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Sample records for inhibit mevalonate biosynthesis

  1. Biosynthesis of sterols from mevalonate in a starfish, Coscinasterias acutispina

    International Nuclear Information System (INIS)

    Teshima, Shin-ichi; Kanazawa, Akio

    1976-01-01

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

  2. Manipulation of isoprenoid biosynthesis as a possible therapeutic option in mevalonate kinase deficiency

    NARCIS (Netherlands)

    Schneiders, Marit S.; Houten, Sander M.; Turkenburg, Marjolein; Wanders, Ronald J. A.; Waterham, Hans R.

    2006-01-01

    OBJECTIVE: In cells from patients with the autoinflammatory disorder mevalonate kinase (MK) deficiency, which includes the hyperimmunoglobulin D with periodic fever syndrome, MK becomes the rate-limiting enzyme in the isoprenoid biosynthesis pathway. This suggests that up-regulation of residual MK

  3. Triterpenoid biosynthesis in Euphorbia lathyris latex

    International Nuclear Information System (INIS)

    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 50 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 I 50 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- 3 H-mevalonic acid and incubating latex with a mixture of this and 14 C-mevalonic acid. From the 3 H/ 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

  4. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

    Science.gov (United States)

    Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

    2016-03-01

    Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

  5. 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.

  6. Mevalonate kinase deficiencies: from mevalonic aciduria to hyperimmunoglobulinemia D syndrome

    Directory of Open Access Journals (Sweden)

    Hoffmann Georg F

    2006-04-01

    Full Text Available Abstract Mevalonic aciduria (MVA and hyperimmunoglobulinemia D syndrome (HIDS represent the two ends of a clinical spectrum of disease caused by deficiency of mevalonate kinase (MVK, the first committed enzyme of cholesterol biosynthesis. At least 30 patients with MVA and 180 patients with HIDS have been reported worldwide. MVA is characterized by psychomotor retardation, failure to thrive, progressive cerebellar ataxia, dysmorphic features, progressive visual impairment and recurrent febrile crises. The febrile episodes are commonly accompanied by hepatosplenomegaly, lymphadenopathy, abdominal symptoms, arthralgia and skin rashes. Life expectancy is often compromised. In HIDS, only febrile attacks are present, but a subgroup of patients may also develop neurological abnormalities of varying degree such as mental retardation, ataxia, ocular symptoms and epilepsy. A reduced activity of MVK and pathogenic mutations in the MVK gene have been demonstrated as the common genetic basis in both disorders. In MVA, the diagnosis is established by detection of highly elevated levels of mevalonic acid excreted in urine. Increased levels of immunoglobulin D (IgD and, in most patients of immunoglobulin A (IgA, in combination with enhanced excretion of mevalonic acid provide strong evidence for HIDS. The diagnosis is confirmed by low activity of mevalonate kinase or by demonstration of disease-causing mutations. Genetic counseling should be offered to families at risk. There is no established successful treatment for MVA. Simvastatin, an inhibitor of HMG-CoA reductase, and anakinra have been shown to have beneficial effect in HIDS.

  7. Possible regulation of sterol biosynthesis by phenolic acids

    International Nuclear Information System (INIS)

    Ranganathan, S.; Ramasarma, T.

    1974-01-01

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

  8. Increasing production yield of tyrosine and mevalonate through inhibition of biomass formation

    DEFF Research Database (Denmark)

    Li, Songyuan; Jendresen, Christian Bille; Nielsen, Alex Toftgaard

    2016-01-01

    , in particular, resulted in an increase in mass yield of mevalonate and tyrosine by 80% and 50%, respectively. By tracking production and biomass concentrations, it was observed that the production was maintained for more than 10 h after inhibition of cell growth, despite cell maintenance requirements...

  9. Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei

    International Nuclear Information System (INIS)

    Zhuang, Ningning; Seo, Kyung Hye; Chen, Cong; Zhou, Jia; Kim, Seon Won; Lee, Kon Ho

    2012-01-01

    Recombinant mevalonate kinase from M. mazei has been crystallized. Diffraction data were collected to 2.08 Å resolution. Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl 2 , 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 97.11, b = 135.92, c = 46.03 Å. Diffraction data were collected to 2.08 Å resolution

  10. Selective inhibition of the demethylation at C-14 in ergosterol biosynthesis by the fungicide, Denmert (S-1358)

    International Nuclear Information System (INIS)

    Kato, Toshiro; Kawase, Yasuo

    1976-01-01

    A direct evidence of the inhibitory effect in a cell-free system of S. cerevisiae was experimentally studied, and the site of action of Denmert (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate) in sterol biosynthesis was examined. 14 C-labeled lanosterol and 14-desmethyl-lanosterol were biosynthetically prepared. DL-mevalonate-2- 14 C was incubated with yeast cell-free homogenates for 3 hr at 28 deg C while being shaked vigorously in atmospheric oxygen. The resultant 14 C-labeled sterol was extracted and chromatographed on a silicic acid-Hyflo Super Cel column. 4,4-dimethyl sterol thus obtained was acetylated with acetic anhydride and pyridine. The separation of lanosteryl acetate and 14-desmethyl lanosteryl acetate was accomplished on alumina thin-layer plates. After the saponification of each steryl acetate, the quantity of the sterol was assessed by gas chromatography with cholesterol as an internal standard. The incubation of the 14 C-labeled sterol was achieved under the same conditions as those for the DL-mevalonate-2- 14 C except the addition of the substrate which was dispersed in 0.1M phosphate buffer. Denmert inhibited the conversion of 14 C-labeled lanosterol to 4-desmethyl sterol, while the conversion of 14 C-labeled 14-desmethyl lanosterol to 4-desmethyl sterol was hardly affected by the fungicide. Therefore, Denmert is a potent selective inhibitor of the demethylation at the C-14 position in ergosterol biosynthesis. The fungicide, triarimol, exhibited the same effect on sterol biosynthesis as that of Denmert. (Iwakiri, K.)

  11. Regulation of the Mevalonate Pathway for the Prevention of Breast Cancer

    National Research Council Canada - National Science Library

    Archer, Michael

    2000-01-01

    ...) can be accounted for by their inhibitory effect on the cholesterol biosynthesis (mevalonate) pathway. In Task 1, we have shown that the decrease in mammary gland HMG-CoA reductase seen in LDL-R -/- mice compared...

  12. Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium.

    Science.gov (United States)

    Bledsoe, C S

    1978-11-01

    The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [(14)C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [(14)C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [(14)C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable (14)C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated (14)C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments.

  13. Investigations on the isoprenoid biosynthesis in the green alga Scenedesmus obliquus by using the 13C-labelling technique

    International Nuclear Information System (INIS)

    Schwender, J.

    1995-01-01

    The biosynthesis of several prenyllipids (isoprenoid lipids) of the green alga Scendesmus obliquus was investigated. The aim was to verify, whether the biosynthesis of isopentenyl diphosphate (IPP) in Scenedesmus proceeds according to the classical acetate mevalonate pathway or to an alternative pathway. An alternative pathway for IPP formation has recently been detected in some eubacteria by the group of Prof. M. Rohmer. Some inhibition tests were performed with mevinolin, a specific inhibitor of HMG-CoA reductase which yields mevalonic acid. Mevinolin should block the biosynthesis of such isoprenoids which are formed via the acetate mevalonate pathway. Scenedesmus was grown heterotrophically on 13 C-labelled glucose or acetate. After isolation and purification of 13 C-labelled phytol (side chains of chlorophylls), β-carotene, lutein, plastoquinone-9 and three sterol compounds, the enrichment of 13 C at different carbon-positions of the labelled compounds was determined. This was achieved by the 13 C-NMR technique in cooperation with Miriam Seemann of the group of Prof. M. Rohmer in Mullhouse/France. (orig.) [de

  14. The Regulation of the Mevalonate Pathway for the Prevention of Breast Cancer

    National Research Council Canada - National Science Library

    Archer, Michael

    2001-01-01

    ...)can be accounted for by their inhibitory effect on the cholesterol biosynthesis (mevalonate) pathway. In Task 1, we have shown that the decrease in mammary gland HMG-CoA redustase seen in LDL-R -/- mice compared...

  15. Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium 1

    Science.gov (United States)

    Bledsoe, Caroline S.; Ross, Cleon W.

    1978-01-01

    The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [14C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [14C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [14C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable 14C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated 14C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments. ImagesFig. 1 PMID:16660583

  16. Cholesterol biosynthesis in polychlorinated biphenyl-treated rats

    International Nuclear Information System (INIS)

    Kling, D.; Gamble, W.

    1982-01-01

    After administration of polychlorinated biphenly (PCB) at 0.055 (w/w) of the diet to Wistar rats for 30 days, followed by intraperitioneal injection of tritiated water, [ 14 C]mevalonate, and [ 14 C]acetate, there was a decrease in cholesterol biosynthesis in rat liver. No significant change in cholesterol formation was observed when PCB was administered at 0.01% (w/w) of the diet. In vitro inhibition of cholesterol synthesis by rat liver microsomes was observed with PCB. Squalene 2,3-oxidocyclase activity of rat liver microsomes was not significantly altered. Desmosterol delta 24 reductase activity was inhibited only at relatively high concentrations of PCB. There was increased incorporation of radioactivity into squalene and lanosterol, in vitro, in the presence of PCB. The primary inhibition of cholesterol biosynthesis appears to be at the demethylation and rearrangement reactions between lanosterol and cholesterol in the biosynthetic pathway

  17. Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase

    Directory of Open Access Journals (Sweden)

    Hunter William N

    2007-03-01

    Full Text Available Abstract Background Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results Putative mevalonate kinase encoding genes from Leishmania major (LmMK and Trypanosoma brucei (TbMK have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R- over (S-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix α2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion Our new structural

  18. Mevalonate Pathway Antagonist Inhibits Proliferation of Serous Tubal Intraepithelial Carcinoma and Ovarian Carcinoma in Mouse Models

    Science.gov (United States)

    Kobayashi, Yusuke; Kashima, Hiroyasu; Wu, Ren-Chin; Jung, Jin- Gyoung; Kuan, Jen-Chun; Gu, Jinghua; Xuan, Jianhua; Sokoll, Lori; Visvanathan, Kala; Shih, Ie-Ming; Wang, Tian-Li

    2015-01-01

    Purpose Statins are among the most frequently prescribed drugs because of their efficacy and low toxicity in treating hypercholesterolemia. Recently, statins have been reported to inhibit the proliferative activity of cancer cells, especially those with TP53 mutations. Since TP53 mutations occur in almost all of the ovarian high-grade serous carcinoma, we determined if statins suppressed tumor growth in animal models of ovarian cancer. Experimental Design Two ovarian cancer mouse models were employed. The first one was a genetically engineered model, mogp-TAg, in which the promoter of oviduct glycoprotein-1 was used to drive the expression of SV40 T-antigen in gynecologic tissues. These mice spontaneously develop serous tubal intraepithelial carcinomas (STICs), which are known as ovarian cancer precursor lesions. The second model was a xenograft tumor model in which human ovarian cancer cells were inoculated into immunocompromised mice. Mice in both models were treated with lovastatin, and effects on tumor growth were monitored. The molecular mechanisms underlying the anti-tumor effects of lovastatin were also investigated. Results Lovastatin significantly reduced the development of STICs in mogp-TAg mice and inhibited ovarian tumor growth in the mouse xenograft model. Knockdown of prenylation enzymes in the mevalonate pathway recapitulated the lovastatin-induced anti-proliferative phenotype. Transcriptome analysis indicated that lovastatin affected the expression of genes associated with DNA replication, Rho/PLC signaling, glycolysis, and cholesterol biosynthesis pathways, suggesting that statins have pleiotropic effects on tumor cells. Conclusion The above results suggest that repurposing statin drugs for ovarian cancer may provide a promising strategy to prevent and manage this devastating disease. PMID:26109099

  19. Isoprenoid biosynthesis in hereditary periodic fever syndromes and inflammation

    NARCIS (Netherlands)

    Houten, S. M.; Frenkel, J.; Waterham, H. R.

    2003-01-01

    Mevalonate kinase (MK) is an essential enzyme in the isoprenoid biosynthesis pathway which produces numerous biomolecules (isoprenoids) involved in a variety of cellular processes. The indispensability of MK and isoprenoid biosynthesis for human health is demonstrated by the identification of its

  20. Liquid chromatography-tandem mass spectrometry method for the measurement of serum mevalonic acid: a novel marker of hydroxymethylglutaryl coenzyme A reductase inhibition by statins.

    Science.gov (United States)

    Waldron, Jenna; Webster, Craig

    2011-05-01

    Mevalonic acid (MVA) is synthesized at an early and rate-limiting step in the biosynthesis of cholesterol by the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, and is a useful measure of statin efficacy or treatment. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of serum MVA has been developed. Following the in vitro conversion of MVA to mevalonic acid lactone (MVAL) in the serum, MVAL and a deuterated internal standard were extracted using an online solid-phase extraction procedure. Chromatographic separation was achieved using a Luna PFP column (Phenomenex), with enhanced selectivity and improved resolution for polar compounds. A gradient system was used, with mobile phase comprising methanol and water (5 mmol/L ammonium formate buffer, pH 2.5). Analysis was performed using an API 5000 tandem mass spectrometer (Applied Biosystems) in positive electrospray ionization mode. The method showed excellent recoveries (98 ± 8%) and imprecision (intra-assay coefficient of variation of 2.2% [6.5 ng/mL] and 2.6% [10.5 ng/mL], and inter-assay coefficient of variation of 9% [10.5 ng/mL]). The assay provides a calibration range up to 50 ng/mL with a limit of detection at 0.1 ng/mL. A simple, rapid and analytically specific method has been developed for the measurement of serum MVA, in the form of MVAL. The high analytical sensitivity of the method allows for accurate quantitation of MVAL in serum samples, both at the endogenous levels found in healthy individuals and in statin-treated patients where normal levels are expected to be greatly reduced through the inhibition of HMG-CoA reductase.

  1. Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation.

    Science.gov (United States)

    Zeidler, J; Lichtenthaler, H K

    2001-06-01

    The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

  2. 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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  4. Terpenoid biosynthesis in Euphorbia lathyris and Copaifera spp

    Energy Technology Data Exchange (ETDEWEB)

    Skrukrud, C.L.

    1987-07-01

    Biosynthesis of triterpenoids by isolated latex of Euphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid into triterpenoids was thirty times greater than acetate incorporation indicating that the rate-limiting step in the pathway occurs prior to mevalonate. Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex. HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken from Copaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessibility to the different substrates and/or reflecting the metabolic controls on carbon allocation to the terpenes. Mevalonate incorporation by Copaifera langsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. 119 refs., 58 figs., 16 tabs.

  5. Terpenoid biosynthesis in Euphorbia lathyris and Copaifera spp

    International Nuclear Information System (INIS)

    Skrukrud, C.L.

    1987-07-01

    Biosynthesis of triterpenoids by isolated latex of Euphorbia lathyris was investigated. The rate of in vitro incorporation of mevalonic acid into triterpenoids was thirty times greater than acetate incorporation indicating that the rate-limiting step in the pathway occurs prior to mevalonate. Both HMG-CoA reductase (EC 1.1.1.34) and HMG-CoA lyase (EC 4.1.3.4) activities were detected in isolated latex. HMG-CoA reductase was localized to a membrane-bound fraction of a 5000g pellet of latex. The rate of conversion of HMG-CoA to mevalonate by this enzyme is comparable to the overall rate of acetate incorporation into the triterpenoids suggesting that this enzyme is rate-determining in the biosynthesis of triterpenoids in E. lathyris latex. HMG-CoA reductase of E. lathyris vegetative tissue was localized to the membrane-bound portion of a particulate fraction (18,000g), and was solubilized by treatment with 2% polyoxyethylene ether W-1. Differences in the optimal pH for activity of HMG-CoA reductase from the latex and vegetative tissue suggest that isozymes of the enzyme may be present in the two tissue types. Studies of the incorporation of various precursors into leaf discs and cuttings taken from Copaifera spp. show differences in the rate of incorporation into Copaifera sesquiterpenes suggesting that the site of sesquiterpene biosynthesis may differ in its accessibility to the different substrates and/or reflecting the metabolic controls on carbon allocation to the terpenes. Mevalonate incorporation by Copaifera langsdorfii cuttings into sesquiterpenes was a hundred-fold greater than either acetate or glucose incorporation, however, its incorporation into squalene and triterpenoids was also a hundred-fold greater than the incorporation into sesquiterpenes. 119 refs., 58 figs., 16 tabs

  6. Biosynthesis of furanochromones in Pimpinella monoica

    Indian Academy of Sciences (India)

    polyketide origin of their aromatic and pyrone rings while the furan ring originates via an acetate-mevalonate pathway. The plant also utilises glycine and leucine as substrate via acetate. Biotransformation of 3-H-visnagin to (6) but not to (2) was also observed. Keywords. Biosynthesis; furochromones; polyketide origin; ...

  7. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  8. Mevalonosomes: specific vacuoles containing the mevalonate pathway in Plocamium brasiliense cortical cells (Rhodophyta).

    Science.gov (United States)

    Paradas, Wladimir Costa; Crespo, Thalita Mendes; Salgado, Leonardo Tavares; de Andrade, Leonardo Rodrigues; Soares, Angélica Ribeiro; Hellio, Claire; Paranhos, Ricardo Rogers; Hill, Lilian Jorge; de Souza, Geysa Marinho; Kelecom, Alphonse Germaine Albert Charles; Da Gama, Bernardo Antônio Perez; Pereira, Renato Crespo; Amado-Filho, Gilberto Menezes

    2015-04-01

    This paper has identified, for the first time in a member of the Rhodophyta, a vacuolar organelle containing enzymes that are involved in the mevalonate pathway-an important step in red algal isoprenoid biosynthesis. These organelles were named mevalonosomes (Mev) and were found in the cortical cells (CC) of Plocamium brasiliense, a marine macroalgae that synthesizes several halogenated monoterpenes. P. brasiliense specimens were submitted to a cytochemical analysis of the activity of the 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). Using transmission electron microscopy (TEM), we confirmed the presence of HMGS activity within the Mev. Because HMGS is necessary for the biosynthesis of halogenated monoterpenes, we isolated a hexanic fraction (HF) rich in halogenated monoterpenes from P. brasiliense that contained a pentachlorinated monoterpene as a major metabolite. Because terpenes are often related to chemical defense, the antifouling (AF) activity of pentachlorinated monoterpene was tested. We found that the settlement of the mussel Perna perna was reduced by HF treatment (2.25 times less than control; 40% and 90% of fouled surface, respectively; P = 0.001; F9,9 = 1.13). The HF (at 10 μg · mL(-1) ) also inhibited three species of fouling microalgae (Chlorarachnion reptans, Cylindrotheca cloisterium, and Exanthemachrysis gayraliae), while at a higher concentration (50 μg · mL(-1) ), it inhibited the bacteria Halomonas marina, Polaribacter irgensii, Pseudoalteromonas elyakovii, Shewanella putrefaciens, and Vibrio aestuarianus. The AF activity of P. brasiliense halogenated monoterpenes and the localization of HMGS activity inside Mev suggest that this cellular structure found in CC may play a role in thallus protection against biofouling. © 2015 Phycological Society of America.

  9. Simvastatin inhibits Candida albicans biofilm in vitro.

    Science.gov (United States)

    Liu, Geoffrey; Vellucci, Vincent F; Kyc, Stephanie; Hostetter, Margaret K

    2009-12-01

    By inhibiting the conversion of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) to mevalonate, statins impair cholesterol metabolism in humans. We reasoned that statins might similarly interfere with the biosynthesis of ergosterol, the major sterol of the yeast cell membrane. As assessed by spectrophotometric and microscopic analysis, significant inhibition of biofilm production was noted after 16-h incubation with 1, 2.5, and 5 muM simvastatin, concentrations that did not affect growth, adhesion, or hyphal formation by C. albicans in vitro. Higher concentrations (10, 20, and 25 muM simvastatin) inhibited biofilm by >90% but also impaired growth. Addition of exogenous ergosterol (90 muM) overcame the effects of 1 and 2.5 muM simvastatin, suggesting that at least one mechanism of inhibition is interference with ergosterol biosynthesis. Clinical isolates from blood, skin, and mucosal surfaces produced biofilms; biofilms from bloodstream isolates were similarly inhibited by simvastatin. In the absence of fungicidal activity, simvastatin's interruption of a critical step in an essential metabolic pathway, highly conserved from yeast to man, has unexpected effects on biofilm production by a eukaryotic pathogen.

  10. The mevalonate pathway in C. Elegans

    Directory of Open Access Journals (Sweden)

    Rauthan Manish

    2011-12-01

    Full Text Available Abstract The mevalonate pathway in human is responsible for the synthesis of cholesterol and other important biomolecules such as coenzyme Q, dolichols and isoprenoids. These molecules are required in the cell for functions ranging from signaling to membrane integrity, protein prenylation and glycosylation, and energy homeostasis. The pathway consists of a main trunk followed by sub-branches that synthesize the different biomolecules. The majority of our knowledge about the mevalonate pathway is currently focused on the cholesterol synthesis branch, which is the target of the cholesterol-lowering statins; less is known about the function and regulation of the non-cholesterol-related branches. To study them, we need a biological system where it is possible to specifically modulate these metabolic branches individually or in groups. The nematode Caenorhabditis elegans (C. elegans is a promising model to study these non-cholesterol branches since its mevalonate pathway seems very well conserved with that in human except that it has no cholesterol synthesis branch. The simple genetic makeup and tractability of C. elegans makes it relatively easy to identify and manipulate key genetic components of the mevalonate pathway, and to evaluate the consequences of tampering with their activity. This general experimental approach should lead to new insights into the physiological roles of the non-cholesterol part of the mevalonate pathway. This review will focus on the current knowledge related to the mevalonate pathway in C. elegans and its possible applications as a model organism to study the non-cholesterol functions of this pathway.

  11. Simvastatin Inhibits Goblet Cell Hyperplasia and Lung Arginase in a Mouse Model of Allergic Asthma: A Novel Treatment for Airway Remodeling?

    Science.gov (United States)

    Zeki, Amir A.; Bratt, Jennifer M.; Rabowsky, Michelle; Last, Jerold A.; Kenyon, Nicholas J.

    2010-01-01

    Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting step of cholesterol biosynthesis in the mevalonate pathway. These drugs have been associated with improved respiratory health and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin-exposed mice would attenuate early features of airway remodeling, by a mevalonate-dependent mechanism. BALB/c mice were initially sensitized to ovalbumin, and then exposed to 1% ovalbumin aerosol for 2 weeks after sensitization for a total of six exposures. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) were injected intraperitoneally before each ovalbumin exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but did not alter airway hydroxyproline content or transforming growth factor-β1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any of the control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Further studies utilizing sub-chronic or chronic allergen exposure models are needed to extend these initial findings. PMID:21078495

  12. Mevalonate Kinase Deficiency and Neuroinflammation: Balance between Apoptosis and Pyroptosis

    Directory of Open Access Journals (Sweden)

    Paola Maura Tricarico

    2013-11-01

    Full Text Available Mevalonic aciduria, a rare autosomal recessive disease, represents the most severe form of the periodic fever, known as Mevalonate Kinase Deficiency. This disease is caused by the mutation of the MVK gene, which codes for the enzyme mevalonate kinase, along the cholesterol pathway. Mevalonic aciduria patients show recurrent fever episodes with associated inflammatory symptoms, severe neurologic impairments, or death, in early childhood. The typical neurodegeneration occurring in mevalonic aciduria is linked both to the intrinsic apoptosis pathway (caspase-3 and -9, which is triggered by mitochondrial damage, and to pyroptosis (caspase-1. These cell death mechanisms seem to be also related to the assembly of the inflammasome, which may, in turn, activate pro-inflammatory cytokines and chemokines. Thus, this particular molecular platform may play a crucial role in neuroinflammation mechanisms. Nowadays, a specific therapy is still lacking and the pathogenic mechanisms involving neuroinflammation and neuronal dysfunction have not yet been completely understood, making mevalonic aciduria an orphan drug disease. This review aims to analyze the relationship among neuroinflammation, mitochondrial damage, programmed cell death, and neurodegeneration. Targeting inflammation and degeneration in the central nervous system might help identify promising treatment approaches for mevalonic aciduria or other diseases in which these mechanisms are involved.

  13. Mevalonate kinase deficiency: Evidence for a phenotypic continuum

    NARCIS (Netherlands)

    Simon, A; Kremer, H P H; Wevers, R A; Scheffer, H; de Jong, J G; van der Meer, J W M; Drenth, J. P.

    2004-01-01

    Both mevalonic aciduria, characterized by psychomotor retardation, cerebellar ataxia, recurrent fever attacks, and death in early childhood, and hyper-immunoglobulin D (hyper-IgD) syndrome, with recurrent fever attacks without neurologic symptoms, are caused by a functional deficiency of mevalonate

  14. Fenarimol, a Pyrimidine-Type Fungicide, Inhibits Brassinosteroid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Keimei Oh

    2015-07-01

    Full Text Available The plant steroid hormone brassinosteroids (BRs are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz, the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM, a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 μM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL but not by gibberellin (GA. Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.

  15. Hyper-IgD syndrome or mevalonate kinase deficiency.

    NARCIS (Netherlands)

    Stoffels, M.; Simon, A.

    2011-01-01

    PURPOSE OF REVIEW: The hyper-IgD and periodic fever syndrome (HIDS) is one of the classical monogenetic hereditary autoinflammatory disorders, and together with the more severe mevalonic aciduria it is also known as 'mevalonate kinase deficiency' (MKD). In this study, we will give an overview of the

  16. Autoinflammatory Reaction in Dogs Treated for Cancer via G6PD Inhibition

    Directory of Open Access Journals (Sweden)

    Jonathan W. Nyce

    2017-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is an oncoprotein that is overexpressed in cancer cells to provide the NADPH required for their increased anabolism. NADPH, sourced from G6PD fuels nucleotide biosynthesis, maintains redox potential of thioredoxin and glutathione and drives the mevalonate pathway that powers many of the basic mechanisms by which cancer cells escape host control. G6PD is thus a target for cancer treatment being addressed by many groups around the world. We have discovered that systemic inhibition of G6PD by high dose dehydroepiandrosterone (DHEA causes a severe autoinflammatory response in dogs, which does not occur in mice or rats. Since dogs more closely model the human adrenal androgen system than do common laboratory animals, this finding is relevant to the design of G6PD-inhibiting drugs for humans. The autoinflammatory reaction observed closely resembles mevalonate kinase deficiency (MKD, a rare autosomal recessive disease in humans characterized by recurrent febrile attacks, arthralgia, skin rash, and aphthous ulcers of mucocutaneous tissues. In a manner comparable to animal models of MKD, the reconstitution of protein geranylgeranylation blocked the autoinflammatory reaction caused by systemic G6PD inhibition. This autoinflammatory response to systemic G6PD inhibition represents an unexpected result that must be taken into consideration when targeting this oncoprotein.

  17. Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

    International Nuclear Information System (INIS)

    Chappell, J.; Nable, R.

    1987-01-01

    Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [ 14 C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity, an enzyme of general isoprenoid metabolism, paralleled the changes in [ 14 C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [ 14 C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [ 3 H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures

  18. Cantharidin biosynthesis in a blister beetle: inhibition by 6-fluoromevalonate causes chemical disarmament.

    Science.gov (United States)

    Carrel, J E; Doom, J P; McCormick, J P

    1986-07-15

    Biosynthesis of cantharidin in a blister beetle, Lytta polita, is effectively inhibited by 6-fluoromevalonate. Inhibition is attributed specifically to the fluorine substituent. Biochemical inhibition has not been demonstrated previously for an arthropod's defensive substance.

  19. Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway*

    Science.gov (United States)

    Onono, Fredrick; Subramanian, Thangaiah; Sunkara, Manjula; Subramanian, Karunai Leela; Spielmann, H. Peter; Morris, Andrew J.

    2013-01-01

    Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition. PMID:23908355

  20. 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. © 2014 Scandinavian Plant Physiology Society.

