimer stimulates FSH secretion. Luteinizing hormone (LH)-secreting pituitary cells and gonadal androgen-producing cells have long been shown to form a closed-loop feedback axis. Based on recent studies demonstrated the FSH stimulation of inhibin biosynthesis by ovarian granulosa and testis Sertoli cells, an additional closed-loop feedback axis exists between pituitary FSH- and gonadal inhibin-producing cells. Because uncharacterized Sertoli cell factors have been suggested to either stimulate or inhibit androgen production by testicular Leydig cells, the authors have tested the intragonadal paracrine actions of heterodimers and homodimers of inhibin subunits. In primary cultures of testis cells, the αβ heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the ββ homodimer suppresses androgen production. The data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops

  15. NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Michelle F Goody

    Full Text Available Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction

  16. Alkane production by the marine cyanobacterium Synechococcus sp. NKBG15041c possessing the α-olefin biosynthesis pathway.

    Science.gov (United States)

    Yoshino, Tomoko; Liang, Yue; Arai, Daichi; Maeda, Yoshiaki; Honda, Toru; Muto, Masaki; Kakunaka, Natsumi; Tanaka, Tsuyoshi

    2015-02-01

    The production of alkanes in a marine cyanobacterium possessing the α-olefin biosynthesis pathway was achieved by introducing an exogenous alkane biosynthesis pathway. Cyanobacterial hydrocarbons are synthesized via two separate pathways: the acyl-acyl carrier protein (ACP) reductase/aldehyde-deformylating oxygenase (AAR/ADO) pathway for the alkane biosynthesis and the α-olefin synthase (OLS) pathway for the α-olefin biosynthesis. Coexistence of these pathways has not yet been reported. In this study, the marine cyanobacterium Synechococcus sp. NKBG15041c was shown to produce α-olefins similar to those of Synechococcus sp. PCC7002 via the α-olefin biosynthesis pathway. The production of heptadecane in Synechococcus sp. NKBG15041c was achieved by expressing the AAR/ADO pathway genes from Synechococcus elongatus PCC 7942. The production yields of heptadecane in Synechococcus sp. NKBG15041c varied with the expression level of the aar and ado genes. The maximal yield of heptadecane was 4.2 ± 1.2 μg/g of dried cell weight in the transformant carrying a homologous promoter. Our results also suggested that the effective activation of ADO may be more important for the enhancement of alkane production by cyanobacteria.

  17. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    Directory of Open Access Journals (Sweden)

    Gazi Sakir Hossain

    Full Text Available α-Keto-γ-methylthiobutyric acid (KMTB, a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3 and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]. Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  18. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    Science.gov (United States)

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  19. Benzylisoquinoline alkaloid biosynthesis in opium poppy.

    Science.gov (United States)

    Beaudoin, Guillaume A W; Facchini, Peter J

    2014-07-01

    Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy. PMID:24671624

  20. BIOSYNTHESIS AND PROPERTIES OF ANTIBIOTIC BATUMIN

    Directory of Open Access Journals (Sweden)

    V. V. Klochko

    2014-12-01

    Full Text Available Biosynthesis of antistaphylococcal antibiotic batumin under periodic conditions of Pseudomonas batumici growth has been studied. Antibiotic synthesis in fermenter occurred across the culture growth and achieved its maximal value after 50–55 hours. The active oxygen utilization by the producing strain was observed during 20–55 hours of fermentation with maximum after 40–45 hours. Antibiotic yield was 175–180 mg/l and depended on intensity of aeration. contrast to «freshly isolated» antibiotic after fermentation the long-term kept batumin has shown two identical by molecular mass peaks according to the chromato-mass spectrometric analysis. Taking into account of batumin molecule structure the conclusion has been made that the most probable isomerization type is keto-enolic tautomerism. At the same time batumin is diastereoisomer of kalimantacin A which has the same chemical structure. The optic rotation angle is [α]d25 = +56.3° for kalimantacin and [α]d25 = –13.5° for batumin. The simultaneous P. batumici growth and antibiotic biosynthesis and the ability of this molecule to optical isomerisation and keto-enolic forms formation allow us to suppose that batumin plays a certain role in metabolism of the producing strain.

  1. Initiation of methylglucose lipopolysaccharide biosynthesis in mycobacteria.

    Directory of Open Access Journals (Sweden)

    Devinder Kaur

    Full Text Available BACKGROUND: Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides -- the methylglucose lipopolysaccharides (MGLPs and the methylmannose polysaccharides (MMPs -- the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways. METHODOLOGY/PRINCIPAL FINDINGS: We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084 as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis. CONCLUSIONS/SIGNIFICANCE: This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.

  2. Insights on the evolution of trehalose biosynthesis

    Directory of Open Access Journals (Sweden)

    Morett Enrique

    2006-12-01

    Full Text Available Abstract Background The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes. Results In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS and trehalose-phosphatase (TPP domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein. Conclusion We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

  3. High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

    OpenAIRE

    Li CHEN; Wang, Yemin; Guo, Hang; Xu, Min; Deng, Zixin; Tao, Meifeng

    2012-01-01

    A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators.

  4. It’s a lipid’s world: Bioactive lipid metabolism and signaling in neural stem cell differentiation

    OpenAIRE

    Bieberich, Erhard

    2012-01-01

    Lipids are often considered membrane components whose function is to embed proteins into cell membranes. In the last two decades, studies on brain lipids have unequivocally demonstrated that many lipids have critical cell signaling functions; they are called “bioactive lipids”. Pioneering work in Dr. Robert Ledeen’s laboratory has shown that two bioactive brain sphingolipids, sphingomyelin and the ganglioside GM1 are major signaling lipids in the nuclear envelope. In addition to derivatives o...

  5. Biosynthesis of collagen I, II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue.

    Science.gov (United States)

    Musumeci, Giuseppe; Mobasheri, Ali; Trovato, Francesca Maria; Szychlinska, Marta Anna; Graziano, Adriana Carol Eleonora; Lo Furno, Debora; Avola, Rosanna; Mangano, Sebastiano; Giuffrida, Rosario; Cardile, Venera

    2014-10-01

    The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called 'cell pellets'. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.

  6. Evolution of Lysine Biosynthesis in the Phylum Deinococcus-Thermus

    Directory of Open Access Journals (Sweden)

    Hiromi Nishida

    2012-01-01

    Full Text Available Thermus thermophilus biosynthesizes lysine through the α-aminoadipate (AAA pathway: this observation was the first discovery of lysine biosynthesis through the AAA pathway in archaea and bacteria. Genes homologous to the T. thermophilus lysine biosynthetic genes are widely distributed in bacteria of the Deinococcus-Thermus phylum. Our phylogenetic analyses strongly suggest that a common ancestor of the Deinococcus-Thermus phylum had the ancestral genes for bacterial lysine biosynthesis through the AAA pathway. In addition, our findings suggest that the ancestor lacked genes for lysine biosynthesis through the diaminopimelate (DAP pathway. Interestingly, Deinococcus proteolyticus does not have the genes for lysine biosynthesis through the AAA pathway but does have the genes for lysine biosynthesis through the DAP pathway. Phylogenetic analyses of D. proteolyticus lysine biosynthetic genes showed that the key gene cluster for the DAP pathway was transferred horizontally from a phylogenetically distant organism.

  7. Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Pittet, Martine; Conzelmann, Andreas

    2007-03-01

    Like most other eukaryotes, Saccharomyces cerevisiae harbors a GPI anchoring machinery and uses it to attach proteins to membranes. While a few GPI proteins reside permanently at the plasma membrane, a majority of them gets further processed and is integrated into the cell wall by a covalent attachment to cell wall glucans. The GPI biosynthetic pathway is necessary for growth and survival of yeast cells. The GPI lipids are synthesized in the ER and added onto proteins by a pathway comprising 12 steps, carried out by 23 gene products, 19 of which are essential. Some of the estimated 60 GPI proteins predicted from the genome sequence serve enzymatic functions required for the biosynthesis and the continuous shape adaptations of the cell wall, others seem to be structural elements of the cell wall and yet others mediate cell adhesion. Because of its genetic tractability S. cerevisiae is an attractive model organism not only for studying GPI biosynthesis in general, but equally for investigating the intracellular transport of GPI proteins and the peculiar role of GPI anchoring in the elaboration of fungal cell walls.

  8. Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts.