  1. Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

    2017-10-12

    The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

  2. Activity of mevalonate pathway inhibitors against breast and ovarian cancers in the ATP-based tumour chemosensitivity assay

    International Nuclear Information System (INIS)

    Knight, Louise A; Kurbacher, Christian M; Glaysher, Sharon; Fernando, Augusta; Reichelt, Ralf; Dexel, Susanne; Reinhold, Uwe; Cree, Ian A

    2009-01-01

    Previous data suggest that lipophilic statins such as fluvastatin and N-bisphosphonates such as zoledronic acid, both inhibitors of the mevalonate metabolic pathway, have anti-cancer effects in vitro and in patients. We have examined the effect of fluvastatin alone and in combination with zoledronic acid in the ATP-based tumour chemosensitivity assay (ATP-TCA) for effects on breast and ovarian cancer tumour-derived cells. Both zoledronic acid and fluvastatin showed activity in the ATP-TCA against breast and ovarian cancer, though fluvastatin alone was less active, particularly against breast cancer. The combination of zoledronic acid and fluvastatin was more active than either single agent in the ATP-TCA with some synergy against breast and ovarian cancer tumour-derived cells. Sequential drug experiments showed that pre-treatment of ovarian tumour cells with fluvastatin resulted in decreased sensitivity to zoledronic acid. Addition of mevalonate pathway components with zoledronic acid with or without fluvastatin showed little effect, while mevalonate did reduced inhibition due to fluvastatin. These data suggest that the combination of zoledronic acid and fluvastatin may have activity against breast and ovarian cancer based on direct anti-cancer cell effects. A clinical trial to test this is in preparation

  3. Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

    Directory of Open Access Journals (Sweden)

    Zhiliang Yu

    2015-01-01

    Full Text Available Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab, L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

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

    Science.gov (United States)

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

    2014-12-01

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

  5. Mevalonate Pathway Antagonist Suppresses Formation of Serous Tubal Intraepithelial Carcinoma and Ovarian Carcinoma in Mouse Models.

    Science.gov (United States)

    Kobayashi, Yusuke; Kashima, Hiroyasu; Wu, Ren-Chin; Jung, Jin-Gyoung; Kuan, Jen-Chun; Gu, Jinghua; Xuan, Jianhua; Sokoll, Lori; Visvanathan, Kala; Shih, Ie-Ming; Wang, Tian-Li

    2015-10-15

    Statins are among the most frequently prescribed drugs because of their efficacy and low toxicity in treating hypercholesterolemia. Recently, statins have been reported to inhibit the proliferative activity of cancer cells, especially those with TP53 mutations. Because TP53 mutations occur in almost all ovarian high-grade serous carcinoma (HGSC), we determined whether statins suppressed tumor growth in animal models of ovarian cancer. Two ovarian cancer mouse models were used. The first one was a genetically engineered model, mogp-TAg, in which the promoter of oviduct glycoprotein-1 was used to drive the expression of SV40 T-antigen in gynecologic tissues. These mice spontaneously developed serous tubal intraepithelial carcinomas (STICs), which are known as ovarian cancer precursor lesions. The second model was a xenograft tumor model in which human ovarian cancer cells were inoculated into immunocompromised mice. Mice in both models were treated with lovastatin, and effects on tumor growth were monitored. The molecular mechanisms underlying the antitumor effects of lovastatin were also investigated. Lovastatin significantly reduced the development of STICs in mogp-TAg mice and inhibited ovarian tumor growth in the mouse xenograft model. Knockdown of prenylation enzymes in the mevalonate pathway recapitulated the lovastatin-induced antiproliferative phenotype. Transcriptome analysis indicated that lovastatin affected the expression of genes associated with DNA replication, Rho/PLC signaling, glycolysis, and cholesterol biosynthesis pathways, suggesting that statins have pleiotropic effects on tumor cells. The above results suggest that repurposing statin drugs for ovarian cancer may provide a promising strategy to prevent and manage this devastating disease. ©2015 American Association for Cancer Research.

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

    NARCIS (Netherlands)

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

    1999-01-01

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

  7. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  8. 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

  9. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  10. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

    International Nuclear Information System (INIS)

    Nishijima, M.; Kuge, O.; Akamatsu, Y.

    1986-01-01

    The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32 Pi, the incorporation of 32 Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32 Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32 Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [ 3 H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U- 14 C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine

  11. Effect of inhibition of biosynthesis of phenylpropanoids on sessile oak somatic embryogenesis

    Czech Academy of Sciences Publication Activity Database

    Cvikrová, Milena; Malá, J.; Hrubcová, Marie; Eder, Josef; Zoń, J.; Macháčková, Ivana

    2003-01-01

    Roč. 41, č. 3 (2003), s. 251-259 ISSN 0981-9428 R&D Projects: GA MŠk LN00A081 Institutional research plan: CEZ:AV0Z5038910 Keywords : Inhibition of phenylpropanoid biosynthesis * Somatic embryogenesis * Oak Subject RIV: CE - Biochemistry Impact factor: 1.729, year: 2003

  12. Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS

    NARCIS (Netherlands)

    Henneman, Linda; van Cruchten, Arno G.; Kulik, Willem; Waterham, Hans R.

    2011-01-01

    The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In

  13. Ultraviolet light inhibition of phytochrome-induced flavonoid biosynthesis and DNA photolyase formation in mustard cotyledons (Sinapis alba L.)

    International Nuclear Information System (INIS)

    Buchholz, G.; Ehmann, B.; Wellmann, E.

    1995-01-01

    In cotyledons of etiolated mustard (Sinapis alba L.) seedlings, phytochrome-far-red-absorbing form-induced flavonoid biosynthesis was found to be inhibited by short-term ultraviolet (UV) irradiations. UV inhibition was shown for the synthesis of quercetin, anthocyanin, and also for the accumulation of the mRNA for chalcone synthase, the key enzyme of this pathway. The UV effect was more pronounced on flavonoid biosynthesis, a process that selectively occurs in the epidermal layers, than on the synthesis of mRNA for chlorophyll a/b-binding protein localized in the mesophyll tissue. These UV inhibitory effects were accompanied by cyclobutane pyrimidine dimer (CPD) formation showing a linear fluence-response relationship. CPD formation and UV inhibition of flavonoid biosynthesis was found to be partially reversible by blue/UV-A light via DNA photolyase (PRE), allowing photoreactivation of the DNA by splitting of CPDs, which are the cause of the UV effect. Like flavonoid formation PRE was also induced by the far-red-absorbing form of phytochrome and induction was inhibited by UV. A potential risk of inhibition, in response to solar UV-B irradiation, was shown for anthocyanin formation. This inhibition, however, occurred only if photoreactivation was experimentally reduced. The PRE activity present in the etiolated seedlings (further increasing about 5-fold during light acclimatization) appears to be sufficient to prevent the persistence of CPDs even under conditions of high solar irradiation

  14. Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159

    Directory of Open Access Journals (Sweden)

    Soon-Nang Park

    2015-06-01

    Full Text Available In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

  15. Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

    Science.gov (United States)

    Montero-Villegas, Sandra; Polo, Mónica; Galle, Marianela; Rodenak-Kladniew, Boris; Castro, María; Ves-Losada, Ana; Crespo, Rosana; García de Bravo, Margarita

    2017-01-01

    The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [ 14 C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [ 14 C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

  16. Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

    Science.gov (United States)

    Guo, Chang; Ma, Jingfan; Zhong, Qionghong; Zhao, Mengyuan; Hu, Tianxing; Chen, Tong; Qiu, Longxin; Wen, Longping

    2017-08-01

    Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy. Copyright © 2017. Published by Elsevier Inc.

  17. Trichoderma harzianum: Inhibition of mycotoxin producing fungi and toxin biosynthesis.

    Science.gov (United States)

    Braun, H; Woitsch, L; Hetzer, B; Geisen, R; Zange, B; Schmidt-Heydt, M

    2018-04-19

    A quarter of the world-wide crop is spoiled by filamentous fungi and their mycotoxins and weather extremes associated with the climate change lead to further deterioration of the situation. The ingestion of mycotoxins causes several health issues leading in the worst case to cancer in humans and animals. Common intervention strategies against mycotoxin producing fungi, such as the application of fungicides, may result in undesirable residues and in some cases to a stress induction of mycotoxin biosynthesis. Moreover, development of fungicide resistances has greatly impacted pre- and postharvest fungal diseases. Hence there is the need to develop alternative strategies to reduce fungal infestation and thus mycotoxin contamination in the food chain. Such a strategy for natural competition of important plant-pathogenic and mycotoxin producing fungi could be Trichoderma harzianum, a mycoparasitic fungus. Especially in direct comparison to certain tested fungicides, the inhibition of different tested fungal species by T. harzianum was comparable, more sustainable and in some cases more effective, too. Besides substantially reduced growth rates, a transcriptional based inhibition of mycotoxin biosynthesis in the competed Aspergillus species could be shown. Furthermore it could be clearly observed by high-resolution Scanning Electron Microscopy (SEM) that T. harzianum actively attaches to the competitor species followed by subsequent enzymatic lysis of those mycelial filaments. The analyzed isolate of T. harzianum MRI349 is not known to produce mycotoxins. In this study it could be successfully proven that T. harzianum as a biological competitor is an effective complement to the use of fungicides. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

    International Nuclear Information System (INIS)

    Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

    2005-01-01

    Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

  19. Biosynthesis of steroidal alkaloids in Solanaceae plants: involvement of an aldehyde intermediate during C-26 amination.

    Science.gov (United States)

    Ohyama, Kiyoshi; Okawa, Akiko; Moriuchi, Yuka; Fujimoto, Yoshinori

    2013-05-01

    The C-26 amino group of steroidal alkaloids, such as tomatine, is introduced during an early step of their biosynthesis from cholesterol. In the present study, the mechanism of C-26 amination was reinvestigated by administering stable isotope labeled compounds, such as (26,26,26,27,27,27-(2)H6)cholesterol during biosynthesis of tomatine, solanine and solasonine. The chemical compositions of tomatine and solanine so obtained were analyzed by LC-MS after administering the d6-cholesterol to a tomato seedling and a potato shoot, respectively. The resulting spectra indicated that two deuterium atoms were eliminated from C-26 of cholesterol during biosynthesis. Furthermore, administration of (6-(13)C(2)H3)mevalonate in combination with lovastatin to an eggplant seedling, followed by GC-MS analysis of solasodine after TMS derivatization established that two deuterium atoms were eliminated from C-26 of cholesterol during solasonine biosynthesis. These findings are in contrast to an earlier observation that one hydrogen atom was lost from C-26 during tomatidine biosynthesis, and suggest that C-26 nitrogen atom addition involves an aldehyde intermediate. Thus, it is proposed that the C-26 amination reaction that occurs during steroidal alkaloid biosynthesis proceeds by way of a transamination mechanism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

    Science.gov (United States)

    van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

    2012-01-01

    SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

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

    Science.gov (United States)

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

    2013-01-01

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

  2. A new cholesterol biosynthesis and absorption disorder associated with epilepsy, hypogonadism, and cerebro-cerebello-bulbar degeneration.

    Science.gov (United States)

    Korematsu, Seigo; Uchiyama, Shin-ichi; Honda, Akira; Izumi, Tatsuro

    2014-06-01

    Cholesterol is one of the main components of human cell membranes and constitutes an essential substance in the central nervous system, endocrine system, and its hormones, including sex hormones. A 19-year-old male patient presented with failure to thrive, psychomotor deterioration, intractable epilepsy, hypogonadism, and cerebro-cerebello-bulbar degeneration. His serum level of cholesterol was low, ranging from 78.7 to 116.5 mg/dL. The serum concentrations of intermediates in the cholesterol biosynthesis pathway, such as 7-dehydrocholesterol, 8-dehydrocholesterol, desmosterol, lathosterol, and dihydrolanosterol, were not increased. In addition, the levels of the urinary cholesterol biosynthesis marker mevalonic acid, the serum cholesterol absorption markers, campesterol and sitosterol, and the serum cholesterol catabolism marker, 7α-hydroxycholesterol, were all low. A serum biomarker analysis indicated that the patient's basic abnormality differed from that of Smith-Lemli-Opitz syndrome and other known disorders of cholesterol metabolism. Therefore, this individual may have a new metabolic disorder with hypocholesterolemia because of decreased biosynthesis and absorption of cholesterol. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells.

    Science.gov (United States)

    Laureys, F; Dewitte, W; Witters, E; Van Montagu, M; Inzé, D; Van Onckelen, H

    1998-04-10

    The importance of N6-isoprenoid cytokinins in the G2-M transition of Nicotiana tabacum BY-2 cells was investigated. Both cytokinin biosynthesis and entry in mitosis were partially blocked by application at early or late G2 of lovastatin (10 microM), an inhibitor of mevalonic acid synthesis. LC-MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG-CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well-defined cytokinin (here, zeatin) in the G2-M transition of tobacco BY-2 cells.

  4. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    Science.gov (United States)

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

    Directory of Open Access Journals (Sweden)

    Stephen A Brose

    2016-11-01

    Full Text Available Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA biosynthesis (FAS followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FAS maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FAS inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FAS inhibitors, TOFA (inhibits Acetyl-CoA carboxylase and cerulenin (inhibits FA synthase, increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FAS inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

  6. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia.

    Science.gov (United States)

    Brose, Stephen A; Golovko, Svetlana A; Golovko, Mikhail Y

    2016-01-01

    Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

  7. Host-Tree Monoterpenes and Biosynthesis of Aggregation Pheromones in the Bark Beetle Ips paraconfusus

    Directory of Open Access Journals (Sweden)

    John A. Byers

    2012-01-01

    Full Text Available A paradigm developed in the 1970s that Ips bark beetles biosynthesize their aggregation pheromone components ipsenol and ipsdienol by hydroxylating myrcene, a host tree monoterpene. Similarly, host α-pinene was hydroxylated to a third pheromone component cis-verbenol. In 1990, however, we reported that amounts of ipsenol and ipsdienol produced by male Ips paraconfusus (Coleoptera: Scolytinae feeding in five host pine species were nearly the same, even though no detectable myrcene precursor was detected in one of these pines (Pinus sabiniana. Subsequent research showed ipsenol and ipsdienol are also biosynthesized from smaller precursors such as acetate and mevalonate, and this de novo pathway is the major one, while host tree myrcene conversion by the beetle is the minor one. We report concentrations of myrcene, α-pinene and other major monoterpenes in five pine hosts (Pinus ponderosa, P. lambertiana, P. jeffreyi, P. sabiniana, and P. contorta of I. paraconfusus. A scheme for biosynthesis of ipsdienol and ipsenol from myrcene and possible metabolites such as ipsenone is presented. Mass spectra and quantities of ipsenone are reported and its possible role in biosynthesis of aggregation pheromone. Coevolution of bark beetles and host trees is discussed in relation to pheromone biosynthesis, host plant selection/suitability, and plant resistance.

  8. Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis.

    Science.gov (United States)

    Hwang, Hwan-Su; Lee, Hyoshin; Choi, Yong Eui

    2015-03-14

    Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

  9. Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

    Science.gov (United States)

    Agarwal, Aditya Vikram; Singh, Deeksha; Dhar, Yogeshwar Vikram; Michael, Rahul; Gupta, Parul; Chandra, Deepak; Trivedi, Prabodh Kumar

    2018-02-01

    Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

  10. Cyclosporine A suppresses immunoglobulin G biosynthesis via inhibition of cyclophilin B in murine hybridomas and B cells.

    Science.gov (United States)

    Lee, Jisun; Choi, Tae Gyu; Ha, Joohun; Kim, Sung Soo

    2012-01-01

    Immunoglubulin G (IgG) is a major isotype of antibody, which is predominantly involved in immune response. The complete tetramer is needed to fold and assemble in endoplasmic reticulum (ER) prior to secretion from cells. Protein quality control guided by ER chaperons is most essential for full biological activity. Cyclophilin B (CypB) was initially identified as a high-affinity binding protein for the immunosuppressive drug Cyclosporine A (CsA). CsA suppresses organ rejection by halting productions of pro-inflammatory molecules in T cell and abolishes the enzymatic property of CypB that accelerates the folding of proteins by catalysing the isomerization of peptidyl-proline bonds in ER. Here, we reported that CsA significantly inhibited IgG biosynthesis at posttranslational level in antibody secreting cells. Moreover, CsA stimulated the extracellular secretion of CypB and induced ROS generation, leading to expressions of ER stress markers. In addition, the absence of intracellular CypB impaired the formation of ER multiprotein complex, which is most important for resisting ER stress. Interestingly, CsA interrupted IgG folding via occupying the PPIase domain of CypB in ER. Eventually, unfolded IgG is degraded via Herp-dependent ERAD pathway. Furthermore, IgG biosynthesis was really abrogated by inhibition of CypB in primary B cells. We established for the first time the immunosuppressive effect of CsA on B cells. Conclusively, the combined results of the current study suggest that CypB is a pivotal molecule for IgG biosynthesis in ER quality control. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

    NARCIS (Netherlands)

    Sapir, Amir; Tsur, Assaf; Koorman, Thijs; Ching, Kaitlin; Mishra, Prashant; Bardenheier, Annabelle; Podolsky, Lisa; Bening-Abu-Shach, Ulrike; Boxem, Mike; Chou, Tsui-Fen; Broday, Limor; Sternberg, Paul W

    2014-01-01

    Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as

  12. Metabolic plasticity for isoprenoid biosynthesis in bacteria.

    Science.gov (United States)

    Pérez-Gil, Jordi; Rodríguez-Concepción, Manuel

    2013-05-15

    Isoprenoids are a large family of compounds synthesized by all free-living organisms. In most bacteria, the common precursors of all isoprenoids are produced by the MEP (methylerythritol 4-phosphate) pathway. The MEP pathway is absent from archaea, fungi and animals (including humans), which synthesize their isoprenoid precursors using the completely unrelated MVA (mevalonate) pathway. Because the MEP pathway is essential in most bacterial pathogens (as well as in the malaria parasites), it has been proposed as a promising new target for the development of novel anti-infective agents. However, bacteria show a remarkable plasticity for isoprenoid biosynthesis that should be taken into account when targeting this metabolic pathway for the development of new antibiotics. For example, a few bacteria use the MVA pathway instead of the MEP pathway, whereas others possess the two full pathways, and some parasitic strains lack both the MVA and the MEP pathways (probably because they obtain their isoprenoids from host cells). Moreover, alternative enzymes and metabolic intermediates to those of the canonical MVA or MEP pathways exist in some organisms. Recent work has also shown that resistance to a block of the first steps of the MEP pathway can easily be developed because several enzymes unrelated to isoprenoid biosynthesis can produce pathway intermediates upon spontaneous mutations. In the present review, we discuss the major advances in our knowledge of the biochemical toolbox exploited by bacteria to synthesize the universal precursors for their essential isoprenoids.

  13. 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.

    2015-01-01

    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. PMID:26299433

  14. Block of the Mevalonate Pathway Triggers Oxidative and Inflammatory Molecular Mechanisms Modulated by Exogenous Isoprenoid Compounds

    Directory of Open Access Journals (Sweden)

    Paola Maura Tricarico

    2014-04-01

    Full Text Available Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD. One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3, cytokines and nitric oxide (NO]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD.

  15. Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica

    Directory of Open Access Journals (Sweden)

    Cory Schwartz

    2017-11-01

    Full Text Available Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.

  16. Expression of the mevalonate pathway enzymes in the Lutzomyia longipalpis (Diptera: Psychodidae) sex pheromone gland demonstrated by an integrated proteomic approach.

    Science.gov (United States)

    González-Caballero, Natalia; Rodríguez-Vega, Andrés; Dias-Lopes, Geovane; Valenzuela, Jesus G; Ribeiro, Jose M C; Carvalho, Paulo Costa; Valente, Richard H; Brazil, Reginaldo P; Cuervo, Patricia

    2014-01-16

    In Latin America, Lutzomyia longipalpis is the main vector of the protozoan parasite Leishmania infantum, which is the causal agent of American Visceral Leishmaniasis. This insect uses male-produced pheromones for mate recognition. Elucidation of pheromone biogenesis or its regulation may enable molecular strategies for mating disruption and, consequently, the vector's population management. Motivated by our recent results of the transcriptomic characterization of the L. longipalpis pheromone gland, we performed a proteomic analysis of this tissue combining SDS-PAGE, and mass spectrometry followed by an integrative data analysis. Considering that annotated genome sequences of this sand fly are not available, we designed an alternative workflow searching MS/MS data against two customized databases using three search engines: Mascot, OMSSA and ProLuCID. A total of 542 proteins were confidently characterized, 445 of them using a Uniref100-insect protein database, and 97 using a transcript translated database. In addition, use of PEAKS for de novo peptide sequencing of MS/MS data confirmed ~90% identifications made with the combination of the three search engines. Our results include the identification of six of the seven enzymes of the mevalonate-pathway, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. L. longipalpis is the main vector of the protozoan parasite L. infantum, which is the causal agent of American Visceral Leishmaniasis. One of the control measures of such disease is focused on vector population control. As this insect uses male-produced pheromones for mate recognition, the elucidation of pheromone biogenesis or its regulating process may enable molecular strategies for mating disruption and, consequently, this vector's population management. On this regard, in this manuscript we report expression evidence, at the protein level, of several molecules potentially

  17. Metabolomics Analysis of the Toxic Effects of the Production of Lycopene and Its Precursors

    Directory of Open Access Journals (Sweden)

    April M. Miguez

    2018-05-01

    behaviors and may be linked to the growth inhibition seen when the mevalonate pathway is induced overnight, suggesting potential future work that may be useful in engineering increased lycopene biosynthesis.

  18. Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes.

    Science.gov (United States)

    Hashimoto, F; Taira, S; Hayashi, H

    2000-05-15

    We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

    The effects of β-chloroalanine (β-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 β-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 β-Cl-alanine and this treatment inhibited [ 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 β-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [ 14 C]serine uptake was not blocked, total lipid biosynthesis from [ 14 C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [ 3 H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [ 3 H]leucine, which was only decreased by 14%. Although β-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 β-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis

  20. The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula.

    Science.gov (United States)

    Mertens, Jan; Pollier, Jacob; Vanden Bossche, Robin; Lopez-Vidriero, Irene; Franco-Zorrilla, José Manuel; Goossens, Alain

    2016-01-01

    Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula. © 2016 American Society of Plant Biologists. All Rights Reserved.

  1. The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis.

    Science.gov (United States)

    Ashikhmin, Aleksandr; Makhneva, Zoya; Moskalenko, Andrey

    2014-03-01

    The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (≥99 %) at 71 μM DPA. Considering "empty" Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 μM DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 μM DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect. haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 μM DPA (as traces) and at 71 μM DPA. (3) DPA may affect both phytoene synthase (at ≤71 μM DPA) and phytoene desaturase (at ≥53.25 μM DPA). (4) The assembly of LH2 complex does occur without any colored Cars.

  2. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  3. Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, H.; Ohki, A.; Sato, Y.; Wakabayashi, K. [Tamagawa Univ., Tokyo (Japan). Graduate School of Agricultural Science; Kanzaki, M. [Regulatory Affairs Dept., Chugai Pharmaceutical Co. Ltd., Tokyo (Japan); Tanaka, A. [Showa Univ., Tokyo (Japan). School of Pharmaceutical Sciences; Matthes, B.; Boeger, P. [Konstanz Univ. (Germany). Lehrstuhl fuer Physiologie und Biochemie der Pflanzen

    2001-10-01

    The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1-{sup 14}C]-oleate and [2-{sup 14}C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C{sub 18}. For a strong VLCFA biosynthesis inhibition an -SO{sub 2}- linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation. (orig.)

  4. Very-long-chain fatty acid biosynthesis is inhibited by cafenstrole, N,N-diethyl-3-mesitylsulfonyl-1H-1,2,4-triazole-1-carboxamide and its analogs

    International Nuclear Information System (INIS)

    Takahashi, H.; Ohki, A.; Sato, Y.; Wakabayashi, K.; Tanaka, A.; Matthes, B.; Boeger, P.

    2001-01-01

    The rice herbicide cafenstrole and its analogs inhibited the incorporation of [1- 14 C]-oleate and [2- 14 C]-malonate into very-long-chain fatty acids (VLCFAs), using Scenedesmus cells and leek microsomes from Allium porrum. Although the precise mode of interaction of cafenstrole at the molecular level is not completely clarified by the present study, it is concluded that cafenstrole acts as a specific inhibitor of the microsomal elongase enzyme involved in the biosynthesis of fatty acids with alkyl chains longer than C 18 . For a strong VLCFA biosynthesis inhibition an -SO 2 - linkage of the 1,2,4-triazole-1-carboxamides was required. Furthermore, N,N-dialkyl substitution of the carbamoyl nitrogen and electron-donating groups such as methyl at the benzene ring of 1,2,4-triazole-1-carboxamides produced a strong inhibition of VLCFA formation. A correlation was found between the phytotoxic effect against barnyardgrass (Echinochloa oryzicola) and impaired VLCFA formation. (orig.)

  5. Inhibitory effect of luteolin on estrogen biosynthesis in human ovarian granulosa cells by suppression of aromatase (CYP19).

    Science.gov (United States)

    Lu, Dan-feng; Yang, Li-juan; Wang, Fei; Zhang, Guo-lin

    2012-08-29

    Inhibition of aromatase, the key enzyme in estrogen biosynthesis, is an important strategy in the treatment of breast cancer. Several dietary flavonoids show aromatase inhibitory activity, but their tissue specificity and mechanism remain unclear. This study found that the dietary flavonoid luteolin potently inhibited estrogen biosynthesis in a dose- and time-dependent manner in KGN cells derived from human ovarian granulosa cells, the major source of estrogens in premenopausal women. Luteolin decreased aromatase mRNA and protein expression in KGN cells. Luteolin also promoted aromatase protein degradation and inhibited estrogen biosynthesis in aromatase-expressing HEK293A cells, but had no effect on recombinant expressed aromatase. Estrogen biosynthesis in KGN cells was inhibited with differing potencies by extracts of onion and bird chili and by four other dietary flavonoids: kaempferol, quercetin, myricetin, and isorhamnetin. The present study suggests that luteolin inhibits estrogen biosynthesis by decreasing aromatase expression and destabilizing aromatase protein, and it warrants further investigation as a potential treatment for estrogen-dependent cancers.

  6. Isoprenoid biosynthesis and mevalonate kinase deficiency

    NARCIS (Netherlands)

    Henneman, L.

    2011-01-01

    Mevalonaat Kinase Deficiëntie (MKD) is een aangeboren ziekte geassocieerd met heftige koortsaanvallen die drie tot vier dagen aanhouden en gepaard gaan met koude rillingen, gewrichtsklachten, huiduitslag, hoofdpijn, duizeligheid, buikpijn, braken en diarree. De koortsaanvallen treden gemiddeld eens

  7. Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency

    Directory of Open Access Journals (Sweden)

    Paola Maura Tricarico

    2017-03-01

    Full Text Available Background/Aims: Mevalonate Kinase Deficiency (MKD, is a hereditary disease due to mutations in mevalonate kinase gene (MVK. MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA, the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. Methods: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. Results: MVK mutants’ over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. Conclusions: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

  8. Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

    Science.gov (United States)

    Tricarico, Paola Maura; Romeo, Alessandra; Gratton, Rossella; Crovella, Sergio; Celsi, Fulvio

    2017-01-01

    Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation. © 2017 The Author(s)Published by S. Karger AG, Basel.

  9. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    Science.gov (United States)

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  10. Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

    DEFF Research Database (Denmark)

    Mendez-Perez, Daniel; Alonso-Gutierrez, Jorge; Hu, Qijun

    2017-01-01

    ). FPP biosynthesis diverts the carbon flux from monoterpene production to C15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate...

  11. Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis.

    Science.gov (United States)

    Tomita, Hiroya; Imanaka, Tadayuki; Atomi, Haruyuki

    2013-10-01

    Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20-29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea. © 2013 John Wiley & Sons Ltd.

  12. P53- and mevalonate pathway–driven malignancies require Arf6 for metastasis and drug resistance

    Science.gov (United States)

    Hashimoto, Ari; Oikawa, Tsukasa; Hashimoto, Shigeru; Sugino, Hirokazu; Yoshikawa, Ayumu; Otsuka, Yutaro; Handa, Haruka; Onodera, Yasuhito; Nam, Jin-Min; Oneyama, Chitose; Okada, Masato; Fukuda, Mitsunori

    2016-01-01

    Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy. PMID:27044891

  13. Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

    Science.gov (United States)

    Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

    2017-06-01

    The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. © 2017 Scandinavian Plant Physiology Society.