    Science.gov (United States)

    Jabłońska-Trypuć, Agata; Pankiewicz, Walentyn; Czerpak, Romuald

    2016-09-01

    Traumatic acid (TA) is a plant hormone (cytokinin) that in terms of chemical structure belongs to the group of fatty acids derivatives. It was isolated from Phaseolus vulgaris. TA activity and its influence on human cells and organism has not previously been the subject of research. The aim of this study was to examine the effects of TA on collagen content and basic oxidative stress parameters, such as antioxidative enzyme activity, reduced glutathione, thiol group content, and lipid peroxidation in physiological conditions. The results show a stimulatory effect of TA on tested parameters. TA caused a decrease in membrane phospholipid peroxidation and exhibited protective properties against ROS production. It also increases protein and collagen biosynthesis and its secretion into the culture medium. The present findings reveal that TA exhibits multiple and complex activity in fibroblast cells in vitro. TA, with its activity similar to unsaturated fatty acids, shows antioxidant and stimulatory effects on collagen biosynthesis. It is a potentially powerful agent with applications in the treatment of many skin diseases connected with oxidative stress and collagen biosynthesis disorders. PMID:27423205

  9. Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity.

    Directory of Open Access Journals (Sweden)

    Marianne Lucas-Hourani

    Full Text Available Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

  10. Effects of orally administered fumonisin B₁ (FB₁), partially hydrolysed FB₁, hydrolysed FB₁ and N-(1-deoxy-D-fructos-1-yl) FB₁ on the sphingolipid metabolism in rats.

    Science.gov (United States)

    Hahn, Irene; Nagl, Veronika; Schwartz-Zimmermann, Heidi Elisabeth; Varga, Elisabeth; Schwarz, Christiane; Slavik, Veronika; Reisinger, Nicole; Malachová, Alexandra; Cirlini, Martina; Generotti, Silvia; Dall'Asta, Chiara; Krska, Rudolf; Moll, Wulf-Dieter; Berthiller, Franz

    2015-02-01

    Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC-MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans.

  11. Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Lihua Shi; Yusuf A Hannun; Jianru Zuo; Jacek Bielawski; Jinye Mu; Haili Dong; Chong Teng; Jian Zhang; Xiaohui Yang; Nario Tomishige; Kentaro Hanada

    2007-01-01

    Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin Bl resistant11-1 (fbr11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B1 (FB1), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain basel (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbrll-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB1 By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1 -phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

  12. The expanding roles of c-di-GMP in the biosynthesis of exopolysaccharides and secondary metabolites.

    Science.gov (United States)

    Liang, Zhao-Xun

    2015-05-01

    The cyclic dinucleotide c-di-GMP has emerged in the last decade as a prevalent intracellular messenger that orchestrates the transition between the motile and sessile lifestyles of many bacterial species. The motile-to-sessile transition is often associated with the formation of extracellular matrix-encased biofilm, an organized community of bacterial cells that often contributes to antibiotic resistance and host-pathogen interaction. It is increasingly clear that c-di-GMP controls motility, biofilm formation and bacterial pathogenicity partially through regulating the production of exopolysaccharides (EPS) and small-molecule secondary metabolites. This review summarizes our current understanding of the regulation of EPS biosynthesis by c-di-GMP in a diversity of bacterial species and highlights the emerging role of c-di-GMP in the biosynthesis of small-molecule secondary metabolites.

  13. Chronic exposure to free fatty acid reduces pancreatic beta cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation.

    OpenAIRE

    Bollheimer, L. C.; Skelly, R H; Chester, M W; McGarry, J D; Rhodes, C. J.

    1998-01-01

    The pancreatic beta cell normally maintains a stable balance among insulin secretion, insulin production, and insulin degradation to keep optimal intracellular stores of the hormone. Elevated levels of FFA markedly enhance insulin secretion; however, the effects of FFA on insulin production and intracellular stores remain unclear. In this study, twofold elevation in total circulating FFA effected by infusion of lard oil and heparin into rats for 6 h under normoglycemic conditions resulted in ...

  14. Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?

    Science.gov (United States)

    Cook, Sam D; Nichols, David S; Smith, Jason; Chourey, Prem S; McAdam, Erin L; Quittenden, Laura; Ross, John J

    2016-06-01

    The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway. PMID:27208245

  15. Biosurfactant Mediated Biosynthesis of Selected Metallic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Grażyna A. Płaza

    2014-08-01

    Full Text Available Developing a reliable experimental protocol for the synthesis of nanomaterials is one of the challenging topics in current nanotechnology particularly in the context of the recent drive to promote green technologies in their synthesis. The increasing need to develop clean, nontoxic and environmentally safe production processes for nanoparticles to reduce environmental impact, minimize waste and increase energy efficiency has become essential in this field. Consequently, recent studies on the use of microorganisms in the synthesis of selected nanoparticles are gaining increased interest as they represent an exciting area of research with considerable development potential. Microorganisms are known to be capable of synthesizing inorganic molecules that are deposited either intra- or extracellularly. This review presents a brief overview of current research on the use of biosurfactants in the biosynthesis of selected metallic nanoparticles and their potential importance.