  14. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by β-Cl-alanine

    International Nuclear Information System (INIS)

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

    1987-01-01

    Serine palmitoyltransferase (SPT) is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the first committed step of long-chain base (LCB) synthesis. Inhibition of SPT activity and de novo biosynthesis of sphinganine and sphingosine was observed in vitro and in intact Chinese hamster ovary cells (CHO). In vitro studies revealed that inhibition was irreversible and concentration- and time-dependent, which are characteristics of suicide inhibition. Incubation of intact CHO cells with 5 mM β-Cl-alanine for 15 min completely inhibited SPT activity and LCB synthesis from [ 14 C]serine. The concentration dependences of inhibition of SPT activity and LCB formation were identical. There was no loss of viability of recovery of SPT activity over the 2 hour time course of these experiments. The synthesis of several other lipids was not affected by the same treatment. These results establish the association between the activity of SPT and the cellular rate of LCB formation and indicate that β-Cl-alanine can be used to study alterations in cellular LCB synthesis

  15. Inhibition of aflatoxin biosynthesis in Aspergillus flavus by phenolic compounds extracted of Piper betle L.

    Science.gov (United States)

    Yazdani, Darab; Mior Ahmad, Zainal Abidin; Yee How, Tan; Jaganath, Indu Bala; Shahnazi, Sahar

    2013-12-01

    Food contamination by aflatoxins is an important food safety concern for agricultural products. In order to identify and develop novel antifungal agents, several plant extracts and isolated compounds have been evaluated for their bioactivities. Anti-infectious activity of Piper betle used in traditional medicine of Malaysia has been reported previously. Crude methanol extract from P. betel powdered leaves was partitioned between chloroform and water. The fractions were tested against A. flavus UPMC 89, a strong aflatoxin producing strain. Inhibition of mycelial growth and aflatoxin biosynthesis were tested by disk diffusion and macrodillution techniques, respectively. The presence of aflatoxin was determined by thin-layer chromatography (TLC) and fluorescence spectroscopy techniques using AFB1 standard. The chloroform soluble compounds were identified using HPLC-Tandem mass spectrometry technique. The results, evaluated by measuring the mycelial growth and quantification of aflatoxin B1(AFLB1) production in broth medium revealed that chloroform soluble compounds extract from P. betle dried leaves was able to block the aflatoxin biosynthesis pathway at concentration of 500μg/ml without a significant effect on mycelium growth. In analyzing of this effective fractions using HPLC-MS(2) with ESI ionization technique, 11 phenolic compounds were identified. The results showed that the certain phenolic compounds are able to decline the aflatoxin production in A. flavus with no significant effect on the fungus mycelia growth. The result also suggested P. betle could be used as potential antitoxin product.

  16. Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis.

    Science.gov (United States)

    Matsushima, K; Chang, P K; Yu, J; Abe, K; Bhatnagar, D; Cleveland, T E

    2001-05-01

    The aflR gene product is the main transcriptional regulator of aflatoxin biosynthesis in Aspergillus parasiticus and Aspergillus flavus. Although A. sojae strains do not produce aflatoxins, they do have an aflR homologue. When compared with the aflR of A. parasiticus, the A. sojae gene contains two mutations: an HAHA motif and a premature stop codon. To investigate the functionality of the A. sojae aflR gene product, we used a GAL4 one-hybrid system in yeast. The transcription-activating activity of AflR from A. sojae was 15% of that from A. parasiticus. The introduction of an additional aflR from A. sojae into an A. parasiticus strain did not affect aflatoxin productivity. A hybrid aflR comprising the amino-terminal region of A. sojae aflR and the carboxy-terminal region of A. parasiticus aflR suppressed the effect associated with pre-termination of the A. sojae AflR. We conclude that the premature stop codon of the A. sojae aflR is the key to its functionality and leads to prevention of aflatoxin biosynthesis through loss of the transcription of aflatoxin biosynthesis-related genes.

  17. Arginine Deprivation Inhibits the Warburg Effect and Upregulates Glutamine Anaplerosis and Serine Biosynthesis in ASS1-Deficient Cancers

    Directory of Open Access Journals (Sweden)

    Jeff Charles Kremer

    2017-01-01

    Full Text Available Targeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1 is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

  18. 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

  19. Isoprenoid biosynthesis in eukaryotic phototrophs: A spotlight on algae

    Energy Technology Data Exchange (ETDEWEB)

    Lohr M.; Schwender J.; Polle, J. E. W.

    2012-04-01

    Isoprenoids are one of the largest groups of natural compounds and have a variety of important functions in the primary metabolism of land plants and algae. In recent years, our understanding of the numerous facets of isoprenoid metabolism in land plants has been rapidly increasing, while knowledge on the metabolic network of isoprenoids in algae still lags behind. Here, current views on the biochemistry and genetics of the core isoprenoid metabolism in land plants and in the major algal phyla are compared and some of the most pressing open questions are highlighted. Based on the different evolutionary histories of the various groups of eukaryotic phototrophs, we discuss the distribution and regulation of the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in land plants and algae and the potential consequences of the loss of the MVA pathway in groups such as the green algae. For the prenyltransferases, serving as gatekeepers to the various branches of terpenoid biosynthesis in land plants and algae, we explore the minimal inventory necessary for the formation of primary isoprenoids and present a preliminary analysis of their occurrence and phylogeny in algae with primary and secondary plastids. The review concludes with some perspectives on genetic engineering of the isoprenoid metabolism in algae.

  20. Priming by Hexanoic acid induce activation of mevalonic and linolenic pathways and promotes the emission of plant volatiles.

    Directory of Open Access Journals (Sweden)

    Eugenio eLlorens

    2016-04-01

    Full Text Available Hexanoic acid is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of hexanoic acid in response to the challenge pathogen Alternaria alternata, focusing on the response of the plant. Moreover, we used 13C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of 13C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than two hundred molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by hexanoic acid. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of hexanoic acid this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application.

  1. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    Science.gov (United States)

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-01-01

    Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

  2. Alteration of Mevalonate Pathway in Rat Splenic Lymphocytes: Possible Role in Cytokines Secretion Regulated by L-Theanine

    Directory of Open Access Journals (Sweden)

    Chengjian Li

    2018-01-01

    Full Text Available L-Theanine is a nonprotein amino acid in tea, and its immunomodulatory function has been confirmed. This study aimed to investigate the effect of L-theanine addition on cytokines secretion in rat splenic lymphocytes and explore its potential immunomodulatory effects on the mevalonate biosynthetic pathway. Our results showed that L-theanine treatment did not influence the proliferation and division indexes of the splenic lymphocytes subsets. Interestingly, L-theanine treatment had regulated the contents of IFN-γ, IL-2, IL-4, IL-10, IL-12, and TNF-α  (P<0.001 except IL-6 and upregulated the mRNA and protein expression of Ras-related protein Rap-1A (Rap1A, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR, and farnesyl diphosphate synthase (FDPs (P<0.001. Additionally, there was a positive correlation between Rap1A and HMGCR proteins expression and IFN-γ, IL-4, and IL-6 levels. In conclusion, L-theanine regulated the secretion of cytokines probably by activating expression of Rap1A and HMGCR proteins involved in the mevalonate biosynthetic pathway in rat splenic lymphocytes. Therefore, L-theanine might be a promising potential drug candidate as immunopotentiator.

  3. Phosphatidylcholine (PC) biosynthesis in pancreatic islets of Langerhans

    International Nuclear Information System (INIS)

    Hoffman, J.M.; Laychock, S.G.

    1986-01-01

    Islets of Langerhans isolated from rat pancreata were incubated with [ 14 C]choline to determine the biosynthesis of PC by the CDP choline to determine the biosynthesis of PC by the CDPcholine pathway. Recovery of [ 14 C]PC in islet membranes was time-related, and stimulated by glucose (17mM) during 60 min. The rate of PC synthesis was constant during 60 min with glucose stimulation. In contrast, the sulfonylurea tolbutamide (2 mM) reduced the recovery of [ 14 C]choline in PC, and 8-bromo-cyclic AMP (5 mM) did not significantly affect [ 14 C]PC recovery. Incubation of islets in Ca 2+ -free medium enhanced glucose-stimulated recovery of [ 14 C]choline-labeled PC due to the inhibition of phospholipase and phospholipid hydrolysis. Inhibition of CTP:phosphocholine cytidylyltransferase with 5-deoxy-5'-isobutylthioadenosine (SIBA) reduced [ 14 C]PC levels and insulin release in a concentration dependent manner. Treatment with SIBA also reduced Mg 2+ -dependent Ca 2+ -ATPase activity in islet microsomes. Quantitation of membrane PC showed that glucose stimulation did not alter islet P levels. Thus, islet PC biosynthesis is linked to glucose stimulation and contributes to the maintenance of PC levels in membranes undergoing exocytosis and phospholipid hydrolysis. Adequate PC levels support Ca 2+ pump activity and secretory mechanisms

  4. High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

    DEFF Research Database (Denmark)

    Kang, Aram; Meadows, Corey W.; Canu, Nicolas

    2017-01-01

    Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high...... ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass� isopentenol pathway using the promiscuous activity...

  5. Purine biosynthesis in L1210 leukemia cells is inhibited by 7-hydroxymethotrexate (7-OH-MTX) polyglutamates (PGS)

    International Nuclear Information System (INIS)

    Seither, R.L.; Matherly, L.H.; Goldman, I.D.

    1986-01-01

    The biochemical basis for 7-OH-MTX cytotoxicity was examined in L1210 tumor cells. Cells were exposed to 100 μM 7-OH-MTX (approx. 50% growth inhibition) or 10 μM methotrexate (MTX) (approx. 95% growth inhibition) for 6 hrs to allow high levels of PGS to accumulate. Dihydrofolate reductase (DHFR) activity was assessed by dihydrofolate (FH 2 ) pools labeled with 5-formyl-[ 3 H]-tetrahydrofolate (5μM) or 3 H-folic acid (1 μM). FH 2 was not elevated above control levels in 7-OH-MTX treated cells, in contrast to MTX treated cells in which FH 2 increased 4- to 7-fold. 3 H-Deoxyuridine incorporation into DNA was not inhibited in cells containing high levels (11.5 nmol/g dry wt.) of 7-OH-MTX tetraglutamate (7-OH-4-NH 2 -10-CH 3 -PteGlu 4 ), well in excess of the DHFR-binding capacity (7.3 +/- 0.9 nmol/g), indicating a normal rate of thymidylate synthesis. Although small amounts of 7-OH-MTX and its PGS were bound to DHFR in L1210 cells, as assessed by gel filtration, there was evidence for the preferential binding of 7-OH-MTX tetraglutamate. In all cases this was well below the DHFR binding capacity, consistent with normal rates of deoxyuridine metabolism and FH 2 levels in the cell. Incorporation of 14 C-formate (60 min) into thymidylate and amino acids was unaffected by 7-OH-MTX, yet incorporation into purines was inhibited over 50%, supporting a block(s) in de novo purine biosynthesis

  6. Inhibitors of amino acids biosynthesis as antifungal agents.

    Science.gov (United States)

    Jastrzębowska, Kamila; Gabriel, Iwona

    2015-02-01

    Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

  7. Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

    Directory of Open Access Journals (Sweden)

    Yang Xing

    2009-07-01

    Full Text Available Abstract Background 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary. Methods Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement. Results In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly. Conclusion The present study proves for the first time that ovarian androgen

  8. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.

    Science.gov (United States)

    Yang, Chen; Gao, Xiang; Jiang, Yu; Sun, Bingbing; Gao, Fang; Yang, Sheng

    2016-09-01

    Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The (13)C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  9. Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Takla Griss

    2015-12-01

    Full Text Available Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC. However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity.

  10. Inhibition of ethylene production by putrescine alleviates aluminium-induced root inhibition in wheat plants.

    Science.gov (United States)

    Yu, Yan; Jin, Chongwei; Sun, Chengliang; Wang, Jinghong; Ye, Yiquan; Zhou, Weiwei; Lu, Lingli; Lin, Xianyong

    2016-01-08

    Inhibition of root elongation is one of the most distinct symptoms of aluminium (Al) toxicity. Although putrescine (Put) has been identified as an important signaling molecule involved in Al tolerance, it is yet unknown how Put mitigates Al-induced root inhibition. Here, the possible mechanism was investigated by using two wheat genotypes differing in Al resistance: Al-tolerant Xi Aimai-1 and Al-sensitive Yangmai-5. Aluminium caused more root inhibition in Yangmai-5 and increased ethylene production at the root apices compared to Xi Aimai-1, whereas the effects were significantly reversed by ethylene biosynthesis inhibitors. The simultaneous exposure of wheat seedlings to Al and ethylene donor, ethephon, or ethylene biosynthesis precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), increased ethylene production and aggravated root inhibition, which was more pronounced in Xi Aimai-1. In contrast, Put treatment decreased ethylene production and alleviated Al-induced root inhibition in both genotypes, and the effects were more conspicuous in Yangmai-5. Furthermore, our results indicated that Al-induced ethylene production was mediated by ACC synthase (ACS) and ACC oxidase, and that Put decreased ethylene production by inhibiting ACS. Altogether, these findings indicate that ethylene is involved in Al-induced root inhibition and this process could be alleviated by Put through inhibiting ACS activity.

  11. [MVK gene abnormality and new approach to treatment of hyper IgD syndrome and periodic fever syndrome].

    Science.gov (United States)

    Naruto, Takuya

    2007-04-01

    Hyper IgD and periodic fever syndrome (HIDS; OMIM 260920) is one of the hereditary autoinflammatory syndromes characterized by recurrent episodes of fever and inflammation.. HIDS is an autosomal recessive disorder characterized by recurrent fever attacks in early childhood. HIDS caused by mevalonate kinase (MK) mutations, also that is the gene of mevalonic aciduria (OMIM 251170). During febrile episodes, urinary mevalonate concentrations were found to be significantly elevated in patients. Diagnosis of HIDS was retrieving gene or measurement of the enzyme activity in peripheral blood lymphocyte in general. This of HIDS is an activity decline of MK, and a complete deficiency of MK becomes a mevalonic aciduria with a nervous symptom. The relation between the fever and inflammation of mevalonate or isoprenoid products are uncertain. The therapy attempt with statins, which is inhibited the next enzyme after HMG-CoA reductase, or inhibit the proinflammatory cytokines.

  12. Zincophorin – biosynthesis in Streptomyces griseus and antibiotic properties

    Directory of Open Access Journals (Sweden)

    Walther, Elisabeth

    2016-11-01

    Full Text Available Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium . The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.

  13. Purine biosynthesis de novo by lymphocytes in gout

    International Nuclear Information System (INIS)

    Kamoun, P.; Chanard, J.; Brami, M.; Funck-Brentano, J.L.

    1978-01-01

    A method of measurement in vitro of purine biosynthesis de novo in human circulating blood lymphocytes is proposed. The rate of early reactions of purine biosynthesis de novo was determined by the incorporation of [ 14 C]formate into N-formyl glycinamide ribonucleotide when the subsequent reactions of the metabolic pathway were completely inhibited by the antibiotic azaserine. Synthesis of 14 C-labelled N-formyl glycinamide ribonucleotide by lymphocytes was measured in healthy control subjects and patients with primary gout or hyperuricaemia secondary to renal failure, with or without allopurinol therapy. The average synthesis was higher in gouty patients without therapy than in control subjects, but the values contained overlap the normal range. In secondary hyperuricaemia the synthesis was at same value as in control subjects. These results are in agreement with the inconstant acceleration of purine biosynthesis de novo in gouty patients as seen by others with measurement of [ 14 C]glycine incorporation into urinary uric acid. (author)

  14. Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides

    Directory of Open Access Journals (Sweden)

    Jean-Luc eDo-Rego

    2012-01-01

    Full Text Available The enzymatic pathways leading to the synthesis of bioactive steroids in the brain are now almost completely elucidated in various groups of vertebrates and, during the last decade, the neuronal mechanisms involved in the regulation of neurosteroid production have received increasing attention. This report reviews the current knowledge concerning the effects of neurotransmitters, peptide hormones and neuropeptides on the biosynthesis of neurosteroids. Anatomical studies have been carried out to visualize the neurotransmitter- or neuropeptide-containing fibers contacting steroid-synthesizing neurons as well as the neurotransmitter, peptide hormones or neuropeptide receptors expressed in these neurons. Biochemical experiments have been conducted to investigate the effects of neurotransmitters, peptide hormones or neuropeptides on neurosteroid biosynthesis, and to characterize the type of receptors involved. Thus, it has been found that glutamate, acting through kainate and/or AMPA receptors, rapidly inactivates P450arom, and that melatonin produced by the pineal gland and eye inhibits the biosynthesis of 7-hydroxypregnenolone (7-OH-5P, while prolactin produced by the adenohypophysis enhances the formation of 7-OH-5P. It has also been demonstrated that the biosynthesis of neurosteroids is inhibited by GABA, acting through GABAA receptors, and neuropeptide Y, acting through Y1 receptors. In contrast, it has been shown that the octadecaneuropetide ODN, acting through central-type benzodiazepine receptors, the triakontatetraneuropeptide TTN, acting though peripheral-type benzodiazepine receptors, and vasotocine, acting through V1a-like receptors, stimulate the production of neurosteroids. Since neurosteroids are implicated in the control of various neurophysiological and behavioral processes, these data suggest that some of the neurophysiological effects exerted by neurotransmitters and neuropeptides may be mediated via the regulation

  15. Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation1

    Science.gov (United States)

    Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-01-01

    Germination and thermoinhibition in lettuce (Lactuca sativa ‘Jianyexianfeng No. 1’) seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  16. Effects of a Squalene Epoxidase Inhibitor, Terbinafine, on Ether Lipid Biosyntheses in a Thermoacidophilic Archaeon, Thermoplasma acidophilum

    Science.gov (United States)

    Kon, Takahide; Nemoto, Naoki; Oshima, Tairo; Yamagishi, Akihiko

    2002-01-01

    The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-14C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum. PMID:11844769

  17. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    Grä wert, Tobias; Rohdich, Felix; Span, Lngrid; Backer, Adelbert; Eisenreich, Wolfgang; Eppinger, Jö rg; Groll, Michael

    2009-01-01

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue

  18. Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

    Science.gov (United States)

    Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

    2017-11-18

    Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kirby, James; Dietzel, Kevin L.; Wichmann, Gale

    2016-01-01

    Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP......) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP...... time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway....

  20. Utilization of biodiesel by-product as substrate for high-production of β-farnesene via relatively balanced mevalonate pathway in Escherichia coli.

    Science.gov (United States)

    You, Shengping; Yin, Qingdian; Zhang, Jianye; Zhang, Chengyu; Qi, Wei; Gao, Lan; Tao, Zhiping; Su, Rongxin; He, Zhimin

    2017-11-01

    Farnesene has been identified as suitable jet fuel substitutes and metabolic engineering for microbial production of farnesene is an alternative and attractive route. In this study, due to accumulation of toxic intermediate isopentenyl pyrophosphate (IPP), an engineered Escherichia coli strain harboring heterologous mevalonate pathway produced only 4.11mg/L β-farnesene. Through higher-level expression of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase to minimize the accumulated IPP, another engineered strain with relatively balanced mevalonate pathway was constructed and had the highest production of β-farnesene to date (8.74g/L) by Escherichia coli in a lab bioreactor. Furthermore, this is the first report on utilization of biodiesel by-product (simple purification) as substrate for high-production of β-farnesene by the engineered strain optimized and β-farnesene concentration reached 2.83g/L in a lab bioreactor. Therefore, the engineered strain optimized could be used as a platform host for high-production of other terpenoids using biodiesel by-product as substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Nonorthologous gene displacement of phosphomevalonate kinase

    NARCIS (Netherlands)

    Houten, S. M.; Waterham, H. R.

    2001-01-01

    Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes the phosphorylation of 5-phosphomevalonate into 5-diphosphomevalonate, an essential step in isoprenoid biosynthesis via the mevalonate pathway. So far, two nonorthologous genes encoding PMK have been described, the Saccharomyces cerevisiae ERG8

  2. Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

    Science.gov (United States)

    Creelman, R A; Bell, E; Mullet, J E

    1992-07-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

  3. Biosynthesis of NAD from nicotinic acid and nicotinamide by resting cells of Arthrobacter globiformis

    International Nuclear Information System (INIS)

    Kuwahara, Masaaki

    1978-01-01

    Isotopically labeled nicotinic acid and nicotinamide were incorporated into the metabolites of nicotinic acid-dependent pathway (Preiss-Handler pathway) of the NAD biosynthesis by resting cells of Arthrobacter globiformis. Azaserine and adenosine markedly stimulated the accumulation of NAD in the cells. Radioactive nicotinic acid and nicotinamide were also incorporated into an unknown compound when the cells were incubated in the presence of azaserine. Cell-free extract of the organism showed the NAD synthetase activity, which required ammonium ion and ATP for the amidation of deamido-NAD. Adenosine inhibited the enzyme activity. The organism possessed nicotinamidase, suggesting deamidation is the first step in the biosynthesis of NAD from nicotinamide. The activity was inhibited by NAD, NADP and NMN. (auth.)

  4. Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

    Science.gov (United States)

    Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

    2016-10-01

    Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis

    Directory of Open Access Journals (Sweden)

    Giaever Guri

    2009-01-01

    Full Text Available Abstract Background Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.

  6. Electron microscopy of hydrocarbon production in parthenium argentatum (guayule)

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, Thomas E. [Univ. of California, Berkeley, CA (United States)

    1977-11-01

    The electron microscope was used to study the biological processes involved in hydrocarbon production. The little desert shrub Guayule (Parthenium argentatum) was selected for study. This shrub can produce hydrocarbons (rubber) in concentrations up to 1/4 of its dry weight. It grows on semi-arid land and has been extensively studied. The potential of Guayule is described in detail. Results of an investigation into the morphology of Guayule at the electron microscope level are given. Experiments, which would allow the biosynthesis of hydrocarbon in Guayule to be followed, were designed. In order to do this, knowledge of the biochemistry of rubber formation was used to select a tracer, mevalonic acid. Mevalonic acid is the precursor of all the terpenoids, a large class of hydrocarbons which includes rubber. It was found that when high enough concentrations of mevalonic acid are administered to seedling Guayule plants, build-ups of metabolized products are found within the chloroplasts of the seedlings. Also, tritium labeled mevalonic acid was used as a precursor, and its metabolic progress was followed by using the technique of electron microscope autoradiography. The results of these experiments also implicated chloroplasts of the Guayule plant in hydrocarbon production. The final task was the development of a system to produce three-dimensional stereo reconstructions of organelles suspected of involvement in hydrocarbon biosynthesis in Guayule. The techniques are designed to reconstruct an object from serial sections of that object. The techniques use stereo imaging both to abstract information for computer processing, and also in the computer produced reconstruction.

  7. Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and Aspergillus fumigatus Growth.

    Science.gov (United States)

    Martín Del Campo, Julia S; Vogelaar, Nancy; Tolani, Karishma; Kizjakina, Karina; Harich, Kim; Sobrado, Pablo

    2016-11-18

    Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N 5 -l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosynthesis through SidA inhibiton.

  8. Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

    Science.gov (United States)

    Pisithkul, Tippapha; Jacobson, Tyler B; O'Brien, Thomas J; Stevenson, David M; Amador-Noguez, Daniel

    2015-09-01

    An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. Copyright © 2015, Pisithkul et al.

  9. Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

    Science.gov (United States)

    Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; Stevenson, David M.

    2015-01-01

    An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

  10. Probenazole treatment inhibits anthocyanins biosynthesis via ...

    African Journals Online (AJOL)

    It has been found that anthocyanins were accumulated in Arabidopsis under drought or salt stress. In this study, such accumulation was found to be inhibited by external applied probenazole (3-allyloxy-1, 2-benzisothiazole-1,1-dioxide, PBZ), which is the active ingredient in oryzemate used for the protection of rice from ...

  11. Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic β-Cells

    Directory of Open Access Journals (Sweden)

    Khadija G. Syeda

    2017-10-01

    Full Text Available Background/Aims: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin B undergo posttranslational modifications (e.g., isoprenylation at their C-terminal cysteine residues. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Using human islets, rodent islets, and INS-1 832/13 cells, we recently reported significant metabolic defects under glucotoxic and endoplasmic reticulum (ER stress conditions, including caspase 3 activation and lamin B degradation. The current study is aimed at further understanding the regulatory roles of protein prenylation in the induction of the aforestated metabolic defects. Methods: Subcellular phase partitioning assay was done using Triton X-114. Cell morphology and metabolic cell viability assays were carried out using standard methodologies. Results: We report that exposure of pancreatic β-cells to Simvastatin, an inhibitor of mevalonic acid (MVA biosynthesis, and its downstream isoprenoid derivatives, or FTI-277, an inhibitor of farnesyltransferase that mediates farnesylation of lamins, leads to activation of caspase 3 and lamin B degradation. Furthermore, Simvastatin-treatment increased activation of p38MAPK (a stress kinase and inhibited ERK1/2 (regulator of cell proliferation. Inhibition of farnesylation also resulted in the release of degraded lamin B into the cytosolic fraction and promoted loss in metabolic cell viability. Conclusion: Based on these findings we conclude that protein prenylation plays key roles in islet β-cell function. These findings affirm further support to the hypothesis that defects in prenylation pathway induce caspase-3 activation and nuclear lamin degradation in pancreatic β-cells under the duress of metabolic stress (e.g., glucotoxicity.

  12. Polyamine biosynthesis during germination of yeast ascospores.

    Science.gov (United States)

    Brawley, J V; Ferro, A J

    1979-01-01

    The role of the diamine putrescine during germination and outgrowth of ascospores of Saccharomyces cerevisiae was examined. Ornithine decarboxylase activity increased and declined rapidly during germination and outgrowth; peak activity was attained after the cells had proceeded through the G1 interval of the cell cycle, whereas minimal activity was present at the completion of the first cell division. alpha-Methylornithine inhibited both ornithine decarboxylase activity and the in vivo accumulation of putrescine. In the presence of alpha-methylornithireak dormancy and proceed through one cell division. Subsequent cellular growth, however, was retarded but not completely inhibited. The supplementation of Methylglyoxal bis(guanylhydrazone) to sporulation medium greatly inhibited this sexual process. These data suggest that the synthesis of putrescine is not required for the breaking of spore dormancy, but that polyamine biosynthesis may be essential for meiosis and sporulation. PMID:387744

  13. Involvement of a Lipoxygenase-Like Enzyme in Abscisic Acid Biosynthesis 1

    Science.gov (United States)

    Creelman, Robert A.; Bell, Erin; Mullet, John E.

    1992-01-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. 18O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID:16668998

  14. Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

    Directory of Open Access Journals (Sweden)

    Adelene Ai-Lian Song

    Full Text Available Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus. A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain's endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR, an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

  15. Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells.

    OpenAIRE

    Seppänen, P; Fagerström, R; Alhonen-Hongisto, L; Elo, H; Lumme, P; Jänne, J

    1984-01-01

    Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diami...

  16. Synthesis of the mevalonic acid labelled with "1"4C, "1"3C and "3H

    International Nuclear Information System (INIS)

    Rousseau, Bernard

    1982-01-01

    This thesis describes five new methods of synthesis of the (R,S) mevalonic acid adapted to the labelling with "1"4C and "1"3C in positions 4,5 or 5 or 3', or with tritium in position 3'. Three of them use the tri-oxa-2,4,10 adamantyl group as masked carboxyl function. The two others take benefit from the regioselectivity of the bis-hydro-boration of terminal acetylenics by the 9-borabicyclo [3-3-1]nonane. The acylation of the bis-trimethylsilyl lithiomalonate, and the chemistry of dithiannes are also involved. Acetylene and methyl iodide labelled with isotopes are used as cheap base products [fr

  17. Inhibitory effect of isoprenoid-substituted flavonoids isolated from Artocarpus heterophyllus on melanin biosynthesis.

    Science.gov (United States)

    Arung, Enos Tangke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2006-07-01

    Isoprenoid-substituted flavonoids were isolated from the wood of Artocarpus heterophyllus by means of activity-guided fractionation. Artocarpin (1), cudraflavone C (2), 6-prenylapigenin (3), kuwanon C (4), norartocarpin (5) and albanin A (6) inhibited melanin biosynthesis in B16 melanoma cells without inhibiting tyrosinase. A structure-activity investigation indicated that the presence of the isoprenoid-substituted moiety enhanced the inhibitory activity on melanin production in B16 melanoma cells.

  18. 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.

  19. Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Seker, Tamay; Møller, Kasper; Nielsen, Jens

    2005-01-01

    The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl Co......A, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we...... used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase...

  20. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

    International Nuclear Information System (INIS)

    Johnson, Timothy E.; Zhang, Xiaohua; Bleicher, Kimberly B.; Dysart, Gary; Loughlin, Amy F.; Schaefer, William H.; Umbenhauer, Diane R.

    2004-01-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  2. An insight into structural and functional characteristics of 3-hydoxy 3-methyl glutarylCoA reductase from Ocimum species

    Directory of Open Access Journals (Sweden)

    Shilpi Bansal

    2017-10-01

    Full Text Available Secondary metabolites, the biological compounds secreted by plants as an aid to support their growth and development under stress conditions or as a part of their defense mechanism, now hold equal importance for mankind who employs it immensely for medication, flavorings, aroma, etc. Wide applicability of these compounds instigates one to understand the biosynthesis, structure and regulation of these bioactive molecules. Terpenoids form the largest group of secondary metabolites which comprise of a wide range of structurally and functionally distinct metabolites synthesized either via mevalonate pathway or non-mevalonate pathway. Targeting a key regulatory enzyme of this pathway, modulation of which would alter the carbon flux would be beneficial to enhance our knowledge about the above issue. For this the transcriptome (from SRA of different Ocimum species was mined out for important pathway genes using various bioinformatics approaches. Amongst them 3-hydoxy 3-methyl glutaryl CoA reductase (HMGR was selected which is the rate limiting enzyme in mevalonate pathway which controls the conversion of HMG-CoA to mevalonic acid. Isolation, cloning, protein expression, purification, etc. would be discussed in detail in the meeting. Full length protein was also characterized through bioinformatics tools to study its structure, properties, conserved domains, etc. Increase in secondary metabolite production by alteration of HMGR pool along with transcript modulation studies in planta revealed that HMGR gene governs the biosynthesis of secondary metabolites. Transcriptome mapping of different HMGR homologs which on comparison within member of same genus revealed its divergent nature which could account to its multifunctional role in different plants. Besides, providing a deep insight about the enzyme function combination of such molecular, transgenic and bioinformatics tools would help to develop strategies to engineer the HMGR mediated flux and also

  3. Isoprenylation is required for the processing of the lamin A precursor

    International Nuclear Information System (INIS)

    Beck, L.A.; Hosick, T.J.; Sinensky, M.

    1990-01-01

    The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. Moreover, inhibition of prelamin A processing by mevinolin treatment is rapidly reversed by the addition of exogenous mevalonate. Processing of prelamin A is, therefore, dependent on isoprenoid metabolism. Analysis of the conversion of prelamin A to lamin A by two independent methods, immunoprecipitation and two-dimensional nonequilibrium pH gel electrophoresis, demonstrates that a precursor-product relationship exists between prelamin A and lamin A. Analysis of R,S-[5-3H(N)]mevalonate-labeled cells shows that the rate of turnover of the isoprenoid group from prelamin A is comparable to the rate of conversion of prelamin A to lamin A. These results suggest that during the proteolytic maturation of prelamin A, the isoprenylated moiety is lost. A significant difference between prelamin A processing, and that of p21ras and the B-type lamins that undergo isoprenylation-dependent proteolytic maturation, is that the mature form of lamin A is no longer isoprenylated

  4. Biosynthesis of silver nanoparticles using Sida acuta extract for antimicrobial actions and corrosion inhibition potential.

    Science.gov (United States)

    Idrees, Muhammad; Batool, Saima; Kalsoom, Tanzila; Raina, Sadaf; Sharif, Hafiz Muhammad Adeel; Yasmeen, Summera

    2018-02-12

    Nanotechnology exhibits a multidisciplinary area and gained interests for researchers. Nanoparticles produced via physical and chemical methods affects ecosystem drastically. Green synthesis is the charming technique that is inexpensive and safe for the environment. This study aimed to explore the antibacterial actions of as-synthesized silver nanoparticles (Ag-NPs) against Escherichia coli, Staphylococcus aureus and Streptococcus faecalis. Also, the anti-corrosion actions confirmed that the Ag-NPs proved as good inhibitors. In this way, Ag-NPs were prepared via biosynthesis technique by consuming the ground leaves and stem of 'Sida acuta' as a capping agent. The Ag-NPs were formed by irradiation of the aqueous solution of silver nitrate (AgNO 3 ) with extract of S. acuta stem and leaves. The as-synthesized reaction mixture of Ag-NPs was found to exhibit an absorbance band at 446-447 nm, by an UV/VIS spectrophotometer, which is a characteristic of Ag-NPs due to the surface plasmon resonance absorption band. The X-ray diffraction and transmission electron microscopy (TEM) were used for the confirmation of Ag-NPs' variety dimension, morphology and dispersion. The infrared spectra confirmed the bio-fabrication of the Ag-NPs displayed the existence of conceivable functional groups responsible for the bio-reduction and capping. The antimicrobial actions were measured and the zone of inhibition was compared with standard antibiotics.

  5. Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Nickolas; Bonawitz, Nicholas D.; Nyffeler, Kayleigh E.; Chapple, Clint

    2015-06-05

    Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism.

  6. Hydrocarbons in the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda)—I. In vivo incorporation of 14C-labelled precursors into the hydrocarbon fraction

    NARCIS (Netherlands)

    Oudejans, R.C.H.M.

    1972-01-01

    1. 1. Biosynthesis of hydrocarbons in male and female specimens of the millipede Graphidostreptus tumuliporus was investigated after injection of the following precursors: 1-14C-acetate, 16-14C- and 1-14C-palmitic acid, isoleucine-14C(U), valine-14C(U) and mevalonic-2-14C acid. 2. 2. Both sexes

  7. Hydrocarbons and energy from plants: Final report, 1984-1987

    Energy Technology Data Exchange (ETDEWEB)

    Calvin, M.; Otvos, J.; Taylor, S.E.; Nemethy, E.K.; Skrukrud, C.L.; Hawkins, D.R.; Lago, R.

    1988-08-01

    Plant hydrocarbon (isoprenoid) production was investigated as an alternative source to fossil fuels. Because of their high triterpenoid (hydrocarbon) content of 4--8%, Euphorbia lathyris plants were used as a model system for this study. The structure of the E. lathyris triterpenoids was determined, and triterpenoid biosynthesis studied to better understand the metabolic regulation of isoprenoid production. Triterpenoid biosynthesis occurs in two distinct tissue types in E. lathyris plants: in the latex of the laticifer cells; and in the mesophyll cells of the leaf and stem. The latex has been fractionated by centrifugation, and it has been determined that the later steps of isoprenoid biosynthesis, the conversion of mevalonic acid to the triterpenes, are compartmentized within a vacuole. Also identified was the conversion of hydroxymethyl glutaryl-CoA to mevalonic acid, catalyzed by the enzyme Hydroxymethyl glutaryl-CoA Reductase, as a key rate limiting step in isoprenoid biosynthesis. At least two isozymes of this enzyme, one in the latex and another in the leaf plastids, have been identified. Environmental stress has been applied to plants to study changes in carbon allocation. Salinity stress caused a large decrease in growth, smaller decreases in photosynthesis, resulting in a larger allocation of carbon to both hydrocarbon and sugar production. An increase in Hydroxymethyl glutaryl-CoA Reductase activity was also observed when isoprenoid production increased. Other species where also screened for the production of hydrogen rich products such as isoprenoids and glycerides, and their hydrocarbon composition was determined.

  8. Effects of lead on enzymes of porphyrine biosynthesis in chloroplasts and erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hampp, R.; Kriebitzsch, C.; Ziegler, H.

    1974-01-01

    Two enzymes of the chlorophyll biosynthesis pathway, delta-aminolevulinic acid dehydratase (ALAD) and prophobilinogenase (PBGA), show a pronounced sensitivity to lead ion, as was shown in isolated chloroplasts of spinach. It has been reported by several authors that the activity of ALAD involved in the hemoglobine-biosynthesis in erythrocytes is also very sensitive to lead ions. Spinach chloroplasts were isolated and sonicated and the enzyme activity tested. Calf blood was collected with heparin and kept at 0/sup 0/C until enzyme determination. Hemolyzed erythrocytes (rapid freezing and thawing twice) were used as the source of enzymes. The incubation mixture was the same as for chloroplasts; the hemoglobin content per test was about 44 mg (ALAD) and 91 mg (PBGA). ALAD in erythrocytes is somewhat more sensitive to lead ions than ALAD in chloroplasts. PBGA in erythrocytes is also inhibited by Pb/sup 2 +/ ions, again more than the chloroplast enzyme. At all concentrations of Pb/sup 2 +/ checked in our experiments the percentage of inhibition was higher with PBGA. 3 references, 1 figure.

  9. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation

    Science.gov (United States)

    Duivenvoorden, Raphaël; Tang, Jun; Cormode, David P.; Mieszawska, Aneta J.; Izquierdo-Garcia, David; Ozcan, Canturk; Otten, Maarten J.; Zaidi, Neeha; Lobatto, Mark E.; van Rijs, Sarian M.; Priem, Bram; Kuan, Emma L.; Martel, Catherine; Hewing, Bernd; Sager, Hendrik; Nahrendorf, Matthias; Randolph, Gwendalyn J.; Stroes, Erik S. G.; Fuster, Valentin; Fisher, Edward A.; Fayad, Zahi A.; Mulder, Willem J. M.

    2014-01-01

    Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

  10. Impact of Chemical Analogs of 4-Hydroxybenzoic Acid on Coenzyme Q Biosynthesis: From Inhibition to Bypass of Coenzyme Q Deficiency

    Directory of Open Access Journals (Sweden)

    Fabien Pierrel

    2017-06-01

    Full Text Available Coenzyme Q is a lipid that participates to important physiological functions. Coenzyme Q is synthesized in multiple steps from the precursor 4-hydroxybenzoic acid. Mutations in enzymes that participate to coenzyme Q biosynthesis result in primary coenzyme Q deficiency, a type of mitochondrial disease. Coenzyme Q10 supplementation of patients is the classical treatment but it shows limited efficacy in some cases. The molecular understanding of the coenzyme Q biosynthetic pathway allowed the design of experiments to bypass deficient biosynthetic steps with analogs of 4-hydroxybenzoic acid. These molecules provide the defective chemical group and can reactivate endogenous coenzyme Q biosynthesis as demonstrated recently in yeast, mammalian cell cultures, and mouse models of primary coenzyme Q deficiency. This mini review presents how the chemical properties of various analogs of 4-hydroxybenzoic acid dictate the effect of the molecules on CoQ biosynthesis and how the reactivation of endogenous coenzyme Q biosynthesis may achieve better results than exogenous CoQ10 supplementation.

  11. Cloning and expression analysis of JcAACT, jcMDC and JcFPS, involved in terpenoid biosynthesis in jatropha curcas l

    International Nuclear Information System (INIS)

    Huang, Y.; Wen, J.

    2018-01-01

    To better understand the functions of key genes involved in terpenoid biosynthesis in Jatropha curcas, we cloned and characterized three genes, namely acetyl CoA acyltransferase (JcAACT), diphosphate mevalonate decarboxylase (JcMDC) and farnesyl pyrophosphate synthase (JcFPS). The opening reading frames (ORFs) of JcAACT, JcMDC and JcFPS were 1239 bp,1248 bp and 1029 bp, respectively, encoding a 412-amino acid, 415-amino acid and 342-amino acid polypeptide, respectively. Results of homology analysis showed that JcAACT, JcMDC and JcFPS encoded proteins that all had the highest identity and closest relationship with the corresponding genes in Hevea brasiliensis, with identities of 89%, 92% and 93%, respectively. JcAACT, JcMDC and JcFPS were expressed in all organs tested of J. curcas; the highest expression level for each gene occurred in seeds. In the early growth stage of seeds, the expression level of each of these three genes increased with time, with JcAACT and JcMDC expression level reaching a peak at the late stage of seed development (50 d), while JcFPS expression level reached a peak at the mid-late stage (40 d). Following the peak, the expression of each gene then declined. The expression level of JcAACT was the highest of the three genes, regardless of the organ or the stage of seed growth, indicating its important role in J. curcas. This study lays the foundation for a better understanding of the important role of the JcAACT, JcMDC and JcFPS genes in the terpenoid biosynthesis pathway of J. curcas. (author)

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

    Directory of Open Access Journals (Sweden)

    Rafael Luis Kessler

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

  13. Action of radiation on biosynthesis of hemoglobin and some of its electrophoretic fractions

    International Nuclear Information System (INIS)

    Starodub, N.F.; Kriklivyj, I.A.; Shur'yan, I.M.

    1976-01-01

    Biosynthesis of hemoglobin and some of its electrophoretic fractions in red cells of peripheral blood and spleen of irradiated (650 R) rats has been studied. Hemoglobin synthesis is found to be most drastically inhibited in the first and second fractions on the first and eighth days after irradiation and in the fifth and sixth fractions on the eighth day (less expressed). The synthesis is restored on the twelfth day, the process under study proceeding more slowly in the above-mentioned fractions than in others. In the course of radiation sickness, the biosynthesis of certain hemoglobin fractions varies differently in the hemoglobin-synthesizing cells of peripheral blood than in the cells of spleenic erythroid series

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

    Science.gov (United States)

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

    2017-06-01

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

  15. [The inhibitor of free radical processes decrease of protein biosynthesis in gun short wound tissues and weaken development of the general adaptation syndrome].

    Science.gov (United States)

    Todorov, I N; Bogdanov, G N; Mitrokhin, Iu I; Varfolomeev, V N; Sidorenko, L I; Mishchenko, D V

    2006-01-01

    The dynamics of total protein biosynthesis and procollagen biosynthesis in skeletal muscle of injury tissues with the antioxidant BHT (dibunol) treatment and with common healing were studied. The obtained date indicate that the AO treatment reduce the rate of biosynthesis both the total proteins and procollagen at the 3th day of healing. Dibunol also considerably reduce the protein biosynthesis in adrenals and brake of corticosteroids biogenesis as measured by ESR-signals intensity of reduced adrenodoxine. AO treatment also reduce the protein biosynthesis in thymus, spleen and bone marrow. The lowering of functional activity of endocrine and immune systems indicate that the AO significantly inhibit the systemic reactions of organism induced by acute wound affect. It was suggested that as "primary mediator" of stress-reaction may be considered lipoperoxide radicals and decay products of lipohydroperoide.

  16. Biosynthetic origin of the isoprene units in chromenes of Piper aduncum (Piperaceae)

    International Nuclear Information System (INIS)

    Leite, Ana C.; Lopes, Adriana A.; Bolzani, Vanderlan da S.; Furlan, Maysa; Kato, Massuo J.

    2007-01-01

    Metabolic studies involving the incorporation of [1- 13 C]-D-glucose into intact leaves of Piper aduncum (Piperaceae) have indicated that both the mevalonate (MVA) and the pyruvate-triose (MEP) non-mevalonate pathways are implicated in the biosynthesis of isoprene moieties present in methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl- 2'-butenyl)-2H-1-chromene-6-carboxylate (2). The pattern of incorporation of label from [1- 13 C]-D-glucose into these chromenes was determined by quantitative 13 C NMR spectroscopy. The results confirmed that biosynthetic compartment of 1 and 2 could either be the plastid and/ or the cytosol or, possibly, an additional compartment such as the plastid inter-membrane space. (author)

  17. The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

    International Nuclear Information System (INIS)

    Hayashi, H.; Miwa, A.

    1989-01-01

    The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1- 14 C]butyric acid and [1- 14 C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [ 14 C]lignoceric acid into primary bile acids was approximately four times higher than that of [ 14 C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [ 14 C]lignoceric acid and [ 14 C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis

  18. The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, H.; Miwa, A. (Josai Univ., Saitama (Japan))

    1989-11-01

    The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using (1-{sup 14}C)butyric acid and (1-{sup 14}C)lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of ({sup 14}C)lignoceric acid into primary bile acids was approximately four times higher than that of ({sup 14}C)butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both ({sup 14}C)lignoceric acid and ({sup 14}C)butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.

  19. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Directory of Open Access Journals (Sweden)

    Michael Hartmann

    2013-08-01

    Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

  20. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

    Science.gov (United States)

    Bach, Thomas J

    2013-01-01

    We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

  1. Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

    Science.gov (United States)

    Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

    2017-03-14

    We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

  2. Arabidopsis DREB2C modulates ABA biosynthesis during germination.

    Science.gov (United States)

    Je, Jihyun; Chen, Huan; Song, Chieun; Lim, Chae Oh

    2014-09-12

    Plant dehydration-responsive element binding factors (DREBs) are transcriptional regulators of the APETELA2/Ethylene Responsive element-binding Factor (AP2/ERF) family that control expression of abiotic stress-related genes. We show here that under conditions of mild heat stress, constitutive overexpression seeds of transgenic DREB2C overexpression Arabidopsis exhibit delayed germination and increased abscisic acid (ABA) content compared to untransformed wild-type (WT). Treatment with fluridone, an inhibitor of the ABA biosynthesis abrogated these effects. Expression of an ABA biosynthesis-related gene, 9-cis-epoxycarotenoid dioxygenase 9 (NCED9) was up-regulated in the DREB2C overexpression lines compared to WT. DREB2C was able to trans-activate expression of NCED9 in Arabidopsis leaf protoplasts in vitro. Direct and specific binding of DREB2C to a complete DRE on the NCED9 promoter was observed in electrophoretic mobility shift assays. Exogenous ABA treatment induced DREB2C expression in germinating seeds of WT. Vegetative growth of transgenic DREB2C overexpression lines was more strongly inhibited by exogenous ABA compared to WT. These results suggest that DREB2C is a stress- and ABA-inducible gene that acts as a positive regulator of ABA biosynthesis in germinating seeds through activating NCED9 expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. 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.

  4. Penicillium expansum (compatible) and Penicillium digitatum (non-host) pathogen infection differentially alter ethylene biosynthesis in apple fruit.

    Science.gov (United States)

    Vilanova, Laura; Vall-Llaura, Núria; Torres, Rosario; Usall, Josep; Teixidó, Neus; Larrigaudière, Christian; Giné-Bordonaba, Jordi

    2017-11-01

    The role of ethylene on inducing plant resistance or susceptibility to certain fungal pathogens clearly depends on the plant pathogen interaction with little or no-information available focused on the apple-Penicillium interaction. Taken advantage that Penicillium expansum is the compatible pathogen and P. digitatum is the non-host of apples, the present study aimed at deciphering how each Penicillium spp. could interfere in the fruit ethylene biosynthesis at the biochemical and molecular level. The infection capacity and different aspects related to the ethylene biosynthesis were conducted at different times post-inoculation. The results show that the fruit ethylene biosynthesis was differently altered during the P. expansum infection than in response to other biotic (non-host pathogen P. digitatum) or abiotic stresses (wounding). The first symptoms of the disease due to P. expansum were visible before the initiation of the fruit ethylene climacteric burst. Indeed, the ethylene climacteric burst was reduced in response to P. expansum concomitant to an important induction of MdACO3 gene expression and an inhibition (ca. 3-fold) and overexpression (ca. 2-fold) of ACO (1-Aminocyclopropane-1-carboxylic acid oxidase) and ACS (1-Aminocyclopropane-1-carboxylic acid synthase) enzyme activities, indicating a putative role of MdACO3 in the P. expansum-apple interaction which may, in turn, be related to System-1 ethylene biosynthesis. System-1 is auto-inhibited by ethylene and is characteristic of non-climateric or pre-climacteric fruit. Accordingly, we hypothesise that P. expansum may 'manipulate' the endogenous ethylene biosynthesis in apples, leading to the circumvention or suppression of effective defences hence facilitating its colonization. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Mörck, Catarina; Elmelund-Præstekær, Louise Cathrine Braun; Kurth, Caroline

    2009-01-01

    of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C....... elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER...... and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C...

  6. Inhibition of glycolipid biosynthesis by N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin protects against the inflammatory response in hapten-induced colitis

    NARCIS (Netherlands)

    Shen, Chong; Bullens, Dominique; Kasran, Ahmad; Maerten, Philippe; Boon, Louis; Aerts, Johannes M. F. G.; van Assche, Gert; Geboes, Karel; Rutgeerts, Paul; Ceuppens, Jan L.

    2004-01-01

    Since glycolipid biosynthesis is potentially involved in immunological and inflammatory responses, we tested the effect of a novel inhibitor of intracellular glycolipid biosynthesis N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) in two hapten-induced colitis models: trinitrobenzene

  7. New Insights on the Terpenome of the Red Seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta

    Directory of Open Access Journals (Sweden)

    Louisi Souza de Oliveira

    2015-02-01

    Full Text Available The red seaweeds belonging to the genus Laurencia are well known as halogenated secondary metabolites producers, mainly terpenoids and acetogennins. Several of these chemicals exhibit important ecological roles and biotechnological applications. However, knowledge regarding the genes involved in the biosynthesis of these compounds is still very limited. We detected 20 different genes involved in the biosynthesis of terpenoid precursors, and 21 different genes coding for terpene synthases that are responsible for the chemical modifications of the terpenoid precursors, resulting in a high diversity of carbon chemical skeletons. In addition, we demonstrate through molecular and cytochemical approaches the occurrence of the mevalonate pathway involved in the biosynthesis of terpenes in L. dendroidea. This is the first report on terpene synthase genes in seaweeds, enabling further studies on possible heterologous biosynthesis of terpenes from L. dendroidea exhibiting ecological or biotechnological interest.

  8. New Insights on the terpenome of the red seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta).

    Science.gov (United States)

    de Oliveira, Louisi Souza; Tschoeke, Diogo Antonio; de Oliveira, Aline Santos; Hill, Lilian Jorge; Paradas, Wladimir Costa; Salgado, Leonardo Tavares; Thompson, Cristiane Carneiro; Pereira, Renato Crespo; Thompson, Fabiano L

    2015-02-10

    The red seaweeds belonging to the genus Laurencia are well known as halogenated secondary metabolites producers, mainly terpenoids and acetogennins. Several of these chemicals exhibit important ecological roles and biotechnological applications. However, knowledge regarding the genes involved in the biosynthesis of these compounds is still very limited. We detected 20 different genes involved in the biosynthesis of terpenoid precursors, and 21 different genes coding for terpene synthases that are responsible for the chemical modifications of the terpenoid precursors, resulting in a high diversity of carbon chemical skeletons. In addition, we demonstrate through molecular and cytochemical approaches the occurrence of the mevalonate pathway involved in the biosynthesis of terpenes in L. dendroidea. This is the first report on terpene synthase genes in seaweeds, enabling further studies on possible heterologous biosynthesis of terpenes from L. dendroidea exhibiting ecological or biotechnological interest.

  9. Differential effects of lipid biosynthesis inhibitors on Zika and Semliki Forest viruses.

    Science.gov (United States)

    Royle, Jamie; Donald, Claire L; Merits, Andres; Kohl, Alain; Varjak, Margus

    2017-12-01

    The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Mevalonates, Ras and Breast Cancer

    National Research Council Canada - National Science Library

    White, Michael

    2001-01-01

    .... This selective inhibition appears to be a consequence of expression of oncogenic Ras. Here we are evaluating the ability of Fmev to selectively interfere with proliferation of breast cancer cells...

  11. Viral MicroRNAs Repress the Cholesterol Pathway, and 25-Hydroxycholesterol Inhibits Infection.

    Science.gov (United States)

    Serquiña, Anna K P; Kambach, Diane M; Sarker, Ontara; Ziegelbauer, Joseph M

    2017-07-11

    From various screens, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) is sufficient to reduce intracellular cholesterol levels; however, small interfering RNAs (siRNAs) targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly regulated pathway. We also report here that cholesterol levels were decreased in de novo -infected HUVECs after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo -infected HUVECs but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC. IMPORTANCE A subset of viruses express unique microRNAs (miRNAs), which act like cellular miRNAs to generally repress host gene

  12. Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells.

    Science.gov (United States)

    Stulov, Sergey V; Mankevich, Olga V; Dugin, Nikita O; Novikov, Roman A; Timofeev, Vladimir P; Misharin, Alexander Yu

    2013-04-01

    Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 μM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Effect of interleukin-1 on the biosynthesis of proinsulin and insulin in isolated rat pancreatic islets

    DEFF Research Database (Denmark)

    Hansen, Birgit Sehested; Linde, S; Spinas, G A

    1988-01-01

    Insulin dependent diabetes mellitus (IDDM) is often preceded or associated with lymphocytic infiltration in the islets of Langerhans (insulitis). We recently demonstrated that interleukin-1 (IL-1) produced by activated macrophages exerts a bimodal effect on insulin release and biosynthesis...... in isolated rat islets. In the present study we have further analysed the effect of recombinant human interleukin-1 beta (rIL-1) on the biosynthesis and conversion of proinsulin 1 and 2 in rat islets. By RP-HPLC-analysis of islets labelled with [3H]leucine we found that exposure to 6 ng/ml of IL-1 for 24 h.......1 to 3.4 +/- 0.4, respectively. Pulse-chase experiments with [3H]leucine and [35S]methionine indicated a more marked reduction in the conversion rate of proinsulin-2 compared to that of proinsulin-1. In conclusion these experiments demonstrate that IL-1 inhibits insulin biosynthesis by preferential...

  14. HOG MAP kinase regulation of alternariol biosynthesis in Alternaria alternata is important for substrate colonization.

    Science.gov (United States)

    Graf, Eva; Schmidt-Heydt, Markus; Geisen, Rolf

    2012-07-16

    Strains of the genus Alternaria are ubiquitously present and frequently found on fruits, vegetables and cereals. One of the most commonly found species from this genus is A. alternata which is able to produce the mycotoxin alternariol among others. To date only limited knowledge is available about the regulation of the biosynthesis of alternariol, especially under conditions relevant to food. Tomatoes are a typical substrate of A. alternata and have a high water activity. On the other hand cereals with moderate water activity are also frequently colonized by A. alternata. In the current analysis it was demonstrated that even minor changes in the osmotic status of the substrate affect the alternariol biosynthesis of strains from vegetables resulting in nearly complete inhibition. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade) which is a MAP kinase transduction pathway. The phosphorylation status of the A. alternata HOG (AaHOG) was determined. Various concentrations of NaCl induce the phosphorylation of AaHOG in a concentration, time and strain dependent manner. A strain with a genetically inactivated aahog gene was no longer able to produce alternariol indicating that the activity of the aahog gene is required for alternariol biosynthesis. Further experiments revealed that the biosynthesis of alternariol is important for the fungus to colonize tomato tissue. The tight water activity dependent regulation of alternariol biosynthesis ensures alternariol biosynthesis at conditions which indicate an optimal colonization substrate for the fungus. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Biosynthetic origin of the isoprene units in chromenes of Piper aduncum (Piperaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Leite, Ana C.; Lopes, Adriana A.; Bolzani, Vanderlan da S.; Furlan, Maysa [UNESP, Araraquara, SP (Brazil). Inst. de Quimica]. E-mail: maysaf@iq.unesp.br; Kato, Massuo J. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica

    2007-07-01

    Metabolic studies involving the incorporation of [1-{sup 13}C]-D-glucose into intact leaves of Piper aduncum (Piperaceae) have indicated that both the mevalonate (MVA) and the pyruvate-triose (MEP) non-mevalonate pathways are implicated in the biosynthesis of isoprene moieties present in methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl- 2'-butenyl)-2H-1-chromene-6-carboxylate (2). The pattern of incorporation of label from [1- {sup 13}C]-D-glucose into these chromenes was determined by quantitative {sup 13}C NMR spectroscopy. The results confirmed that biosynthetic compartment of 1 and 2 could either be the plastid and/ or the cytosol or, possibly, an additional compartment such as the plastid inter-membrane space. (author)

  16. On the mechanism for the formation of indole alkalois in Penicillium concavo-rugulosum

    International Nuclear Information System (INIS)

    Ohmomo, Sadahiro; Miyazaki, Kenji; Ohashi, Tsutomu; Abe, Matazo

    1977-01-01

    Experiments on the biosynthesis and microbiological conversion of indole alkaloids in Penicillium concavo-rugulosum were carried out with the growing and resting mycelia, respectively, of a selected strain of the same mold. The former experiments were performed by the use of DL-tryptophan-3- 14 C or DL-mevalonic acid-2- 14 C-lactone as a precursor, while the latter experiments by the use of rugulovasine A- 3 H, dihydrorugulovasine A- 3 H, 4-[γ,γ-dimethylallyl]-tryptophan- 3 H, chanoclavine-[I]- 3 H or the other tritiated ergoline alkaloids. The results of these experiments suggested that in the Penicillium mold employed there exist the following biosynthetic route: tryptophan+mevalonic acid → 4-[γ,γ-dimethylallyl]-tryptophan → rugulovasine A → dihydrorugulovasine A → dihydrorugulovasine A-lactam. (auth.)