  16. The Spatial Organization of Glucosinolate Biosynthesis

    DEFF Research Database (Denmark)

    Nintemann, Sebastian

    in human and animal food sources. The glucosinolate defense system belongs to the best-studied pathways in plant specialized metabolism and the steps involved in their biosynthesis are known, their action as defense compounds is well understood and glucosinolate transport proteins have been......Plants interact with their environment through numerous chemical compounds and because plants are the primary producers of biomass in many ecosystems, a large number of these compounds serve as chemical defenses against herbivores and pathogens. Defense compounds have vast consequences for plant...... resistance and nutritional value and many plant specialized metabolites are of high value due to their health promoting characteristics. Glucosinolates are defense compounds found in many crops from the Brassicaceae family and are of high interest because of their nutritional and antinutritional properties...

  17. Biosynthesis of the phytoalexin pisatin. [Pisum sativum

    Energy Technology Data Exchange (ETDEWEB)

    Preisig, C.L.; Bell, J.N.; Matthews, D.E.; VanEtten, H.D. (Cornell Univ., Ithaca, NY (USA)); Sun, Yuejin; Hrazdina, G. (Cornell Univ., Geneva, NY (USA))

    1990-11-01

    NADPH-dependent reduction of 2{prime},7-dihydroxy-4{prime},5{prime}-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl{sub 2}. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of CuCl{sub 2}-treated seedlings was also observed; three products were radiolabeled when ({sup 3}H)sophorol was the substrate, one of which is tentatively identified as maackiain.

  18. Regulation of Brassinosteroid Biosynthesis and Inactivation

    Institute of Scientific and Technical Information of China (English)

    Baolin Zhao; Jia Li

    2012-01-01

    Brassinosteroids (BRs) are a group of naturally-occurring steroidal phytohormones playing fundamental roles during normal plant growth and development.Using a combination of experimental approaches,including analytical chemistry,genetics,and biochemistry,the major BR biosynthetic pathway has been largely elucidated.The least-understood knowledge in the BR research field is probably the molecular mechanisms controlling the bioactive levels of BRs in response to various developmental and environmental cues.In this review,we focus our discussion on a recently-proposed,8-step predominant BR biosynthetic pathway,several newly-identified transcription factors regulating the expression of key enzymes that catalyze BR biosynthesis,and up-to-date information about the mechanisms that plants use to inactivate unnecessary BRs.

  19. Biosynthesis and function of plant lipids

    International Nuclear Information System (INIS)

    The Sixth Annual Symposium in Botany and Plant Physiology was held January 13-15, 1983, at the University of California, Riverside. This volume comprises the papers that were presented. Subjects discussed at the symposium covered a wide range in the field of plant lipids. Biosynthesis of lipids occupied an important fraction of the presentations at the symposium. Subjects included detailed studies of the enzymes of fatty acid synthesis, several discussions of the incorporation of fatty acids into glycerolipids and the further modification of the fatty acids, and the synthesis of glycerolipids and desaturation of fatty acids in both maturing oilseeds and chloroplasts. The physicochemical studies of glycerolipids and sterols in artificial membranes have led to distinct conclusions about their behaviour which must be relevant in the biological membrane. Results on the functional consequences of modifying the galactolipid composition in the chloroplast were an encouraging sign of progress in the attempts to relate membrane lipid composition to physiological function

  20. Biosynthesis and function of plant lipids

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, W.W.; Mudd, J.B.; Gibbs, M. (eds.)

    1983-01-01

    The Sixth Annual Symposium in Botany and Plant Physiology was held January 13-15, 1983, at the University of California, Riverside. This volume comprises the papers that were presented. Subjects discussed at the symposium covered a wide range in the field of plant lipids. Biosynthesis of lipids occupied an important fraction of the presentations at the symposium. Subjects included detailed studies of the enzymes of fatty acid synthesis, several discussions of the incorporation of fatty acids into glycerolipids and the further modification of the fatty acids, and the synthesis of glycerolipids and desaturation of fatty acids in both maturing oilseeds and chloroplasts. The physicochemical studies of glycerolipids and sterols in artificial membranes have led to distinct conclusions about their behaviour which must be relevant in the biological membrane. Results on the functional consequences of modifying the galactolipid composition in the chloroplast were an encouraging sign of progress in the attempts to relate membrane lipid composition to physiological function.