  17. Inhibitory effects of various oxygenated sterols on the differentiation and function of tumor-specific cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Spangrude, G.J.; Sherris, D.; Daynes, R.A.

    1982-01-01

    Irradiation of skin with ultraviolet light (UVL) is capable of causing many biological and biochemical changes in this complex organ. One early consequence is the oxidation of epidermal plasma membrane cholesterol, causing the induction of a wide variety of photoproducts. It is well recognized that some oxygenated sterols possess potent biological activity on mammalian cells by their ability to inhibit endogeneous mevalonate and cholesterol biosynthesis. In the few immunological systems that have been studied, there is general agreement that lymphocyte function is lacking, as both afferent and efferent blockades have been suggested. These studies were undertaken to determine the effect of various oxygenated sterols (representing a number of known cholesterol-derived photoproducts) on the generation (afferent) and function (efferent) of cytotoxic T lymphocytes (CTLs). Cell-mediated immune responses which result in the generation of both alloantigen-specific and syngeneic tumor-specific CTLs were evaluated

  18. The mevalonate pathway in neurons: It's not just about cholesterol.

    Science.gov (United States)

    Moutinho, Miguel; Nunes, Maria João; Rodrigues, Elsa

    2017-11-01

    Cholesterol homeostasis greatly impacts neuronal function due to the essential role of this sterol in the brain. The mevalonate (MVA) pathway leads to the synthesis of cholesterol, but also supplies cells with many other intermediary molecules crucial for neuronal function. Compelling evidence point to a model in which neurons shutdown cholesterol synthesis, and rely on a shuttle derived from astrocytes to meet their cholesterol needs. Nevertheless, several reports suggest that neurons maintain the MVA pathway active, even with sustained cholesterol supply by astrocytes. Hence, in this review we focus not on cholesterol production, but rather on the role of the MVA pathway in the synthesis of particular intermediaries, namely isoprenoids, and on their role on neuronal function. Isoprenoids act as anchors for membrane association, after being covalently bound to proteins, such as most of the small guanosine triphosphate-binding proteins, which are critical to neuronal cell function. Based on literature, on our own results, and on the analysis of public transcriptomics databases, we raise the idea that in neurons there is a shift of the MVA pathway towards the non-sterol branch, responsible for isoprenoid synthesis, in detriment to post-squalene branch, and that this is ultimately essential for synaptic activity. Nevertheless new tools that facilitate imaging and the biochemical characterization and quantification of the prenylome in neurons and astrocytes are needed to understand the regulation of isoprenoid production and protein prenylation in the brain, and to analyze its differences on diverse physiological or pathological conditions, such as aging and neurodegenerative states. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. 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

    histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of similar to 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus......-and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability...

  20. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: L-glutamine inhibits the generation of L-arginine by cultured endothelial cells

    International Nuclear Information System (INIS)

    Sessa, W.C.; Hecker, M.; Mitchell, J.A.; Vane, J.R.

    1990-01-01

    The mechanism by which L-glutamine (L-Gln) inhibits the release of endothelium-derived factor from bovine aortic cultured endothelial cells was investigated. The intracellular concentration of L-arginine (L-Arg) in Arg-depleted endothelial cells was inversely related to the level of L-Gln. Removal of L-Gln from the culture medium (usually containing L-Gln at 2 mM) abolished the inhibitory effect of the culture medium on L-Arg generation. L-Gln (0.2 and 2 mM) but not D-Gln inhibited the generation of L-Arg by both Arg-depleted and nondepleted endothelial cells. L-Gln did not interfere with the uptake of L-Arg or the metabolism of L-Arg-L-Phe to L-Arg but inhibited the formation of L-Arg from L-citrulline (L-Cit), L-Cit-L-Phe, and N G -monomethyl-L-arginine. L-Gln also inhibited the conversion of L-[ 14 C]Cit to L-[ 14 C]Arg by Arg-depleted endothelial cells. However, L-Gln did not inhibit the conversion of L-argininosuccinic acid to L-Arg by endothelial cell homogenates. Thus, L-Gln interferes with the conversion of L-Cit to L-Arg probably by acting on argininosuccinate synthetase rather than argininosuccinate lyase. L-Gln also inhibited the generation of L-Arg by the monocyte-macrophage cell line J774 but had no effect on the conversion of L-Cit to L-Arg by these cells. As the release of endothelium-derived relaxing factor from cultured and non-cultured endothelial cells is limited by the availability of L-Arg, endogenous L-Gln may play a regulatory role in the biosynthesis of endothelium-derived relaxing factor

  1. The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells

    Science.gov (United States)

    Castella, Barbara; Kopecka, Joanna; Sciancalepore, Patrizia; Mandili, Giorgia; Foglietta, Myriam; Mitro, Nico; Caruso, Donatella; Novelli, Francesco; Riganti, Chiara; Massaia, Massimo

    2017-01-01

    Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. PMID:28580927

  2. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    Science.gov (United States)

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (Pseeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. © 2015 American Society of Plant Biologists. All Rights Reserved.

  3. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  4. Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells.

    Science.gov (United States)

    Seppänen, P; Fagerström, R; Alhonen-Hongisto, L; Elo, H; Lumme, P; Jänne, J

    1984-07-15

    Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in

  5. Inhibition of chitin biosynthesis in cultured imaginal discs: Effects of alpha-amanitin, actinomycin-D, cycloheximide, and puromycin.

    Science.gov (United States)

    Oberlander, Herbert; Ferkovich, Stephen; Leach, Eddie; Van Essen, Frank

    1980-02-01

    Wing imaginal discs isolated from last instar larvae of the Indian meal moth,Plodia interpunctella, produced chitin when incubated in vitro with ≧2×10 -7 M 20-hydroxyecdysone. Chitin biosynthesis was initiated 8 h after the conclusion of a 24-h treatment with hormone. Simulataneous incubation of wing discs with 20-hydroxyecdysone and either inhibitors of RNA synthesis (alpha-amanitin, actinomycin-D) or inhibitors of protein systhesis (cycloheximide, puromycin) prevented chitin biosynthesis. We conclude from our results that RNA and protein synthesis must continue undiminished during the hormone-contact period, and that synthesis of protein, but not of new RNA is required during the posthormone culture period. Our findings are consistent with the hypothesis that ecdysteroids stimulate insect metamorphosis by promoting the synthesis of new RNA and protein during a hormone-dependent phase followed by hormone-independent protein synthesis.

  6. Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

    Science.gov (United States)

    Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

    2016-01-01

    IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

  7. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. RNA interference of pheromone biosynthesis-activating neuropeptide receptor suppresses mating behavior by inhibiting sex pheromone production in Plutella xylostella (L.).

    Science.gov (United States)

    Lee, Dae-Weon; Shrestha, Sony; Kim, A Young; Park, Seok Joo; Yang, Chang Yeol; Kim, Yonggyun; Koh, Young Ho

    2011-04-01

    Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Regulation of cell wall biosynthesis.

    Science.gov (United States)

    Zhong, Ruiqin; Ye, Zheng-Hua

    2007-12-01

    Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

  10. S-Carvone Suppresses Cellulase-Induced Capsidiol Production in Nicotiana tabacum by Interfering with Protein Isoprenylation1[C][W

    Science.gov (United States)

    Huchelmann, Alexandre; Gastaldo, Clément; Veinante, Mickaël; Zeng, Ying; Heintz, Dimitri; Tritsch, Denis; Schaller, Hubert; Rohmer, Michel; Bach, Thomas J.; Hemmerlin, Andréa

    2014-01-01

    S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway–dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism. PMID:24367019

  11. Biosynthesis of tylophora alkaloids

    International Nuclear Information System (INIS)

    Mulchandani, N.B.; Iyer, S.S.; Badheka, L.P.

    1974-01-01

    Using labelled precursors, biosynthesis of the tylophora alkaloids, tylophorine, tylophorinidine and tylophorinide has been investigated in Tylophora asthmatica plants. The radioactive precursors, phenylalanine-2- 14 C, benzoic acid-1- 14 C, benzoic acid-ring 14 C, acetate-2- 14 C, ornithine-5- 14 C, acetate-2- 14 C, ornithine-5- 14 C and cinnamic acid-2- 14 C 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.)

  12. Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

    Science.gov (United States)

    Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

    2017-08-09

    Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

  13. Different biosynthesis patterns among flavonoid 3-glycosides with distinct effects on accumulation of other flavonoid metabolites in pears (Pyrus bretschneideri Rehd..

    Directory of Open Access Journals (Sweden)

    Rui Zhai

    Full Text Available Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear 'Zaosu' (Pyrus bretschneideri Rehd. and its red mutant 'Red Zaosu'. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in 'Zaosu' pear. By comparison with 'Red Zaosu', the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of 'Zaosu' after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in 'Zaosu' pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between 'Zaosu' and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.

  14. Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.

    Science.gov (United States)

    Wang, Yongkang; Song, Xiaodan; Zhang, Yongjun; Wang, Bochu; Zou, Xiang

    2016-08-22

    Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated. A high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated. The level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in

  15. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    Gräwert, Tobias

    2009-07-20

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue of the substrate. Together with mutation studies and theoretical calculations, these data support a mechanism which is analogous to the Birch reduction of allylic alcohols. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

  16. Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation

    International Nuclear Information System (INIS)

    Fernandez, Carlos; Lobo, Maria del Val T.; Gomez-Coronado, Diego; Lasuncion, Miguel A.

    2004-01-01

    As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14α-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells

  17. ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

    Science.gov (United States)

    Benoist, F; Grand-Perret, T

    1996-10-01

    Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

  18. Influence of light, UV-B radiation, and herbicides on wax biosynthesis of cucumber seedlings

    International Nuclear Information System (INIS)

    Tevini, M.; Steinmüller, D.

    1987-01-01

    The behavior of cuticular alkane-1-ols and alkanes were studied in different developmental stages of cucumber seedlings grown in the dark or under white light, with or without UV-B radiation or in presence of wax biosynthesis inhibitors, trichloroacetic acid and metolachlor. Accumulation of alkane-1-ols increased light independently with seedling age. Synthesis of alkanes was strictly light and dose dependent. Addition of UV-B radiation did not alter the amounts of alkanes or alcohols, however, the distribution of homologues was shifted towards shorter chain homologues. Treatments with Cl 3 AcOH resulted in strong inhibition of alkane accumulation, whereas the amount of alkane-1-ols was changed neither at low nor at moderate concentrations of Cl 3 AcOH but their homologue distribution shifted towards longer chain lengths. This shifting was depressed in the presence of UV-B. At high concentrations of Cl 3 Ac0H similar homologue distributions as produced by UV-B (shift to shorter homologues) were observed. Metolachlor treatment resulted in an inhibition of alkane-1-ol production connected with rising amounts of alkanes, predominantly of short chain species. A simple model of wax biosynthesis is proposed which describes the interactions with white light, UV-B radiation and herbicides. (author)

  19. Fluridone and norflurazon, carotenoid-biosynthesis inhibitors, promote seed conditioning and germination of the holoparasite Orobanche minor.

    Science.gov (United States)

    Chae, Sang Heon; Yoneyama, Koichi; Takeuchi, Yasutomo; Joel, Daniel M.

    2004-02-01

    Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor, they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30 degrees C) as well as in the light. Gibberellic acid (GA(3)) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.

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

    International Nuclear Information System (INIS)

    Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1989-01-01

    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

  1. Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

    International Nuclear Information System (INIS)

    Gardner, L.C.; Cox, T.M.

    1988-01-01

    Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular free heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation

  2. Engineering Pseudomonas for phenazine biosynthesis, regulation, and biotechnological applications: a review.

    Science.gov (United States)

    Bilal, Muhammad; Guo, Shuqi; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2017-10-03

    Pseudomonas strains are increasingly attracting considerable attention as a valuable bacterial host both for basic and applied research. It has been considered as a promising candidate to produce a variety of bioactive secondary metabolites, particularly phenazines. Apart from the biotechnological perspective, these aromatic compounds have the notable potential to inhibit plant-pathogenic fungi and thus are useful in controlling plant diseases. Nevertheless, phenazines production is quite low by the wild-type strains that necessitated its yield improvement for large-scale agricultural applications. Metabolic engineering approaches with the advent of plentiful information provided by systems-level genomic and transcriptomic analyses enabled the development of new biological agents functioning as potential cell factories for producing the desired level of value-added bioproducts. This study presents an up-to-date overview of recombinant Pseudomonas strains as the preferred choice of host organisms for the biosynthesis of natural phenazines. The biosynthetic pathway and regulatory mechanism involved in the phenazine biosynthesis are comprehensively discussed. Finally, a summary of biological functionalities and biotechnological applications of the phenazines is also provided.

  3. Involvement of ethylene biosynthesis and signalling in fruit set and early fruit development in zucchini squash (Cucurbita pepo L.).

    Science.gov (United States)

    Martínez, Cecilia; Manzano, Susana; Megías, Zoraida; Garrido, Dolores; Picó, Belén; Jamilena, Manuel

    2013-09-22

    We have identified a kind of parthenocarpy in zucchini squash which is associated with an incomplete andromonoecy, i.e. a partial conversion of female into bisexual flowers. Given that andromonoecy in this and other cucurbit species is caused by a reduction of ethylene production in the female flower, the associated parthenocarpic development of the fruit suggested the involvement of ethylene in fruit set and early fruit development. We have compared the production of ethylene as well as the expression of 13 ethylene biosynthesis and signalling genes in pollinated and unpollinated ovaries/fruits of two cultivars, one of which is parthenocarpic (Cavili), while the other is non-parthenocarpic (Tosca). In the latter, unpollinated ovaries show an induction of ethylene biosynthesis and ethylene signal transduction pathway genes three days after anthesis, which is concomitant with the initiation of fruit abortion and senescence. Fruit set and early fruit development in pollinated flowers of both cultivars and unpollinated flowers of Cavili is coupled with low ethylene biosynthesis and signalling, which would also explain the partial andromonoecy in the parthenocarpic genotype. The reduction of ethylene production in the ovary cosegregates with parthenocarpy and partial andromonoecy in the selfing progeny of Cavili. Moreover, the induction of ethylene in anthesis (by ethephon treatments) reduced the percentage of bisexual parthenocarpic flowers in Cavili, while the inhibition of ethylene biosynthesis or response (by AVG and STS treatments) induces not only andromonoecy but also the parthenocarpic development of the fruit in both cultivars. Results demonstrate that a reduction of ethylene production or signalling in the zucchini flower is able to induce fruit set and early fruit development, and therefore that ethylene is actively involved in fruit set and early fruit development. Auxin and TIBA treatments, inducing fruit set and early fruit development in this species

  4. Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa).

    Science.gov (United States)

    Park, Jong-Sug; Kim, Jung-Bong; Cho, Kang-Jin; Cheon, Choong-Ill; Sung, Mi-Kyung; Choung, Myoung-Gun; Roh, Kyung-Hee

    2008-06-01

    The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.

  5. The Spatial Organization of Glucosinolate Biosynthesis

    DEFF Research Database (Denmark)

    Nintemann, Sebastian

    cells is an open question. Likewise, it is not known how glucosinolate biosynthesis is orchestrated at the subcellular level. These open questions were addressed with several approaches in this project, with the aim of shedding light on the spatial organization of glucosinolate biosynthesis from...... between the individual classes of glucosinolates under constitutive and induced conditions and identified the source tissues of these defense compounds. Protein-protein interaction studies were carried out to investigate the subcellular organization of glucosinolate biosynthesis. We identified a family...

  6. Effects of whole-body γ-irradiation on the biosynthesis of certain serum proteins. Final report, November 29, 1967--June 30, 1976

    International Nuclear Information System (INIS)

    Neuhaus, O.W.

    1976-01-01

    Whole-body exposure of rats to ionizing radiations yielded an increased incorporation of labeled amino acids into serum albumin in in vivo studies suggesting a stimulation of biosynthesis. Actually this may have been caused by an elevated hepatic transport of labeled amino acids (see below). A suppressed biosynthesis of albumin was observed when the experiments were performed in vitro using liver microsomes. Impaired biosynthesis appeared to be caused by a reduced mRNA production. Irradiation stimulated the biosynthesis of acute-phase plasma proteins (stress response) and inhibited the excretion of α/sub 2u/-globulin, the sex-dependent protein of the adult male rat. Exposure of rats to γ-rays stimulated amino acid transport into the liver. This process which is Na + and energy-dependent was studied with α-aminoisobutyric acid, cycloleucine, and L-methionine among others. After irradiation the serum glucagon and insulin, as well as hepatic cAMP levels, were elevated. Amino acid transport may be an important factor in controlling the increased gluconeogenesis and glycogenesis observed in rats following whole-body irradiation

  7. Anthocyanin biosynthesis and degradation mechanisms in Solanaceous vegetables: a review

    Science.gov (United States)

    Liu, Ying; Tikunov, Yury; Schouten, Rob E.; Marcelis, Leo F. M.; Visser, Richard G. F.; Bovy, Arnaud

    2018-03-01

    Anthocyanins are a group of polyphenolic pigments that are ubiquitously found in the plant kingdom. In plants, anthocyanins play a role not only in reproduction, by attracting pollinators and seed dispersers, but also in protection against various abiotic and biotic stresses. There is accumulating evidence that anthocyanins have health-promoting properties, which makes anthocyanin metabolism an interesting target for breeders and researchers. In this review, the state of the art knowledge concerning anthocyanins in the Solanaceous vegetables, i.e. pepper, tomato, eggplant and potato, is discussed, including biochemistry and biological function of anthocyanins, as well as their genetic and environmental regulation. Anthocyanin accumulation is determined by the balance between biosynthesis and degradation. Although the anthocyanin biosynthetic pathway has been well studied in Solanaceous vegetables, more research is needed on the inhibition of biosynthesis and, in particular, the anthocyanin degradation mechanisms if we want to control anthocyanin content of Solanaceous vegetables. In addition, anthocyanin metabolism is distinctly affected by environmental conditions, but the molecular regulation of these effects is poorly understood. Existing knowledge is summarized and current gaps in our understanding are highlighted and discussed, to create opportunities for the development of anthocyanin-rich crops through breeding and environmental management.

  8. Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging

    Science.gov (United States)

    Pina, Edieidia S.; Silva, Denise B.; Teixeira, Simone P.; Coppede, Juliana S.; Furlan, Maysa; França, Suzelei C.; Lopes, Norberto P.; Pereira, Ana Maria S.; Lopes, Adriana A.

    2016-03-01

    Biosynthetic investigation of quinonemethide triterpenoid 22β-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using 13C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22β-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes.

  9. Structure-activity relationship of prenyl-substituted polyphenols from Artocarpus heterophyllus as inhibitors of melanin biosynthesis in cultured melanoma cells.

    Science.gov (United States)

    Arung, Enos Tangke; Shimizu, Kuniyoshi; Kondo, Ryuichiro

    2007-09-01

    A series of prenylated, flavone-based polyphenols, compounds 1-8, were isolated from the wood of Artocarpus heterophyllus. These compounds, which have previously been shown not to inhibit tyrosinase activity, were found to be active inhibitors of the in vivo melanin biosynthesis in B16 melanoma cells, with little or no cytotoxicity. To clarify the structural requirement for inhibition, some structure-activity relationships were studied, in comparison with related compounds lacking prenyl side chains. Our experiments indicate that both prenyl and OH groups, as well as the type of substitution pattern, are crucial for the inhibition of melanin production in B16 melanoma cells.

  10. Surfactin – A Review on Biosynthesis, Fermentation, Purification and Applications

    Directory of Open Access Journals (Sweden)

    Nikhil S. Shaligram

    2010-01-01

    Full Text Available Surfactin, a bacterial cyclic lipopeptide, is produced by various strains of Bacillus subtilis and is primarily recognized as one of the most effective biosurfactants. It has the ability to reduce surface tension of water from 72 to 27 mN/m at a concentration as low as 0.005 %. The structure of surfactin consists of seven amino acids bonded to the carboxyl and hydroxyl groups of a 14-carbon fatty acid. Surfactin possesses a number of biological activities such as the ability to lyse erythrocytes, inhibit clot formation, lyse bacterial spheroplasts and protoplasts, and inhibit cyclic 3',5-monophosphate diesterase. The high cost of production and low yields have limited its use in various commercial applications. Both submerged and solid-state fermentation have been investigated with the mutational approach to improve the productivity. In this review, current state of knowledge on biosynthesis of surfactin, its fermentative production, purification, analytical methods and biomedical applications is presented.

  11. Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

    International Nuclear Information System (INIS)

    Vidyashankar, Satyakumar; Nandakumar, Krishna S.; Patki, Pralhad S.

    2012-01-01

    Highlights: ► Ethanol induced cytotoxicity in HepG2 cells in absence of lipogenesis. ► Ethanol inhibited HMG-CoA reductase activity. ► Ethanol induced HMG-CoA reductase inhibition is due to decreased cell viability. ► Incubation with mevalonate could not increase the cholesterol. ► Cytotoxicity brought about by CoQ10 depletion and increased TNF-alpha. -- Abstract: Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP 450 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP 450 2E1. The results showed that ethanol at 100 mM concentration caused 40% cytotoxicity at 72 h as determined by MTT assay. The incorporation of labeled [2- 14 C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24 h incubation, whereas incorporation of labeled [2- 14 C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q 10 which is detrimental for cell viability. But vitamin E (10 mM) could partially restore coenzyme-Q 10 and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained

  12. Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Kirby, James [Univ. of California, Berkeley, CA (United States). California Institute of Quantitative Biosciences (QB3); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Dietzel, Kevin L. [Amyris, inc., Emeryville, CA (United States); Wichmann, Gale [Amyris, inc., Emeryville, CA (United States); Chan, Rossana [Univ. of California, Berkeley, CA (United States). California Institute of Quantitative Biosciences (QB3); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Antipov, Eugene [Amyris, inc., Emeryville, CA (United States); Moss, Nathan [Amyris, inc., Emeryville, CA (United States); Baidoo, Edward E. K. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Jackson, Peter [Amyris, inc., Emeryville, CA (United States); Gaucher, Sara P. [Amyris, inc., Emeryville, CA (United States); Gottlieb, Shayin [Amyris, inc., Emeryville, CA (United States); LaBarge, Jeremy [Amyris, inc., Emeryville, CA (United States); Mahatdejkul, Tina [Amyris, inc., Emeryville, CA (United States); Hawkins, Kristy M. [Amyris, inc., Emeryville, CA (United States); Muley, Sheela [Amyris, inc., Emeryville, CA (United States); Newman, Jack D. [Amyris, inc., Emeryville, CA (United States); Liu, Pinghua [Boston Univ., MA (United States). Dept. of Chemistry; Keasling, Jay D. [Univ. of California, Berkeley, CA (United States). California Institute of Quantitative Biosciences (QB3); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). Depts. of Chemical & Biomolecular Engineering and Bioengineering; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Technical Univ. of Denmark, Hoesholm (Denmark). Novo Nodisk Foundation Center for Biosustainability; Zhao, Lishan [Amyris, inc., Emeryville, CA (United States)

    2016-10-27

    Isoprenoids are made by all free-living organisms and range from essential metabolites like sterols and quinones to more complex compounds like pinene and rubber. They are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.

  13. The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae.

    Science.gov (United States)

    Carrión, Víctor J; Arrebola, Eva; Cazorla, Francisco M; Murillo, Jesús; de Vicente, Antonio

    2012-01-01

    Mangotoxin is an antimetabolite toxin produced by certain Pseudomonas syringae pv. syringae strains. This toxin is an oligopeptide that inhibits ornithine N-acetyl transferase, a key enzyme in the biosynthesis of ornithine and arginine. Previous studies have reported the involvement of the putative nonribosomal peptide synthetase MgoA in virulence and mangotoxin production. In this study, we analyse a new chromosomal region of P. syringae pv. syringae UMAF0158, which contains six coding sequences arranged as an operon (mbo operon). The mbo operon was detected in only mangotoxin-producing strains, and it was shown to be essential for the biosynthesis of this toxin. Mutants in each of the six ORFs of the mbo operon were partially or completely impaired in the production of the toxin. In addition, Pseudomonas spp. mangotoxin non-producer strains transformed with the mbo operon gained the ability to produce mangotoxin, indicating that this operon contains all the genetic information necessary for mangotoxin biosynthesis. The generation of a single transcript for the mbo operon was confirmed and supported by the allocation of a unique promoter and Rho-independent terminator. The phylogenetic analysis of the P. syringae strains harbouring the mbo operon revealed that these strains clustered together.

  14. The putative glutamate receptor 1.1 (AtGLR1.1) in Arabidopsis thaliana regulates abscisic acid biosynthesis and signaling to control development and water loss.

    Science.gov (United States)

    Kang, Jiman; Mehta, Sohum; Turano, Frank J

    2004-10-01

    The involvement of the putative glutamate receptor 1.1 (AtGLR1.1) gene in the regulation of abscisic acid (ABA) biosynthesis and signaling was investigated in Arabidopsis. Seeds from AtGLR1.1-deficient (antiAtGLR1.1) lines had increased sensitivity to exogenous ABA with regard to the effect of the hormone on the inhibition of seed germination and root growth. Seed germination, which was inhibited by an animal ionotropic glutamate receptor antagonist, 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione, was restored by co-incubation with an inhibitor of ABA biosynthesis, fluridone. These results confirm that germination in antiAtGLR1.1 lines was inhibited by increased ABA. When antiAtGLR1.1 and WT seeds were co-incubated in fluridone and exogenous ABA, the antiAtGLR1.1 seeds were more sensitive to ABA. In addition, the antiAtGLR1.1 lines exhibited altered expression of ABA biosynthetic (ABA) and signaling (ABI) genes, when compared with WT. Combining the physiological and molecular results suggest that ABA biosynthesis and signaling in antiAtGLR1.1 lines are altered. ABA levels in leaves of antiAtGLR1.1 lines are higher than those in WT. In addition, the antiAtGLR1.1 lines had reduced stomatal apertures, and exhibited enhanced drought tolerance due to deceased water loss compared with WT lines. The results from these experiments imply that ABA biosynthesis and signaling can be regulated through AtGLR1.1 to trigger pre- and post-germination arrest and changes in whole plant responses to water stress. Combined with our earlier results, these findings suggest that AtGLR1.1 integrates and regulates the different aspects of C, N and water balance that are required for normal plant growth and development.

  15. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

    1990-01-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  16. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

    International Nuclear Information System (INIS)

    Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

    1988-01-01

    Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14 CO 2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle

  17. Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes.

    Science.gov (United States)

    Kamiya, Regianne Umeko; Höfling, José Francisco; Gonçalves, Reginaldo Bruno

    2008-05-01

    The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I-IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=-0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

  18. Analysis of sublethal arsenic toxicity to Ceratophyllum demersum: subcellular distribution of arsenic and inhibition of chlorophyll biosynthesis

    Czech Academy of Sciences Publication Activity Database

    Mishra, S.; Alfred, M.; Sobotka, Roman; Andresen, E.; Falkenberg, G.; Küpper, Hendrik

    2016-01-01

    Roč. 67, č. 15 (2016), s. 4639-4646 ISSN 0022-0957 R&D Projects: GA ČR GBP501/12/G055; GA MŠk(CZ) LO1416 Institutional support: RVO:61388971 ; RVO:60077344 Keywords : arsenic toxicity * chlorophyll biosynthesis * subcellular distribution of arsenic * synchrotron micro-X-ray fluorescence Subject RIV: EE - Microbiology, Virology; CE - Biochemistry (BC-A) Impact factor: 5.830, year: 2016

  19. Glycoprotein biosynthesis by human normal platelets

    International Nuclear Information System (INIS)

    Rodriguez, P.; Bello, O.; Apitz-Castro, R.

    1987-01-01

    Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

  20. Glycopeptide antibiotic biosynthesis.

    Science.gov (United States)

    Yim, Grace; Thaker, Maulik N; Koteva, Kalinka; Wright, Gerard

    2014-01-01

    Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

  1. WRI1-1, ABI5, NF-YA3 and NF-YC2 increase oil biosynthesis in coordination with hormonal signaling during fruit development in oil palm.

    Science.gov (United States)

    Yeap, Wan-Chin; Lee, Fong-Chin; Shabari Shan, Dilip Kumar; Musa, Hamidah; Appleton, David Ross; Kulaveerasingam, Harikrishna

    2017-07-01

    The oil biosynthesis pathway must be tightly controlled to maximize oil yield. Oil palm accumulates exceptionally high oil content in its mesocarp, suggesting the existence of a unique fruit-specific fatty acid metabolism transcriptional network. We report the complex fruit-specific network of transcription factors responsible for modulation of oil biosynthesis genes in oil palm mesocarp. Transcriptional activation of EgWRI1-1 encoding a key master regulator that activates expression of oil biosynthesis genes, is activated by three ABA-responsive transcription factors, EgNF-YA3, EgNF-YC2 and EgABI5. Overexpression of EgWRI1-1 and its activators in Arabidopsis accelerated flowering, increased seed size and oil content, and altered expression levels of oil biosynthesis genes. Protein-protein interaction experiments demonstrated that EgNF-YA3 interacts directly with EgWRI1-1, forming a transcription complex with EgNF-YC2 and EgABI5 to modulate transcription of oil biosynthesis pathway genes. Furthermore, EgABI5 acts downstream of EgWRKY40, a repressor that interacts with EgWRKY2 to inhibit the transcription of oil biosynthesis genes. We showed that expression of these activators and repressors in oil biosynthesis can be induced by phytohormones coordinating fruit development in oil palm. We propose a model highlighting a hormone signaling network coordinating fruit development and fatty acid biosynthesis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  2. Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

    Science.gov (United States)

    Chen, Kan; Cao, Wanlu; Li, Juan; Sprengers, Dave; Hernanda, Pratika Y; Kong, Xiangdong; van der Laan, Luc JW; Man, Kwan; Kwekkeboom, Jaap; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-01-01

    As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA. PMID:26467706

  3. Biosynthesis of silver nanoparticles synthesized by Aspergillus ...

    Indian Academy of Sciences (India)

    Biotechnology Division, Applied Science Department, University of ... Abstract. In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic ... example of the biosynthesis using fungi was that the cell-.

  4. The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

    Science.gov (United States)

    Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N; Bach, Thomas J

    2009-01-01

    Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

  5. Use of [2-14C]mevalonate and saponin-bound [14C]-3-hydroxy-3-methylglutaric acid for the biosynthesis of terpenoids in leaves of Dioscorea deltoidea

    International Nuclear Information System (INIS)

    Gurielidze, K.G.; Paseshnichenko, V.A.; Vasil'eva, I.S.

    1986-01-01

    After the introduction of [2- 14 C]acetate into leaves of Dioscorea deltoidea, a radioactive furonanalog of deltafolin - protodeltofolin, containing two-thirds of the label in the 3-hydroxy-3-methylglutaryl portion - was isolated from them. Radioactive β-careotene and sterols were isolated from cut young leaves of Dioscorea 24 h after the introduction of [ 14 C] protodeltofolin into them, using chromatography on a column of silica gel and precipitation of sterols in the form of digitonins for this purpose. The incorporation of radioactivity from [ 14 C]-3-hydroxy-3-methyl-glutaric acid, bound in the form of a saponin, and β-carotene came to 0.18-0.80%, while incorporation into sterols came to 0.07-2.86% of the radioactivity of the alcohol extract. Thereby it was shown that 3-hydroxyl-3-methylglutaric acid, bound in the form of the saponin, can be used to form terpenoids in Dioscorea leaves. It was suggested that the binding of hydroxymethylglutaric acid to saponin represents one of the mechanisms of regulation of the rate of terpenoid biosynthesis in Dioscorea leaves

  6. Anthocyanin Biosynthesis and Degradation Mechanisms in Solanaceous Vegetables: A Review

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2018-03-01

    Full Text Available Anthocyanins are a group of polyphenolic pigments that are ubiquitously found in the plant kingdom. In plants, anthocyanins play a role not only in reproduction, by attracting pollinators and seed dispersers, but also in protection against various abiotic and biotic stresses. There is accumulating evidence that anthocyanins have health-promoting properties, which makes anthocyanin metabolism an interesting target for breeders and researchers. In this review, the state of the art knowledge concerning anthocyanins in the Solanaceous vegetables, i.e., pepper, tomato, eggplant, and potato, is discussed, including biochemistry and biological function of anthocyanins, as well as their genetic and environmental regulation. Anthocyanin accumulation is determined by the balance between biosynthesis and degradation. Although the anthocyanin biosynthetic pathway has been well-studied in Solanaceous vegetables, more research is needed on the inhibition of biosynthesis and, in particular, the anthocyanin degradation mechanisms if we want to control anthocyanin content of Solanaceous vegetables. In addition, anthocyanin metabolism is distinctly affected by environmental conditions, but the molecular regulation of these effects is poorly understood. Existing knowledge is summarized and current gaps in our understanding are highlighted and discussed, to create opportunities for the development of anthocyanin-rich crops through breeding and environmental management.

  7. Differential control of the cholesterol biosynthetic pathway in tumor versus liver: evidence for decontrolled tumor cholesterogenesis in a cell-free system

    International Nuclear Information System (INIS)

    Azrolan, N.

    1987-01-01

    Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant (PMS) systems prepared from both normal rat liver and Morris hepatoma 3924A. Per cell, the rate of cholesterol synthesis from either 14 C-citrate of 14 -acetate in the hepatoma system was 9-fold greater than that observed in the liver system. Furthermore, the ratio of sterol-to-fatty acid synthesis rates from 14 C-citrate was more than 3-fold greater in the tumor than in the normal liver system. Incubations using radiolabeled acetate and mevalonate have demonstrated the loss of a normally rate-limiting control site within the early portion of the cholesterol biosynthetic pathway in the tumor system. Upon analysis of the steady-state levels of early lipogenic intermediates, the specific site of decontrol in the tumor was identified as the 3-hydroxy-3-methylglutaryl-CoA → mevalonate site of this pathway. In contrast, this reaction appeared to retain its rate-limiting properties in the cell-free system from normal liver

  8. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

    1988-03-01

    Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  10. Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

    Directory of Open Access Journals (Sweden)

    Weiwei Dai

    2015-06-01

    Full Text Available Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1, S6, and insulin receptor substrate 1 (IRS-1 on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

  11. Natural product inhibitors of fatty acid biosynthesis: synthesis of the marine microbial metabolites pseudopyronines A and B and evaluation of their anti-infective activities

    DEFF Research Database (Denmark)

    Giddens, Anna C.; Nielsen, Lone; Boshoff, Helena I.

    2007-01-01

    of pathogenic microorganisms and were found to exhibit good potency (IC50≥0.46 μg/mL) and selectivity towards Leishmania donovani. Several of the compounds inhibited recombinant fatty acid biosynthesis enzymes from both Plasmodium falciparum and Mycobacterium tuberculosis, validating these targets in the search...

  12. Borrelidin B: isolation, biological activity, and implications for nitrile biosynthesis.

    Science.gov (United States)

    Schulze, Christopher J; Bray, Walter M; Loganzo, Frank; Lam, My-Hanh; Szal, Teresa; Villalobos, Anabella; Koehn, Frank E; Linington, Roger G

    2014-11-26

    Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.

  13. Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

    Science.gov (United States)

    Ma, Zhaoxue; Hu, Xupeng; Cai, Wenjuan; Huang, Weihua; Zhou, Xin; Luo, Qian; Yang, Hongquan; Wang, Jiawei; Huang, Jirong

    2014-01-01

    An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light. PMID:25101599

  14. pH-Signaling Transcription Factor AopacC Regulates Ochratoxin A Biosynthesis in Aspergillus ochraceus.

    Science.gov (United States)

    Wang, Yan; Liu, Fei; Wang, Liuqing; Wang, Qi; Selvaraj, Jonathan Nimal; Zhao, Yueju; Wang, Yun; Xing, Fuguo; Liu, Yang

    2018-05-02

    In Aspergillus and Penicillium species, an essential pH-response transcription factor pacC is involved in growth, pathogenicity, and toxigenicity. To investigate the connection between ochratoxin A (OTA) biosynthesis and ambient pH, the AopacC in Aspergillus ochraceus was functionally characterized using a loss-of-function mutant. The mycelium growth was inhibited under pH 4.5 and 10.0, while the sporulation increased under alkaline condition. A reduction of mycelium growth and an elevation of sporulation was observed in Δ AopacC mutant. Compared to neutral condition, OTA contents were respectively reduced by 71.6 and 79.8% under acidic and alkaline conditions. The expression of AopacC increased with the elevated pH, and deleting AopacC dramatically decreased OTA production and biosynthetic genes Aopks expression. Additionally, the Δ AopacC mutant exhibited attenuated infection ability toward pear fruits. These results suggest that AopacC is an alkaline-induced regulator responsible for growth and OTA biosynthesis in A. ochraceus and this regulatory mechanism might be pH-dependent.

  15. Arabidopsis chlorophyll biosynthesis: an essential balance between the methylerythritol phosphate and tetrapyrrole pathways.

    Science.gov (United States)

    Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

    2013-12-01

    Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)-derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis.

  16. Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

    Directory of Open Access Journals (Sweden)

    Chua Su-Kiat

    2009-05-01

    Full Text Available Abstract Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.

  17. Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

    Science.gov (United States)

    Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

    2016-03-01

    Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in

  18. Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

    International Nuclear Information System (INIS)

    Sekine, Yoshitaka; Furuya, Yosuke; Nishii, Masahiro; Koike, Hidekazu; Matsui, Hiroshi; Suzuki, Kazuhiro

    2008-01-01

    Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment

  19. Synergistic Radioprotection by Gamma-Tocotrienol and Pentoxifylline: Role of cAMP Signaling

    International Nuclear Information System (INIS)

    Kulkarni, Shilpa; Chakraborty, Kushal; Kumar, K. Sree; Kao, Tzu-Cheg; Hauer-Jensen, Martin; Ghosh, Sanchita P.

    2013-01-01

    Purpose. This study was designed to determine the efficacy and mechanisms of radioprotection by the combination of gamma-tocotrienol (GT3) and pentoxifylline (PTX) against acute radiation injury. Materials and Methods. Post-irradiation survival was monitored to determine the most efficacious dose and time of administration of PTX. Dose reduction factor (DRF) was calculated to compare the radioprotective efficacy of the combination. To determine the mechanism of synergistic radioprotection by the combination, mevalonate or calmodulin were coadministered with the GT3-PTX combination. Mevalonate was used to reverse the inhibitory effect of GT3 on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and calmodulin was used to reverse the inhibition of phosphodiesterase (PDE) by PTX. Results. The combination was most effective when 200 mg/kg of PTX was administered 15 min before irradiation along with 200 mg/kg of GT3 (−24 h) and resulted in a DRF of 1.5. White blood cells and neutrophil counts showed accelerated recovery in GT3-PTX-treated groups compared to GT3. Mevalonate had no effect on the radioprotection of GT3-PTX; calmodulin abrogated the synergistic radioprotection by GT3-PTX. Conclusion. The mechanism of radioprotection by GT3-PTX may involve PDE inhibition

  20. Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining.

    Directory of Open Access Journals (Sweden)

    Gang Wang

    2016-07-01

    Full Text Available Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1 of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes, proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.

  1. In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

    Science.gov (United States)

    Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

    2017-10-01

    For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

  2. Biosynthesis of silver nanoparticles by Aspergillus niger , Fusarium ...

    African Journals Online (AJOL)

    ... scanning electron microscope (SEM). Results indicate the synthesis of silver nanoparticles in the reaction mixture. The synthesis of nanoparticles would be suitable for developing a microbial nanotechnology biosynthesis process for mass scale production. Keywords: Silver nanoparticles, biosynthesis, fungi, Aspergillus.

  3. Inhibition of Lipid A Biosynthesis as the Primary Mechanism of CHIR-090 Antibiotic Activity in Escherichia coli

    Science.gov (United States)

    Barb, Adam W.; McClerren, Amanda L.; Snehelatha, Karnem; Reynolds, C. Michael; Zhou, Pei; Raetz, Christian R.H.

    2009-01-01

    The deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against P. aeruginosa and E. coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of Escherichia coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1 and k6 = 0.18 min-1. CHIR-090 at low nM levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including Pseudomonas aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 μM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 μg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway. PMID:17335290

  4. In vitro screening of inhibition of PPAR-γ activity as a first step in identification of potential breast carcinogens

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Lundqvist, J.; Petersen, R. K.

    2015-01-01

    and estrogen biosynthesis ultimately leading to breast cancer. If other organic solvents inhibit PPAR-γ activity, they should also lead to increased oestrogen biosynthesis and thus be potential breast carcinogens. Ten commonly used hydrophilic organic solvents were first tested in a cell-based screening assay...... followed by a well-established steroidogenesis assay for production of sex hormones in exposed H295 R cells may provide a screening tool for potential breast carcinogens. This initial screening thus identified ethylene glycol and possibly ethyl acetate as potential breast carcinogens....

  5. Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules.

    Science.gov (United States)

    Larrainzar, Estíbaliz; Molenaar, Johanna A; Wienkoop, Stefanie; Gil-Quintana, Erena; Alibert, Bénédicte; Limami, Anis M; Arrese-Igor, Cesar; González, Esther M

    2014-09-01

    Symbiotic nitrogen fixation is one of the first physiological processes inhibited in legume plants under water-deficit conditions. Despite the progress made in the last decades, the molecular mechanisms behind this regulation are not fully understood yet. Recent proteomic work carried out in the model legume Medicago truncatula provided the first indications of a possible involvement of nodule methionine (Met) biosynthesis and related pathways in response to water-deficit conditions. To better understand this involvement, the drought-induced changes in expression and content of enzymes involved in the biosynthesis of Met, S-adenosyl-L-methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen-fixing plants were subjected to a progressive water deficit and a subsequent recovery period. Besides the physiological characterization of the plants, the content of total sulphur, sulphate and main S-containing metabolites was measured. Results presented here show that S availability is not a limiting factor in the drought-induced decline of nitrogen fixation rates in M. truncatula plants and provide evidences for a down-regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water-deficit conditions. © 2014 John Wiley & Sons Ltd.

  6. Jasmonate mediates salt-induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Xiaodong Chen

    2016-04-01

    Full Text Available Jasmonate (JA, as an important signal, plays a key role in multiple processes of plant growth, development and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L. are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA-responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a-recognized G-box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.

  7. Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

    Science.gov (United States)

    Flynn, Christopher M; Schmidt-Dannert, Claudia

    2018-06-01

    The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide

  8. CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

    Science.gov (United States)

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-01-01

    NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

  9. The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells[C][W

    Science.gov (United States)

    Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N.; Bach, Thomas J.

    2009-01-01

    Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation. PMID:19136647

  10. Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)

    KAUST Repository

    Quitterer, Felix; Frank, Annika; Wang, Ke; Rao, Guodong; O'Dowd, Bing; Li, Jikun; Guerra, Francisco; Abdel-Azeim, Safwat; Bacher, Adelbert; Eppinger, Jö rg; Oldfield, Eric; Groll, Michael

    2015-01-01

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme’s structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

  11. Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)

    KAUST Repository

    Quitterer, Felix

    2015-04-11

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme’s structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

  12. Inhibition of β-galactosidase and α-glucosidase synthesis in petroleum refinery effluent bacteria by phenolic compounds

    Directory of Open Access Journals (Sweden)

    Gideon C. Okpokwasili

    2011-04-01

    Full Text Available Inhibition of α-glucosidase (EC 3.2.1.20 and β-galactosidase (EC 3.2.1.23 biosynthesis by phenolic compounds (phenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol in Escherichia coli, Bacillus and Pseudomonas species isolated from petroleum refinery wastewater was assessed. At sufficient concentrations, phenols inhibited the induction of α-glucosidase and β-galactosidase. The patterns of these toxic effects can be mathematically described with logistic and sigmoid dose-response models. The median inhibitory concentrations (IC50 varied among the phenols, the bacteria and enzymes. Quantitative structure–activity relationship (QSAR models based on the logarithm of the octanol–water partition coefficient (log10Kow were developed for each bacterium. The correlation coefficients varied between 0.84and 0.99 for the enzymes. The test results indicated α-glucosidase and β-galactosidase biosynthesis as important microbial indices for evaluation of toxicity of phenolic compounds.

  13. [In vitro study over statins effects on cellular growth curves and its reversibility with mevalonate].

    Science.gov (United States)

    Millan Núñez-Cortés, Jesús; Alvarez Rodriguez, Ysmael; Alvarez Novés, Granada; Recarte Garcia-Andrade, Carlos; Alvarez-Sala Walther, Luis

    2014-01-01

    HMG-CoA-Reductase inhibitors, also known as statins, are currently the most powerful cholesterol-lowering drugs available on the market. Clinical trials and experimental evidence suggest that statins have heavy anti-atherosclerotic effects. These are in part consequence of lipid lowering but also result from pleiotropic actions of the drugs. These so-called pleiotropic properties affect various aspects of cell function, inflammation, coagulation, and vasomotor activity. These effects are mediated either indirectly through LDL-c reduction or via a direct effect on cellular functions. Although many of the pleiotropic properties of statins may be a class effect, some may be unique to certain agents and account for differences in their pharmacological activity. So, although statins typically have similar effects on LDL-c levels, differences in chemical structure and pharmacokinetic profile can lead to variations in pleiotropic effects. In this paper we analize the in vitro effects of different statins over different cell lines from cells implicated in atherosclerotic process: endothelial cells, fibroblasts, and vascular muscular cells. In relation with our results we can proof that the effects of different dosis of different statins provides singular effects over growth curves of different cellular lines, a despite of a class-dependent effects. So, pleiotropic effects and its reversibility with mevalonate are different according with the molecule and the dosis. Copyright © 2013 Elsevier España, S.L. y SEA. All rights reserved.

  14. Biosynthesis and function of chondroitin sulfate.

    Science.gov (United States)

    Mikami, Tadahisa; Kitagawa, Hiroshi

    2013-10-01

    Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin: potential therapeutic relevance

    International Nuclear Information System (INIS)

    Martirosyan, Anna; Clendening, James W; Goard, Carolyn A; Penn, Linda Z

    2010-01-01

    Ovarian carcinoma is a rarely curable disease, for which new treatment options are required. As agents that block HMG-CoA reductase and the mevalonate pathway, the statin family of drugs are used in the treatment of hypercholesterolemia and have been shown to trigger apoptosis in a tumor-specific manner. Recent clinical trials show that the addition of statins to traditional chemotherapeutic strategies can increase efficacy of targeting statin-sensitive tumors. Our goal was to assess statin-induced apoptosis of ovarian cancer cells, either alone or in combination with chemotherapeutics, and then determine these mechanisms of action. The effect of lovastatin on ovarian cancer cell lines was evaluated alone and in combination with cisplatin and doxorubicin using several assays (MTT, TUNEL, fixed PI, PARP cleavage) and synergy determined by evaluating the combination index. The mechanisms of action were evaluated using functional, molecular, and pharmacologic approaches. We demonstrate that lovastatin induces apoptosis of ovarian cancer cells in a p53-independent manner and synergizes with doxorubicin, a chemotherapeutic agent used to treat recurrent cases of ovarian cancer. Lovastatin drives ovarian tumor cell death by two mechanisms: first, by blocking HMG-CoA reductase activity, and second, by sensitizing multi-drug resistant cells to doxorubicin by a novel mevalonate-independent mechanism. This inhibition of drug transport, likely through inhibition of P-glycoprotein, potentiates both DNA damage and tumor cell apoptosis. The results of this research provide pre-clinical data to warrant further evaluation of statins as potential anti-cancer agents to treat ovarian carcinoma. Many statins are inexpensive, off-patent generic drugs that are immediately available for use as anti-cancer agents. We provide evidence that lovastatin triggers apoptosis of ovarian cancer cells as a single agent by a mevalonate-dependent mechanism. Moreover, we also show lovastatin synergizes

  16. Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Igarashi, K; Porter, C W; Morris, D R

    1984-11-01

    Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

  17. Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

    Science.gov (United States)

    Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

    2012-01-01

    Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

  18. Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

    Science.gov (United States)

    Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

    2017-01-01

    In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

  19. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

    KAUST Repository

    Meier, Stuart; Tzfadia, Oren; Vallabhaneni, Ratnakar; Gehring, Christoph A; Wurtzel, Eleanore T

    2011-01-01

    Background: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.Results: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of

  20. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

    KAUST Repository

    Meier, Stuart

    2011-05-19

    Background: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.Results: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of

  1. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Vallabhaneni Ratnakar

    2011-05-01

    Full Text Available Abstract Background The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana. Results A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR but was inhibited by abscisic acid (ABA. Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced

  2. Ebselen is a new skin depigmenting agent that inhibits melanin biosynthesis and melanosomal transfer.

    Science.gov (United States)

    Kasraee, Behrooz; Nikolic, Damjan S; Salomon, Denis; Carraux, Pierre; Fontao, Lionel; Piguet, Vincent; Omrani, Gholamhossein R; Sorg, Olivier; Saurat, Jean-Hilaire

    2012-01-01

    We assessed the ability of ebselen, a glutathione peroxidase mimic, to reduce pigmentation in various models. In murine B16 melanocytes, 25 μm ebselen inhibited melanogenesis and induced a depolymerisation of actin filaments. In co-cultures of B16 melanocytes with BDVII keratinocytes, a pretreatment of melanocytes with ebselen resulted in a strong inhibition of melanosome transfer to keratinocytes, as shown under optical and electron microscopy. In reconstructed epidermis, topical 0.5% ebselen led to a twofold decrease of melanin without affecting the density of active melanocytes. A similar result was obtained with topical 0.5% ebselen in black guinea pig ears. Ebselen induced a decrease of epidermal melanin parallel to a localisation of melanin and melanosomes in the basal layer. Ebselen appears as a new depigmenting compound that inhibits melanin synthesis and melanosome transfer to keratinocytes. © 2011 John Wiley & Sons A/S.

  3. Molecular Link between Leaf Coloration and Gene Expression of Flavonoid and Carotenoid Biosynthesis in Camellia sinensis Cultivar ‘Huangjinya’

    Directory of Open Access Journals (Sweden)

    Lubin Song

    2017-05-01

    Full Text Available ‘Huangjinya’ is an excellent albino tea germplasm cultivated in China because of its bright color and high amino acid content. It is light sensitive, with yellow leaves under intense light while green leaves under weak light. As well, the flavonoid and carotenoid levels increased after moderate shading treatment. However, the mechanism underlying this interesting phenomenon remains unclear. In this study, the transcriptome of ‘Huangjinya’ plants exposed to sunlight and shade were analyzed by high-throughput sequencing followed by de novo assembly. Shading ‘Huangjinya’ made its leaf color turn green. De novo assembly showed that the transcriptome of ‘Huangjinya’ leaves comprises of 127,253 unigenes, with an average length of 914 nt. Among the 81,128 functionally annotated unigenes, 207 differentially expressed genes were identified, including 110 up-regulated and 97 down-regulated genes under moderate shading compared to full light. Gene ontology (GO indicated that the differentially expressed genes are mainly involved in protein and ion binding and oxidoreductase activity. Antioxidation-related pathways, including flavonoid and carotenoid biosynthesis, were highly enriched in these functions. Shading inhibited the expression of flavonoid biosynthesis-associated genes and induced carotenoid biosynthesis-related genes. This would suggest that decreased flavonoid biosynthetic gene expression coincides with increased flavonoids (e.g., catechin content upon moderate shading, while carotenoid levels and biosynthetic gene expression are positively correlated in ‘Huangjinya.’ In conclusion, the leaf color changes in ‘Huangjinya’ are largely determined by the combined effects of flavonoid and carotenoid biosynthesis.

  4. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin

    2017-08-10

    The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

  5. A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2.

    Science.gov (United States)

    Ku, Jason T; Lan, Ethan I

    2018-03-01

    Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO 2 is an attractive direction for both sustainability and CO 2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  6. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  7. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    Science.gov (United States)

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Regulatory cross-talks and cascades in rice hormone biosynthesis pathways contribute to stress signaling

    Directory of Open Access Journals (Sweden)

    Arindam Deb

    2016-08-01

    Full Text Available Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each other’s production directly. Thus multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones.

  9. Differential expression of jasmonate biosynthesis genes in cacao genotypes contrasting for resistance against Moniliophthora perniciosa.

    Science.gov (United States)

    Litholdo, Celso G; Leal, Gildemberg A; Albuquerque, Paulo S B; Figueira, Antonio

    2015-10-01

    The resistance mechanism of cacao against M. perniciosa is likely to be mediated by JA/ET-signaling pathways due to the preferential TcAOS and TcSAM induction in a resistant genotype. The basidiomycete Moniliophthora perniciosa causes a serious disease in cacao (Theobroma cacao L.), and the use of resistant varieties is the only sustainable long-term solution. Cacao resistance against M. perniciosa is characterized by pathogen growth inhibition with reduced colonization and an attenuation of disease symptoms, suggesting a regulation by jasmonate (JA)/ethylene (ET) signaling pathways. The hypothesis that genes involved in JA biosynthesis would be active in the interaction of T. cacao and M. perniciosa was tested here. The cacao JA-related genes were evaluated for their relative quantitative expression in susceptible and resistant genotypes upon the exogenous application of ET, methyl-jasmonate (MJ), and salicylic acid (SA), or after M. perniciosa inoculation. MJ treatment triggered changes in the expression of genes involved in JA biosynthesis, indicating that the mechanism of positive regulation by exogenous MJ application occurs in cacao. However, a higher induction of these genes was observed in the susceptible genotype. Further, a contrast in JA-related transcriptional expression was detected between susceptible and resistant plants under M. perniciosa infection, with the induction of the allene oxide synthase gene (TcAOS), which encodes a key enzyme in the JA biosynthesis pathway in the resistant genotype. Altogether, this work provides additional evidences that the JA-dependent signaling pathway is modulating the defense response against M. perniciosa in a cacao-resistant genotype.

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

    Directory of Open Access Journals (Sweden)

    Kevin A Robertson

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Loto Iris

    2012-10-01

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

  13. Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF.

    Science.gov (United States)

    Hrast, Martina; Turk, Samo; Sosič, Izidor; Knez, Damijan; Randall, Christopher P; Barreteau, Hélène; Contreras-Martel, Carlos; Dessen, Andréa; O'Neill, Alex J; Mengin-Lecreulx, Dominique; Blanot, Didier; Gobec, Stanislav

    2013-08-01

    Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  14. FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

    Science.gov (United States)

    Zara, Giacomo; Goffrini, Paola; Lodi, Tiziana; Zara, Severino; Mannazzu, Ilaria; Budroni, Marilena

    2012-11-01

    Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. Effects of inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Catarina M Quinzii

    Full Text Available Coenzyme Q(10 (CoQ(10 is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ(10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10 biosynthesis. We observed a unimodal distribution of ROS production with CoQ(10 deficiency: cells with <20% of CoQ(10 and 50-70% of CoQ(10 did not generate excess ROS while cells with 30-45% of CoQ(10 showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ(10 deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB, an analog of 4-hydroxybenzoate (4-HB and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2 to induce a range of CoQ(10 deficiencies. Our results support the concept that the degree of CoQ(10 deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40-50% residual CoQ(10 produces maximal oxidative stress and cell death.

  16. The expanding universe of alkaloid biosynthesis.

    Science.gov (United States)

    De Luca, V; Laflamme, P

    2001-06-01

    Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

  17. Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells

    International Nuclear Information System (INIS)

    Jacobsson, K.G.; Riesenfeld, J.; Lindahl, U.

    1985-01-01

    Murine mastocytoma cells were incubated in vitro with inorganic [ 35 S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion of components with high affinity for antithrombin. Structural analysis of heparin labeled with [ 3 H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. A polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[ 3 H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell

  18. Genetic Control of Ascorbic Acid Biosynthesis and Recycling in Horticultural Crops

    Directory of Open Access Journals (Sweden)

    Ifigeneia Mellidou

    2017-07-01

    Full Text Available Ascorbic acid (AsA is an essential compound present in almost all living organisms that has important functions in several aspects of plant growth and development, hormone signaling, as well as stress defense networks. In recent years, the genetic regulation of AsA metabolic pathways has received much attention due to its beneficial role in human diet. Despite the great variability within species, genotypes, tissues and developmental stages, AsA accumulation is considered to be controlled by the fine orchestration of net biosynthesis, recycling, degradation/oxidation, and/or intercellular and intracellular transport. To date, several structural genes from the AsA metabolic pathways and transcription factors are considered to significantly affect AsA in plant tissues, either at the level of activity, transcription or translation via feedback inhibition. Yet, all the emerging studies support the notion that the steps proceeding through GDP-L-galactose phosphorylase and to a lesser extent through GDP-D-mannose-3,5-epimerase are control points in governing AsA pool size in several species. In this mini review, we discuss the current consensus of the genetic regulation of AsA biosynthesis and recycling, with a focus on horticultural crops. The aspects of AsA degradation and transport are not discussed herein. Novel insights of how this multifaceted trait is regulated are critical to prioritize candidate genes for follow-up studies toward improving the nutritional value of fruits and vegetables.

  19. 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

  20. Rapid biosynthesis of cadmium sulfide (CdS) nanoparticles using ...

    African Journals Online (AJOL)

    Rapid biosynthesis of cadmium sulfide (CdS) nanoparticles using culture supernatants of Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633 and Lactobacillus ... The process of extracellular and fast biosynthesis may help in the development of an easy and eco-friendly route for the synthesis of CdS nanoparticles.

  1. 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. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  2. Biosynthesis of the major tetrahydroxystilbenes in spruce, astringin and isorhapontin, proceeds via resveratrol and is enhanced by fungal infection.

    Science.gov (United States)

    Hammerbacher, Almuth; Ralph, Steven G; Bohlmann, Joerg; Fenning, Trevor M; Gershenzon, Jonathan; Schmidt, Axel

    2011-10-01

    Stilbenes are dibenzyl polyphenolic compounds produced in several unrelated plant families that appear to protect against various biotic and abiotic stresses. Stilbene biosynthesis has been well described in economically important plants, such as grape (Vitis vinifera), peanut (Arachis hypogaea), and pine (Pinus species). However, very little is known about the biosynthesis and ecological role of stilbenes in spruce (Picea), an important gymnosperm tree genus in temperate and boreal forests. To investigate the biosynthesis of stilbenes in spruce, we identified two similar stilbene synthase (STS) genes in Norway spruce (Picea abies), PaSTS1 and PaSTS2, which had orthologs with high sequence identity in sitka (Picea sitchensis) and white (Picea glauca) spruce. Despite the conservation of STS sequences in these three spruce species, they differed substantially from angiosperm STSs. Several types of in vitro and in vivo assays revealed that the P. abies STSs catalyze the condensation of p-coumaroyl-coenzyme A and three molecules of malonyl-coenzyme A to yield the trihydroxystilbene resveratrol but do not directly form the dominant spruce stilbenes, which are tetrahydroxylated. However, in transgenic Norway spruce overexpressing PaSTS1, significantly higher amounts of the tetrahydroxystilbene glycosides, astringin and isorhapontin, were produced. This result suggests that the first step of stilbene biosynthesis in spruce is the formation of resveratrol, which is further modified by hydroxylation, O-methylation, and O-glucosylation to yield astringin and isorhapontin. Inoculating spruce with fungal mycelium increased STS transcript abundance and tetrahydroxystilbene glycoside production. Extracts from STS-overexpressing lines significantly inhibited fungal growth in vitro compared with extracts from control lines, suggesting that spruce stilbenes have a role in antifungal defense.

  3. De novo Biosynthesis of "Non-Natural" Thaxtomin Phytotoxins.

    Science.gov (United States)

    Winn, Michael; Francis, Daniel; Micklefield, Jason

    2018-03-30

    Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to "non-natural" product target compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

    Science.gov (United States)

    Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frédéric; Nicolè, Florence; Raymond, Olivier; Huguet, Stéphanie; Baltenweck, Raymonde; Meyer, Sophie; Claudel, Patricia; Jeauffre, Julien; Rohmer, Michel; Foucher, Fabrice; Hugueney, Philippe; Bendahmane, Mohammed; Baudino, Sylvie

    2015-07-03

    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 contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta. Copyright © 2015, American Association for the Advancement of Science.

  5. Microbial biosynthesis of nontoxic gold nanoparticles

    International Nuclear Information System (INIS)

    Roy, Swarup; Das, Tapan Kumar; Maiti, Guru Prasad; Basu, Utpal

    2016-01-01

    Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

  6. Microbial biosynthesis of nontoxic gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Swarup, E-mail: swaruproy@klyuniv.ac.in [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Das, Tapan Kumar [Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal (India); Maiti, Guru Prasad [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India); Department of Anesthesiology, Texas Tech University Health science Center, 3601 4th Street, Lubbock, TX 79430 (United States); Basu, Utpal [Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani 741235, West Bengal (India)

    2016-01-15

    Graphical abstract: The manuscript deals with the fungus mediated optimized biologically synthesized GNPs using Aspergillus foetidus and characterization of biosynthesized GNPs using various physico-chemical methods. The fairly stable synthesized nanoparticles have size in the range of 10–40 nm. Cytotoxicity study of biosynthesized GNPs on Human lung cancer cell line A549 showed no significant toxicity of GNPs. - Highlights: • A novel biosynthesis process of GNPs using Aspergillus foetidus. • Biosynthesized GNPs are in the range of 10–40 nm as observed from TEM. • This process of synthesis is an optimized biosynthesis process of GNPs. • Biosynthesized GNPs are noncytotoxic against A549 cell line. - Abstract: We study the extracellular biosynthesis of gold nanoparticles (GNPs) using the fungal species Aspergillus foetidus. The formation of GNPs were initially monitored by visual observation and then characterized with the help of various characterization techniques. X-ray diffraction (XRD) results revealed distinctive formation of face centered cubic crystalline GNPs. From field emission scanning electron microscopy (FESEM) the morphology of the nanoparticles were found to be roughly spherical and within the size range of 30–50 nm. The spherical and polydispersed GNPs in the range of 10–40 nm were observed by transmission electron microscopy (TEM) analysis. It was established that alkaline pH, 1 mM gold salt concentration and 75 °C temperature were the respective optimum parameter for biosynthesis of GNPs. Cell cytotoxicity of GNP was compared with that of normal gold salt solution on A549 cell. The A549 cell growth in presence of GNPs was found to be comparatively less toxic than the gold ion.

  7. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    Science.gov (United States)

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

  8. Rare cause of post-squalene disorder of cholesterol biosynthesis ...

    African Journals Online (AJOL)

    Errors of cholesterol biosynthesis represent a heterogeneous group of metabolic disorders. The aim of the authors of this article is to present a case of a patient with typical symptoms of a rare post-squalene disorder of cholesterol biosynthesis, its diagnostics and progress in neonatal period. The differential diagnosis of a ...

  9. 46_ _267 - 278__Aminu- Biosynthesis

    African Journals Online (AJOL)

    User

    ISSN 2006 – 6996. BIOSYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL STUDY OF .... the excitation of surface Plasmon vibration with. AgNPs. ... Thin films of the sample were prepared on a carbon ... The resulting film on the SEM.

  10. Proteome analysis provides insight into the regulation of bioactive metabolites in Hericium erinaceus.

    Science.gov (United States)

    Zeng, Xu; Ling, Hong; Yang, Jianwen; Chen, Juan; Guo, Shunxing

    2018-05-05

    Hericium erinaceus, a famous edible mushroom, is also a well-known traditional medicinal fungus. To date, a large number of bioactive metabolites with antitumor, antibacterial, and immune-boosting effects were isolated from the free-living mycelium and fruiting body of H. erinaceus. Here we used the proteomic approach to explore proteins involved in the regulation of bioactive metabolites, including terpenoid, polyketide, sterol and etc. RESULTS: Using mass spectrometry, a total of 2543 unique proteins were identified using H. erinaceus genome, of which 2449, 1855, 1533 and 690 proteins were successfully annotated in Nr, KOG, KEGG and GO databases. Among them, 722 proteins were differentially expressed (528 up- and 194 down-regulated) in fruiting body compared with mycelium. Most of differentially expressed proteins were putatively involved in energy metabolism, molecular signaling, and secondary metabolism. Additionally, numerous proteins involved in terpenoid, polyketide, and sterol biosynthesis were identified. Our data revealed that proteins involved in polyketide biosynthesis were up-regulated in the fruiting body, while some proteins in mevalonate (MEP) pathway from terpenoid biosynthesis were generally up-regulated in mycelium. The present study suggested that the differential regulation of biosynthesis genes could produce various bioactive metabolites with pharmacological effects in H. erinaceus. Copyright © 2017. Published by Elsevier B.V.

  11. Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001.

    Science.gov (United States)

    Gumel, A M; Annuar, M S M; Heidelberg, T

    2014-01-01

    Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 77 kDa.

  12. Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001

    Directory of Open Access Journals (Sweden)

    A.M. Gumel

    2014-06-01

    Full Text Available Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1 and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w and PHA yields ranging from 10.12 g L-1 to 15.45 g L-1, respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7 monomer was also detected when C18:1 was fed. Polymer showed melting temperature (Tm of 42.0 (± 0.2 °C, glass transition temperature (Tg of -1.0 (± 0.2 °C and endothermic melting enthalpy of fusion (ΔHf of 110.3 (± 0.1 J g-1. The molecular weight (Mw range of the polymer was relatively narrow between 55 to 77 kDa.

  13. Recent advances in combinatorial biosynthesis for drug discovery

    Directory of Open Access Journals (Sweden)

    Sun H

    2015-02-01

    Full Text Available Huihua Sun,1,* Zihe Liu,1,* Huimin Zhao,1,2 Ee Lui Ang1 1Metabolic Engineering Research Laboratory, Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research, Singapore; 2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA *These authors contributed equally to this work Abstract: Because of extraordinary structural diversity and broad biological activities, natural products have played a significant role in drug discovery. These therapeutically important secondary metabolites are assembled and modified by dedicated biosynthetic pathways in their host living organisms. Traditionally, chemists have attempted to synthesize natural product analogs that are important sources of new drugs. However, the extraordinary structural complexity of natural products sometimes makes it challenging for traditional chemical synthesis, which usually involves multiple steps, harsh conditions, toxic organic solvents, and byproduct wastes. In contrast, combinatorial biosynthesis exploits substrate promiscuity and employs engineered enzymes and pathways to produce novel “unnatural” natural products, substantially expanding the structural diversity of natural products with potential pharmaceutical value. Thus, combinatorial biosynthesis provides an environmentally friendly way to produce natural product analogs. Efficient expression of the combinatorial biosynthetic pathway in genetically tractable heterologous hosts can increase the titer of the compound, eventually resulting in less expensive drugs. In this review, we will discuss three major strategies for combinatorial biosynthesis: 1 precursor-directed biosynthesis; 2 enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution; 3 pathway-level recombination. Recent examples of combinatorial biosynthesis employing these

  14. Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

    Science.gov (United States)

    McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

    2017-07-07

    NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Effects of drugs inhibiting prostaglandin or leukotriene biosynthesis on postirradiation haematopoiesis in mouse

    International Nuclear Information System (INIS)

    Kozubik, A.; Hofmanova, J.; Pospisil, M.; Netikova, J.; Hola, J.; Lojek, A.

    1994-01-01

    Two non-steroidal anti-inflammatory drugs, i.e. indomethacin (INDO), an inhibitor of prostaglandin production, and esculetin (ESCUL), an inhibitor of leukotriene production, were tested for their ability to modify haematopoiesis in three experimental systems: (a) in vitro clonal proliferation of marrow GM-CFC from the irradiated mouse was found to be augmented by addition of INDO at a low concentration, and inhibited by ESCUL in a dose-dependent manner; (b) in the lethally irradiated and bone marrow-transplanted mice treated with the drugs in the postirradiation period, stimulatory effects of INDO on CFU-S and GM-CFC populations and an inhibitory effect of ESCUL on GM-CFC were observed; and (c) when the drugs were administered i.p. to mice 1 h before 5-Gy irradiation, INDO enhanced the postirradiation recovery of haematopoietic indices such the numbers of CFU-S, GM-CFC, peripheral blood granuloctyes, and nucleated bone marrow cells, while ESCUL had no effect or even inhibited the recovery of these indices. (author)

  16. Effects of drugs inhibiting prostaglandin or leukotriene biosynthesis on postirradiation haematopoiesis in mouse

    Energy Technology Data Exchange (ETDEWEB)

    Kozubik, A.; Hofmanova, J.; Pospisil, M.; Netikova, J.; Hola, J.; Lojek, A. (Ceskoslovenska Akademie Ved, Brno (Czech Republic). Biofysikalni Ustav)

    1994-03-01

    Two non-steroidal anti-inflammatory drugs, i.e. indomethacin (INDO), an inhibitor of prostaglandin production, and esculetin (ESCUL), an inhibitor of leukotriene production, were tested for their ability to modify haematopoiesis in three experimental systems: (a) in vitro clonal proliferation of marrow GM-CFC from the irradiated mouse was found to be augmented by addition of INDO at a low concentration, and inhibited by ESCUL in a dose-dependent manner; (b) in the lethally irradiated and bone marrow-transplanted mice treated with the drugs in the postirradiation period, stimulatory effects of INDO on CFU-S and GM-CFC populations and an inhibitory effect of ESCUL on GM-CFC were observed; and (c) when the drugs were administered i.p. to mice 1 h before 5-Gy irradiation, INDO enhanced the postirradiation recovery of haematopoietic indices such the numbers of CFU-S, GM-CFC, peripheral blood granuloctyes, and nucleated bone marrow cells, while ESCUL had no effect or even inhibited the recovery of these indices. (author).

  17. Downregulation of monocytic differentiation via modulation of CD147 by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

    Directory of Open Access Journals (Sweden)

    Manda V Sasidhar

    Full Text Available CD147 is an activation induced glycoprotein that promotes the secretion and activation of matrix metalloproteinases (MMPs and is upregulated during the differentiation of macrophages. Interestingly, some of the molecular functions of CD147 rely on its glycosylation status: the highly glycosylated forms of CD147 induce MMPs whereas the lowly glycosylated forms inhibit MMP activation. Statins are hydroxy-methylglutaryl coenzyme A reductase inhibitors that block the synthesis of mevalonate, thereby inhibiting all mevalonate-dependent pathways, including isoprenylation, N-glycosylation and cholesterol synthesis. In this study, we investigated the role of statins in the inhibition of macrophage differentiation and the associated process of MMP secretion through modulation of CD147. We observed that differentiation of the human monocytic cell line THP-1 to a macrophage phenotype led to upregulation of CD147 and CD14 and that this effect was inhibited by statins. At the molecular level, statins altered CD147 expression, structure and function by inhibiting isoprenylation and N-glycosylation. In addition, statins induced a shift of CD147 from its highly glycosylated form to its lowly glycosylated form. This shift in N-glycosylation status was accompanied by a decrease in the production and functional activity of MMP-2 and MMP-9. In conclusion, these findings describe a novel molecular mechanism of immune regulation by statins, making them interesting candidates for autoimmune disease therapy.

  18. Downregulation of monocytic differentiation via modulation of CD147 by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

    Science.gov (United States)

    Sasidhar, Manda V; Chevooru, Sai Krishnaveni; Eickelberg, Oliver; Hartung, Hans-Peter; Neuhaus, Oliver

    2017-01-01

    CD147 is an activation induced glycoprotein that promotes the secretion and activation of matrix metalloproteinases (MMPs) and is upregulated during the differentiation of macrophages. Interestingly, some of the molecular functions of CD147 rely on its glycosylation status: the highly glycosylated forms of CD147 induce MMPs whereas the lowly glycosylated forms inhibit MMP activation. Statins are hydroxy-methylglutaryl coenzyme A reductase inhibitors that block the synthesis of mevalonate, thereby inhibiting all mevalonate-dependent pathways, including isoprenylation, N-glycosylation and cholesterol synthesis. In this study, we investigated the role of statins in the inhibition of macrophage differentiation and the associated process of MMP secretion through modulation of CD147. We observed that differentiation of the human monocytic cell line THP-1 to a macrophage phenotype led to upregulation of CD147 and CD14 and that this effect was inhibited by statins. At the molecular level, statins altered CD147 expression, structure and function by inhibiting isoprenylation and N-glycosylation. In addition, statins induced a shift of CD147 from its highly glycosylated form to its lowly glycosylated form. This shift in N-glycosylation status was accompanied by a decrease in the production and functional activity of MMP-2 and MMP-9. In conclusion, these findings describe a novel molecular mechanism of immune regulation by statins, making them interesting candidates for autoimmune disease therapy.

  19. A fluorescent probe distinguishes between inhibition of early and late steps of lipopolysaccharide biogenesis in whole cells

    Science.gov (United States)

    Moison, Eileen; Xie, Ran; Zhang, Ge; Lebar, Matthew D.; Meredith, Timothy C.; Kahne, Daniel

    2017-01-01

    Lipopolysaccharide (LPS) biogenesis in Gram-negative organisms involves its biosynthesis in the cytoplasm and subsequent transport across three cellular compartments to the cell surface. We developed a fluorescent probe that allows us to determine the spatial distribution of LPS in whole cells. We show that polymyxin B nonapeptide (PMBN) containing a dansyl fluorophore specifically binds to LPS in membranes. We show that this probe detects decreases in LPS levels on the cell surface when LPS biosynthesis is inhibited at an early step. We also can detect accumulation of LPS in particular subcellular locations when LPS assembly is blocked during transport, allowing us to differentiate inhibitors targeting early and late stages of LPS biogenesis. PMID:28248483

  20. Transcriptomic analysis of the red seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta and its microbiome

    Directory of Open Access Journals (Sweden)

    de Oliveira Louisi

    2012-09-01

    Full Text Available Abstract Background Seaweeds of the Laurencia genus have a broad geographic distribution and are largely recognized as important sources of secondary metabolites, mainly halogenated compounds exhibiting diverse potential pharmacological activities and relevant ecological role as anti-epibiosis. Host-microbe interaction is a driving force for co-evolution in the marine environment, but molecular studies of seaweed-associated microbial communities are still rare. Despite the large amount of research describing the chemical compositions of Laurencia species, the genetic knowledge regarding this genus is currently restricted to taxonomic markers and general genome features. In this work we analyze the transcriptomic profile of L. dendroidea J. Agardh, unveil the genes involved on the biosynthesis of terpenoid compounds in this seaweed and explore the interactions between this host and its associated microbiome. Results A total of 6 transcriptomes were obtained from specimens of L. dendroidea sampled in three different coastal locations of the Rio de Janeiro state. Functional annotations revealed predominantly basic cellular metabolic pathways. Bacteria was the dominant active group in the microbiome of L. dendroidea, standing out nitrogen fixing Cyanobacteria and aerobic heterotrophic Proteobacteria. The analysis of the relative contribution of each domain highlighted bacterial features related to glycolysis, lipid and polysaccharide breakdown, and also recognition of seaweed surface and establishment of biofilm. Eukaryotic transcripts, on the other hand, were associated with photosynthesis, synthesis of carbohydrate reserves, and defense mechanisms, including the biosynthesis of terpenoids through the mevalonate-independent pathway. Conclusions This work describes the first transcriptomic profile of the red seaweed L. dendroidea, increasing the knowledge about ESTs from the Florideophyceae algal class. Our data suggest an important role for L

  1. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    Energy Technology Data Exchange (ETDEWEB)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-08-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: (/sup 3/H)thymidine incorporation decreased by 80% and (/sup 14/C)leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of (/sup 3/H)histidine/(/sup 14/C)leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

  2. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    International Nuclear Information System (INIS)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-01-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: [ 3 H]thymidine incorporation decreased by 80% and [ 14 C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [ 3 H]histidine/[ 14 C]leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine

  3. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    Directory of Open Access Journals (Sweden)

    Noreen F Rizvi

    Full Text Available The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs, including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs with the plant hormone, methyl jasmonate (MJ, while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM. However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str, illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

  4. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Effect of chlorpromazine on rat arterial lipid synthesis, in vitro

    International Nuclear Information System (INIS)

    Bell, F.P.; Hubert, E.V.

    1982-01-01

    The effect of chlorpromazine, a major tranquilizer, on arterial lipid metabolism was studied in vitro in rat aortas incubated with [ 14 C]acetate and [ 14 C]mevalonate as lipid precursors. Chlorpromazine at a level of 0.25 mM in the incubation medium significantly reduced the incorporation of [ 14 C]acetate into free fatty acids (p less than 0.01) and total phospholipids (p less than 0.001) but not triglycerides. Chlorpromazine also altered the pattern of arterial phospholipids synthesized from [ 14 C]acetate by significantly increasing the relative proportion of phosphatidylinositol plus phosphatidylserine (p less than 0.02) and reducing the relative proportion of sphingomyelin (p less than 0.001). [ 14 C] Acetate incorporation into the combined fractions of steryl esters plus hydrocarbons and sterols plus diglycerides was also significantly reduced (p less than 0.001) by 0.25 mM chlorpromazine. Studies with [ 14 C]mevalonate showed that chlorpromazine is also an inhibitor of sterol biosynthesis in arterial tissues as evidenced by 35-40% reductions (p less than 0.05) in the formation of 14 C-labeled squalene and C27 sterols

  6. Investigations on the mechanism of oxygen-dependent plant processes: ethylene biosynthesis and cyanide-resistant respiration

    International Nuclear Information System (INIS)

    Stegink, S.J.

    1985-01-01

    Two oxygen-dependent plant processes were investigated. A cell-free preparation from pea (Pisum sativum L., cv. Alaska) was used to study ethylene biosynthesis from 1-aminocyclopropane-1-carboxylic acid. Mitochondrial cyanide-resistant respiration was investigated in studies with 14 C-butyl gallate and other respiratory effectors. Ethylene biosynthesis was not due to a specific enzyme, or oxygen radicals. Rather, hydrogen peroxide, generated at low levels, coupled with endogenous manganese produced ethylene. 14 C-butyl gallate bound specifically to mitochondria from cyanide-sensitive and -resistant higher plants and Neurospora crassa mitochondria. The amount of gallate bound was similar for all higher plant mitochondria. Rat liver mitochondria bound very little 14 C-butyl gallate. Plant mitochondria in which cyanide-resistance was induced bound as much 14 C-butyl gallate as before induction. However mitochondria from recently harvested white potato tubers did not bind the gallate. The observations suggest that an engaging factor couples with a gallate binding site in the mitochondrial membrane. With skunk cabbage spadix mitochondria the I 5 0 for antimycin A inhibition of oxygen uptake was decreased by salicylhydroxamic acid pretreatment; this was also true for reverse order additions. No shift was observed with mung bean hypocotyl or Jerusalem artichoke tuber mitochondria

  7. Structure, Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids.

    Science.gov (United States)

    Schimming, Olivia; Challinor, Victoria L; Tobias, Nicholas J; Adihou, Hélène; Grün, Peter; Pöschel, Laura; Richter, Christian; Schwalbe, Harald; Bode, Helge B

    2015-10-19

    Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

    Science.gov (United States)

    Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

    2008-05-01

    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

  9. Cellulose synthesis inhibition, cell expansion, and patterns of cell wall deposition in Nitella internodes

    International Nuclear Information System (INIS)

    Richmond, P.A.; Metraux, J.P.

    1984-01-01

    The authors have investigated the pattern of wall deposition and maturation and correlated it with cell expansion and cellulose biosynthesis. The herbicide 2,6-dichlorobenzonitrile (DCB) was found to be a potent inhibitor of cellulose synthesis, but not of cell expansion in Nitella internodal cells. Although cellulose synthesis is inhibited during DCB treatment, matrix substances continue to be synthesized and deposited. The inhibition of cellulose microfibril deposition can be demonstrated by various techniques. These results demonstrate that matrix deposition is by apposition, not by intussusception, and that the previously deposited wall moves progressively outward while stretching and thinning as a result of cell expansion

  10. Statins: pros and cons.

    Science.gov (United States)

    Pinal-Fernandez, Iago; Casal-Dominguez, Maria; Mammen, Andrew L

    2018-05-23

    Statins inhibit the critical step of cholesterol synthesis in which 3-hydroxy-3-methylglutaryl coenzyme A (HMGC) is transformed to mevalonate by the enzyme HMGC reductase. By doing so, they have a potent lipid-lowering effect that reduces cardiovascular risk and decreases mortality. Since the mevalonate pathway also influences endothelial function, the inflammatory response, and coagulation, the effects of statins reach well beyond their cholesterol lowering properties. As with all drugs, statins may have adverse effects; these include musculoskeletal symptoms, increased risk of diabetes, and higher rates of hemorrhagic stroke. However, the frequency of adverse effects is extremely low and, in selected patient populations, the benefits of statins considerably outweigh the potential risks. Published by Elsevier España, S.L.U.

  11. Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

    NARCIS (Netherlands)

    S. de Weert; P.J. Punt; Christien Lokman; C.A. van den Hondel; A.C. Franken; A.F. Ram

    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

  12. 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

  13. Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, Andrea; Flores, Carmen-Lisset

    2004-01-01

    3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eu...

  14. Structure and mechanism of a bacterial t6A biosynthesis system

    OpenAIRE

    Luthra, Amit; Swinehart, William; Bayooz, Susan; Phan, Phuc; Stec, Boguslaw; Iwata-Reuyl, Dirk; Swairjo, Manal A

    2018-01-01

    Abstract The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t6A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals...

  15. Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

    International Nuclear Information System (INIS)

    Corradino, R.A.; Fullmer, C.S.

    1980-01-01

    Embryonic chick duodenum maintained in organ culture responds to 1 α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+ (3-5 x 10 -5 M) or Zn 2+ (10 -5 -10 -4 M) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109 Cd-binding activity ( 109 CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35 S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35 S-labeled CdBP - the only protein significantly labeled under the conditions employed - migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+ - or Zn 2+ -stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35 S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organ-cultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3 , in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions

  16. The enzymology of polyether biosynthesis.

    Science.gov (United States)

    Liu, Tiangang; Cane, David E; Deng, Zixin

    2009-01-01

    Polyether ionophore antibiotics are a special class of polyketides widely used in veterinary medicine, and as food additives in animal husbandry. In this article, we review current knowledge about the mechanism of polyether biosynthesis, and the genetic and biochemical strategies used for its study. Several clear differences distinguish it from traditional type I modular polyketide biosynthesis: polyether backbones are assembled by modular polyketide synthases but are modified by two key enzymes, epoxidase and epoxide hydrolase, to generate the product. All double bonds involved in the oxidative cyclization in the polyketide backbone are of E geometry. Chain release in the polyether biosynthetic pathway requires a special type II thioesterase which specifically hydrolyzes the polyether thioester. All these discoveries should be very helpful for a deep understanding of the biosynthetic mechanism of this class of important natural compounds, and for the targeted engineering of polyether derivatives.

  17. Analysis of ethylene biosynthesis and perception during postharvest cold storage of Marsh and Star Ruby grapefruits.

    Science.gov (United States)

    Lado, Joanna; Rodrigo, María Jesús; Zacarías, Lorenzo

    2015-10-01

    Grapefruits are among the citrus species more sensitive to cold and develop chilling injury symptoms during prolonged postharvest storage at temperatures lower than 8 ℃-10 ℃. The plant hormone ethylene has been described either to protect or potentiate chilling injury development in citrus whereas little is known about transcriptional regulation of ethylene biosynthesis, perception and response during cold storage and how the hormone is regulating its own perception and signaling cascade. Then, the objective of the present study was to explore the transcriptional changes in the expression of ethylene biosynthesis, receptors and response genes during cold storage of the white Marsh and the red Star Ruby grapefruits. The effect of the ethylene action inhibitor, 1-MCP, was evaluated to investigate the involvement of ethylene in the regulation of the genes of its own biosynthesis and perception pathway. Ethylene production was very low at the harvest time in fruits of both varieties and experienced only minor changes during storage. By contrast, inhibition of ethylene perception by 1-MCP markedly induced ethylene production, and this increase was highly stimulated during shelf-life at 20 ℃, as well as transcription of ACS and ACO. These results support the auto-inhibitory regulation of ethylene in grapefruits, which acts mainly at the transcriptional level of ACS and ACO genes. Moreover, ethylene receptor1 and ethylene receptor3 were induced by cold while no clear role of ethylene was observed in the induction of ethylene receptors. However, ethylene appears to be implicated in the transcriptional regulation of ERFs both under cold storage and shelf-life. © The Author(s) 2014.

  18. Methoxypyrazines biosynthesis and metabolism in grape: A review.

    Science.gov (United States)

    Lei, Yujuan; Xie, Sha; Guan, Xueqiang; Song, Changzheng; Zhang, Zhenwen; Meng, Jiangfei

    2018-04-15

    This review summarizes research on the discovery, biosynthesis, accumulation, transport, and metabolism of 3-alkyl-2-methoxypyrazines (MPs) in grape. The MPs are a family of potent volatile compounds distributed throughout biological kingdoms. These compounds impart herbaceous/green/vegetal sensory attributes to certain varieties of wine. Generally, high levels of MPs in wine are derived mainly from the corresponding grapes. Although two pathways for MPs biosynthesis have been proposed, only the final step and the enzymes that catalyze it has been confirmed in grape, and the metabolic intermediates and key enzymes involved in other steps are still unknown. The limited understanding of MPs metabolism has restricted research on these compounds, and some empirical results cannot be explained by the current knowledge of MPs metabolism. This review provides insights into research on MPs biosynthesis and metabolism, and proposes directions for further research on this important class of flavour/odour compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Exploring the fungal protein cadre in the biosynthesis of PbSe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, Jaya Mary; Sharma, Sumit; Balakrishnan, Raj Mohan, E-mail: rajmohanbala@gmail.com

    2017-02-15

    Highlights: • Pb and Se stress activates specific metal detoxification surge in the fungus. • Fungus releases phytochelatins, metallothioneins, super oxide dismutases etc. • These mechanisms capacitate the fungi as bio-factories for synthesis of PbSe QDs. • A pathway for PbSe QD biosynthesis by marine Aspergillus terreus was elucidated - Abstract: While a large number of microbial sources have recently emerged as potent sources for biosynthesis of chalcogenide quantum dots (QDs), studies regarding their biomimetic strategies that initiate QD biosynthesis are scarce. The present study describes several mechanistic aspects of PbSe QD biosynthesis using marine Aspergillus terreus. Scanning electron microscopic (SEM) studies indicated distinctive morphological features such as abrasion and agglomeration on the fungal biomass after the biosynthesis reaction. Further, the biomass subsequent to the heavy metal/metalloid precursor was characterized with spectral signatures typical to primary and secondary stress factors such as thiol compounds and oxalic acid using Fourier Transform Infra-Red Spectroscopic (FTIR) analysis. An increase in the total protein content in the reaction mixture after biosynthesis was another noteworthy observation. Further, metal-phytochelatins were identified as the prominent metal-ion trafficking components in the reaction mixture using Liquid Chromatography Mass Spectroscopic analysis (LCMS). Subsequent assays confirmed the involvement of metal binding peptides namely metallothioneins and other anti-oxidant enzymes that might have played a prominent role in the microbial metal detoxification system for the biosynthesis of PbSe QDs. Based on these findings a possible mechanism for the biosynthesis of PbSe QDs by marine A. terreus has been elucidated.

  20. The Non-Mevalonate Pathway to Isoprenoid Biosynthesis : A Potential Source of New Drug Targets

    NARCIS (Netherlands)

    Hirsch, Anna K.H.; Diederich, François

    2008-01-01

    Isoprenoids are an essential class of natural products with a myriad of biological functions. All isoprenoids are assembled using two common five-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are biosynthesized via two completely independent routes: the

  1. Acetylglutamate synthase in Neurospora crassa: characterization, localization, and genetic behavior of a regulatory enzyme of arginine biosynthesis

    International Nuclear Information System (INIS)

    Jacobson, J.A.

    1988-01-01

    This study describes the characterization and localization of the first enzyme of arginine biosynthesis in Neurospora crassa. A radioactive assay was developed to detect this enzyme whereby radioactive substrate and product molecules could be separated by ion-exchange chromatography. The enzyme was found to have a pH optimum of 9.0 and K/sub m/ values for glutamate and acetyl-CoA of approximately 4.7 and 0.45 mM, respectively. The enzyme was shown to be feedback inhibited by arginine. Half-maximal inhibition was observed at 0.13 mM arginine, a concentration which is similar to be in vivo cytosolic concentration of 0.2 mM. Arginine was found to act as a competitive inhibitor with respect to acetyl-CoA. Acetylglutamate synthase was localized to the mitochondrion. However, in contrast to the mitochondrial matrix location of the other ornithine biosynthetic enzymes, this enzyme was found to reside on the mitochondrial inner membrane

  2. Convergent Evolution of Ergothioneine Biosynthesis in Cyanobacteria.

    Science.gov (United States)

    Liao, Cangsong; Seebeck, Florian P

    2017-11-02

    Biosynthesis of N-α-trimethyl-2-thiohistidine (ergothioneine) is a frequent trait in cyanobacteria. This sulfur compound may provide essential relief from oxidative stress related to oxygenic photosynthesis. The central steps in ergothioneine biosynthesis are catalyzed by a histidine methyltransferase and an iron-dependent sulfoxide synthase. In this report, we present evidence that some cyanobacteria recruited and adapted a sulfoxide synthase from a different biosynthetic pathway to make ergothioneine. The discovery of a second origin of ergothioneine production underscores the physiological importance of this metabolite and highlights the evolutionary malleability of the thiohistidine biosynthetic machinery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans.

    Science.gov (United States)

    Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A; Wilensky, Robert L; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A

    2012-04-01

    The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

  4. FUNCTIONAL SPECIALIZATION OF DUPLICATED FLAVONOID BIOSYNTHESIS GENES IN WHEAT

    Directory of Open Access Journals (Sweden)

    Khlestkina E.

    2012-08-01

    Full Text Available Gene duplication followed by subfunctionalization and neofunctionalization is of a great evolutionary importance. In plant genomes, duplicated genes may result from either polyploidization (homoeologous genes or segmental chromosome duplications (paralogous genes. In allohexaploid wheat Triticum aestivum L. (2n=6x=42, genome BBAADD, both homoeologous and paralogous copies were found for the regulatory gene Myc encoding MYC-like transcriptional factor in the biosynthesis of flavonoid pigments, anthocyanins, and for the structural gene F3h encoding one of the key enzymes of flavonoid biosynthesis, flavanone 3-hydroxylase. From the 5 copies (3 homoeologous and 2 paralogous of the Myc gene found in T. aestivum, only one plays a regulatory role in anthocyanin biosynthesis, interacting complementary with another transcriptional factor (MYB-like to confer purple pigmentation of grain pericarp in wheat. The role and functionality of the other 4 copies of the Myc gene remain unknown. From the 4 functional copies of the F3h gene in T. aestivum, three homoeologues have similar function. They are expressed in wheat organs colored with anthocyanins or in the endosperm, participating there in biosynthesis of uncolored flavonoid substances. The fourth copy (the B-genomic paralogue is transcribed neither in wheat organs colored with anthocyanins nor in seeds, however, it’s expression has been noticed in roots of aluminium-stressed plants, where the three homoeologous copies are not active. Functional diversification of the duplicated flavonoid biosynthesis genes in wheat may be a reason for maintenance of the duplicated copies and preventing them from pseudogenization.The study was supported by RFBR (11-04-92707. We also thank Ms. Galina Generalova for technical assistance.

  5. Induction of dopamine biosynthesis by l-DOPA in PC12 cells: implications of L-DOPA influx and cyclic AMP.

    Science.gov (United States)

    Jin, Chun Mei; Yang, Yoo Jung; Huang, Hai Shan; Lim, Sung Cil; Kai, Masaaki; Lee, Myung Koo

    2008-09-04

    The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).

  6. Biosynthesis and Characterization of Silver Nanoparticles by Aspergillus Species

    Science.gov (United States)

    Pourshahid, Seyedmohammad; Mehryar, Pouyan; Pakshir, Keyvan; Rahimi, Mohammad Javad; Arabi Monfared, Ali

    2016-01-01

    Currently, researchers turn to natural processes such as using biological microorganisms in order to develop reliable and ecofriendly methods for the synthesis of metallic nanoparticles. In this study, we have investigated extracellular biosynthesis of silver nanoparticles using four Aspergillus species including A. fumigatus, A. clavatus, A. niger, and A. flavus. We have also analyzed nitrate reductase activity in the studied species in order to determine the probable role of this enzyme in the biosynthesis of silver nanoparticles. The formation of silver nanoparticles in the cell filtrates was confirmed by the passage of laser light, change in the color of cell filtrates, absorption peak at 430 nm in UV-Vis spectra, and atomic force microscopy (AFM). There was a logical relationship between the efficiencies of studied Aspergillus species in the production of silver nanoparticles and their nitrate reductase activity. A. fumigatus as the most efficient species showed the highest nitrate reductase activity among the studied species while A. flavus exhibited the lowest capacity in the biosynthesis of silver nanoparticles which was in accord with its low nitrate reductase activity. The present study showed that Aspergillus species had potential for the biosynthesis of silver nanoparticles depending on their nitrate reductase activity. PMID:27652264

  7. CoQ10 and L-carnitine for statin myalgia?

    Science.gov (United States)

    DiNicolantonio, James J

    2012-10-01

    Statins are a standard of care in many clinical settings such as acute myocardial infarction and for patients having or at risk of cardiovascular (CV) disease. This is based on a plethora of data showing reductions in CV events and mortality. The CV benefit of statins can be partly explained by their ability to inhibit of HMG-CoA reductase, which subsequently lowers cholesterol and decreases the formation of mevalonate. However, the inhibition of the mevalonate pathway decreases the formation of coenzyme Q10 (CoQ10) within the body. It has been a long-standing theory that statin-associated muscle pain (myalgia) is caused, or at least partly contributed by, a reduction in CoQ10 levels in muscle mitochondria. One of the main side effects of statins is myalgia, which causes the patient to either stop their statin or significantly reduce the dose of their statin. The question of whether CoQ10 can help treat statin myopathy is a common one encountered by clinicians in current day practice.

  8. Method for determining heterologous biosynthesis pathways

    KAUST Repository

    Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

    2017-01-01

    suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

  9. Melatonin is involved in skotomorphogenesis by regulating brassinosteroids biosynthesis in rice plants.

    Science.gov (United States)

    Hwang, Ok Jin; Back, Kyoungwhan

    2018-04-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis catalyzing the conversion of serotonin into N-acetylserotonin. In plants, SNAT is encoded by two isogenes of which SNAT1 is constitutively expressed and its overexpression confers increased yield in rice. However, the role of SNAT2 remains to be clarified. In contrast to SNAT1, the diurnal rhythm of SNAT2 mRNA expression peaks at night. In this study, transgenic rice plants in which SNAT2 expression was suppressed by RNAi technology showed a decrease in melatonin and a dwarf phenotype with erect leaves, reminiscent of brassinosteroids (BRs)-deficient mutants. Of note, the dwarf phenotype was dependent on the presence of dark, suggesting that melatonin is involved in dark growth (skotomorphogenesis). In support of this suggestion, SNAT2 RNAi lines exhibited photomorphogenic phenotypes such as inhibition of internodes and increased expression of light-inducible CAB genes in the dark. The causative gene for the melatonin-mediated BRs biosynthetic gene was DWARF4, a rate limiting BRs biosynthetic gene. Exogenous melatonin treatment induced several BRs biosynthetic genes, including DWARF4, D11, and RAVL1. As expected from the erect leaves, the SNAT2 RNAi lines produced less BRs than the wild type. Our results show for the first time that melatonin is a positive regulator of dark growth or shade outgrowth by regulating BR biosynthesis in plants. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Dose-dependent dual effects of cholesterol and desmosterol on J774 macrophage proliferation

    International Nuclear Information System (INIS)

    Rodriguez-Acebes, Sara; Cueva, Paloma de la; Ferruelo, Antonio J.; Fernandez-Hernando, Carlos; Lasuncion, Miguel A.; Martinez-Botas, Javier; Gomez-Coronado, Diego

    2008-01-01

    We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 μM lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.

  11. Dose-dependent dual effects of cholesterol and desmosterol on J774 macrophage proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Acebes, Sara [Servicio de Bioquimica-Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar, km 9, 28034 Madrid (Spain); CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid (Spain); Cueva, Paloma de la; Ferruelo, Antonio J; Fernandez-Hernando, Carlos [Servicio de Bioquimica-Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar, km 9, 28034 Madrid (Spain); Lasuncion, Miguel A [Servicio de Bioquimica-Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar, km 9, 28034 Madrid (Spain); CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid (Spain); Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, Alcala de Henares (Spain); Martinez-Botas, Javier [Servicio de Bioquimica-Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar, km 9, 28034 Madrid (Spain); CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid (Spain); Gomez-Coronado, Diego [Servicio de Bioquimica-Investigacion, Hospital Ramon y Cajal, Carretera de Colmenar, km 9, 28034 Madrid (Spain); CIBER de Fisiopatologia de la Obesidad y Nutricion (CIBEROBN), Instituto de Salud Carlos III, Madrid (Spain)], E-mail: diego.gomez@hrc.es

    2008-12-12

    We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 {mu}M lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.

  12. NAD+ biosynthesis, aging, and disease [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sean Johnson

    2018-02-01

    Full Text Available Nicotinamide adenine dinucleotide (NAD+ biosynthesis and its regulation have recently been attracting markedly increasing interest. Aging is marked by a systemic decrease in NAD+ across multiple tissues. The dysfunction of NAD+ biosynthesis plays a critical role in the pathophysiologies of multiple diseases, including age-associated metabolic disorders, neurodegenerative diseases, and mental disorders. As downstream effectors, NAD+-dependent enzymes, such as sirtuins, are involved in the progression of such disorders. These recent studies implicate NAD+ biosynthesis as a potential target for preventing and treating age-associated diseases. Indeed, new studies have demonstrated the therapeutic potential of supplementing NAD+ intermediates, such as nicotinamide mononucleotide and nicotinamide riboside, providing a proof of concept for the development of an effective anti-aging intervention.

  13. Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

    KAUST Repository

    Butt, Haroon

    2018-01-28

    Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

  14. Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

    KAUST Repository

    Butt, Haroon; Jamil, Muhammad; Wang, Jian You; Al-Babili, Salim; Mahfouz, Magdy M.

    2018-01-01

    Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

  15. Herbicide glufosinate inhibits yeast growth and extends longevity during wine fermentation.

    Science.gov (United States)

    Vallejo, Beatriz; Picazo, Cecilia; Orozco, Helena; Matallana, Emilia; Aranda, Agustín

    2017-09-29

    Glufosinate ammonium (GA) is a widely used herbicide that inhibits glutamine synthetase. This inhibition leads to internal amino acid starvation which, in turn, causes the activation of different nutrient sensing pathways. GA also inhibits the enzyme of the yeast Saccharomyces cerevisiae in such a way that, although it is not used as a fungicide, it may alter yeast performance in industrial processes like winemaking. We describe herein how GA indeed inhibits the yeast growth of a wine strain during the fermentation of grape juice. In turn, GA extends longevity in a variety of growth media. The biochemical analysis indicates that GA partially inhibits the nutrient sensing TORC1 pathway, which may explain these phenotypes. The GCN2 kinase mutant is hypersensitive to GA. Hence the control of translation and amino acid biosynthesis is required to also deal with the damaging effects of this pesticide. A global metabolomics analysis under winemaking conditions indicated that an increase in amino acid and in polyamines occurred. In conclusion, GA affects many different biochemical processes during winemaking, which provides us with some insights into both the effect of this herbicide on yeast physiology and into the relevance of the metabolic step for connecting nitrogen and carbon metabolism.

  16. Phylogenomic reconstruction of archaeal fatty acid metabolism

    Science.gov (United States)

    Dibrova, Daria V.; Galperin, Michael Y.; Mulkidjanian, Armen Y.

    2014-01-01

    While certain archaea appear to synthesize and/or metabolize fatty acids, the respective pathways still remain obscure. By analyzing the genomic distribution of the key lipid-related enzymes, we were able to identify the likely components of the archaeal pathway of fatty acid metabolism, namely, a combination of the enzymes of bacterial-type β-oxidation of fatty acids (acyl-CoA-dehydrogenase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase) with paralogs of the archaeal acetyl-CoA C-acetyltransferase, an enzyme of the mevalonate biosynthesis pathway. These three β-oxidation enzymes working in the reverse direction could potentially catalyze biosynthesis of fatty acids, with paralogs of acetyl-CoA C-acetyltransferase performing addition of C2 fragments. The presence in archaea of the genes for energy-transducing membrane enzyme complexes, such as cytochrome bc complex, cytochrome c oxidase, and diverse rhodopsins, was found to correlate with the presence of the proposed system of fatty acid biosynthesis. We speculate that because these membrane complexes functionally depend on fatty acid chains, their genes could have been acquired via lateral gene transfer from bacteria only by those archaea that already possessed a system of fatty acid biosynthesis. The proposed pathway of archaeal fatty acid metabolism operates in extreme conditions and therefore might be of interest in the context of biofuel production and other industrial applications. PMID:24818264

  17. Niacin and biosynthesis of PGD₂ by platelet COX-1 in mice and humans

    DEFF Research Database (Denmark)

    Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela

    2012-01-01

    during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis....... Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased...... thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular...

  18. Evolution of the biosynthesis of the branched-chain amino acids

    Science.gov (United States)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  19. The effects of photosensitizing antibiotics and ultraviolet irradiation on the biosynthesis of prostaglandins

    International Nuclear Information System (INIS)

    Lord, J.T.; Ziboh, V.A.; Blick, G.; Poitier, J.; Kursunoglu, I.; Penneys, N.S.

    1978-01-01

    Oxygenation of arachidonic acid in vitro by calf skin microsomal acetone powder was enhanced by UV-irradiation at wavelengths of 254 and 360 nm. Further enhancement of the oxygenation reaction was observed in the presence of two photosensitizing cyclic antibiotics, tetracycline and demethylchlortetracycline. To test whether or not the oxygenation of arachidonic acid was related to the biosynthesis of prostaglandins, [I- 14 C]-arachidonic acid was incubated with calf skin acetone powder in the presence of UV-irradiation and the cyclic antibiotics. Prostaglandin biosynthesis from arachidonic acid by the calf skin microsomal acetone powder was enhanced after exposure to UV-irradiation at 254 nm and moderately at 360 nm. Incubation in the presence of demethylchlortetra-cycline (0.2 mM) increased prostaglandin biosynthesis approximately 95% over control by UV-irradiation at 254 nm. No significant stimulation of prostaglandin biosynthesis was observed at 360 nm. Non-photosensitizing antibiotics had no effect either on the oxygenation of arachidonic acid or on the biosynthesis of prostaglandin with or without UV-irradiation. It is suggested that the inflammatory reactions associated with these photo-reactive antibiotics may in part, be via the biosynthesis and release of the prostaglandins which are known to produce cutaneous inflammatory reactions. (author)

  20. Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape.

    Science.gov (United States)

    Koyama, Kazuya; Ikeda, Hiroko; Poudel, Puspa Raj; Goto-Yamamoto, Nami

    2012-06-01

    Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49 days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related gene more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    International Nuclear Information System (INIS)

    Sandhu, Navdeep; Vijayan, Mathilakath M.

    2011-01-01

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  2. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, Navdeep [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Vijayan, Mathilakath M., E-mail: mvijayan@uwaterloo.ca [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada)

    2011-05-15

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  3. Pseudopterosin Biosynthesis: Aromatization of the Diterpene Cyclase Product, Elisabethatriene

    Directory of Open Access Journals (Sweden)

    Amber C. Kohl

    2003-11-01

    Full Text Available Abstract: Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10 and erogorgiaene (11, have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A-D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.

  4. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway.

    Science.gov (United States)

    Kunjapur, Aditya M; Hyun, Jason C; Prather, Kristala L J

    2016-04-11

    Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin. Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and L-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-L-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33% (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25% increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability. Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or

  5. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants

    KAUST Repository

    Chen, Hao

    2010-08-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed. © 2010 Blackwell Publishing Ltd.

  6. Biosynthesis of Tropolones in Streptomyces spp: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on Tropone Ring.

    Science.gov (United States)

    Chen, Xuefei; Xu, Min; Lü, Jin; Xu, Jianguo; Wang, Yemin; Lin, Shuangjun; Deng, Zixin; Tao, Meifeng

    2018-04-13

    Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. Herein, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and bioconversion. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes ( paa ) located outside of the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl-CoA hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it towards the formation of heptacyclic intermediates. TrlB is a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces Six seven-membered carbocyclic compounds were identified from the gene deletion mutants of trlC , trlD , trlE , and trlF Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. Monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to afford DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp. IMPORTANCE Tropolonoids are promising drug lead compounds because of their versatile bioactivities attributed to

  7. Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

    International Nuclear Information System (INIS)

    Lichtenthaler, H.K.; Kobek, K.

    1989-01-01

    Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from 14 C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis

  8. Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli.

    Science.gov (United States)

    Liu, Joyce; Zhu, Xuejun; Seipke, Ryan F; Zhang, Wenjun

    2015-05-15

    Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.

  9. 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.

  10. In vitro biosynthesis of unnatural enterocin and wailupemycin polyketides.

    Science.gov (United States)

    Kalaitzis, John A; Cheng, Qian; Thomas, Paul M; Kelleher, Neil L; Moore, Bradley S

    2009-03-27

    Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways.

  11. Induction of SA-signaling pathway and ethylene biosynthesis in Trichoderma harzianum-treated tomato plants after infection of the root-knot nematode Meloidogyne incognita.

    Science.gov (United States)

    Leonetti, Paola; Zonno, Maria Chiara; Molinari, Sergio; Altomare, Claudio

    2017-04-01

    Salicylic acid-signaling pathway and ethylene biosynthesis were induced in tomato treated with Trichoderma harzianum when infected by root-knot nematodes and limited the infection by activation of SAR and ethylene production. Soil pre-treatment with Trichoderma harzianum (Th) strains ITEM 908 (T908) and T908-5 decreased susceptibility of tomato to Meloidogyne incognita, as assessed by restriction in nematode reproduction and development. The effect of T. harzianum treatments on plant defense was detected by monitoring the expression of the genes PR-1/PR-5 and JERF3/ACO, markers of the SA- and JA/ET-dependent signaling pathways, respectively. The compatible nematode-plant interaction in absence of fungi caused a marked suppression of PR-1, PR-5, and ACO gene expressions, either locally or systemically, whilst expression of JERF3 gene resulted unaffected. Conversely, when plants were pre-treated with Th-strains, over-expression of PR-1, PR-5, and ACO genes was observed in roots 5 days after nematode inoculation. JERF3 gene expression did not change in Th-colonized plants challenged with nematodes. In the absence of nematodes, Trichoderma-root interaction was characterized by the inhibition of both SA-dependent signaling pathway and ET biosynthesis, and, in the case of PR-1 and ACO genes, this inhibition was systemic. JERF3 gene expression was systemically restricted only at the very early stages of plant-fungi interaction. Data presented indicate that Th-colonization primed roots for Systemic Acquired Resistance (SAR) against root-knot nematodes and reacted to nematode infection more efficiently than untreated plants. Such a response probably involves also activation of ET production, through an augmented transcription of the ACO gene, which encodes for the enzyme catalyzing the last step of ET biosynthesis. JA signaling and Induced Systemic Resistance (ISR) do not seem to be involved in the biocontrol action of the tested Th-strains against RKNs.

  12. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

    KAUST Repository

    Wang, Zhen-Yu; Gehring, Christoph A; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

    2014-01-01

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

  13. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

    KAUST Repository

    Wang, Zhen-Yu

    2014-11-21

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

  14. Prostaglandin E(2) synthase inhibition as a therapeutic target.

    Science.gov (United States)

    Iyer, Jitesh P; Srivastava, Punit K; Dev, Rishabh; Dastidar, Sunanda G; Ray, Abhijit

    2009-07-01

    Most NSAIDs function by inhibiting biosynthesis of PGE(2) by inhibition of COX-1 and/or COX-2. Since COX-1 has a protective function in the gastro-intestinal tract (GIT), non-selective inhibition of both cycloxy genases leads to moderate to severe gastro-intestinal intolerance. Attempts to identify selective inhibitors of COX-2, led to the identification of celecoxib and rofecoxib. However, long-term use of these drugs has serious adverse effects of sudden myocardial infarction and thrombosis. Drug-mediated imbalance in the levels of prostaglandin I(2) (PGI(2)) and thromboxane A(2) (TXA(2)) with a bias towards TXA(2) may be the primary reason for these events. This resulted in the drugs being withdrawn from the market, leaving a need for an effective and safe anti-inflammatory drug. Recently, the focus of research has shifted to enzymes downstream of COX in the prosta glandin biosynthetic pathway such as prostaglandin E(2) synthases. Microsomal prostaglandin E(2) synthase-1 (mPGES-1) specifically isomerizes PGH(2) to PGE(2), under inflammatory conditions. In this review, we examine the biology of mPGES-1 and its role in disease. Progress in designing molecules that can selectively inhibit mPGES-1 is reviewed. mPGES-1 has the potential to be a target for anti-inflammatory therapy, devoid of adverse GIT and cardiac effects and warrants further investigation.

  15. Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

    Science.gov (United States)

    Minami, Atsushi; Shimaya, Mayu; Suzuki, Gaku; Migita, Akira; Shinde, Sandip S; Sato, Kyohei; Watanabe, Kenji; Tamura, Tomohiro; Oguri, Hiroki; Oikawa, Hideaki

    2012-05-02

    Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis. © 2012 American Chemical Society

  16. Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

    Science.gov (United States)

    Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

    2014-01-01

    Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

  17. Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin

    NARCIS (Netherlands)

    Tytgat, K. M.; Büller, H. A.; Opdam, F. J.; Kim, Y. S.; Einerhand, A. W.; Dekker, J.

    1994-01-01

    Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic

  18. Rapid biosynthesis and characterization of silver nanoparticles: an assessment of antibacterial and antimycotic activity

    Science.gov (United States)

    Kanawaria, Sajjan Kumar; Sankhla, Aryan; Jatav, Pradeep Kumar; Yadav, Raghvendra Singh; Verma, Kumar Sambhav; Velraj, Parthiban; Kachhwaha, Sumita; Kothari, Shanker Lal

    2018-04-01

    Bioassisted synthesis provides a facile, convenient, and promising approach to produce many inorganic nanostructures. Herein, we report a rapid biosynthesis of silver nanoparticles (AgNPs) using Thuja occidentalis (L.) leaf extract with an emphasis on their antibacterial and antimycotic activity. Interestingly, the synthesis of AgNPs was completed in a short duration of 35-40 min. The electron micrographs showed AgNPs with particles Bacillus subtilis. An AgNP solution with 30 µg/ml concentration arrested the growth of bacterial strains making a zone of inhibition > 15 mm. The antimycotic activity against Aspergillus niger, Fusarium spp., and Alternaria alternata species increased monotonically with nanoparticle concentration in the growth media. A 10 ppm solution of AgNP was detrimental to fungal growth. Thus, the technique provides an avenue to synthesize antibiotic AgNPs without use of other external agents.

  19. Genome-wide identification of genes involved in polyamine biosynthesis and the role of exogenous polyamines in Malus hupehensis Rehd. under alkaline stress.

    Science.gov (United States)

    Gong, Xiaoqing; Dou, Fangfang; Cheng, Xi; Zhou, Jing; Zou, Yangjun; Ma, Fengwang

    2018-08-30

    Polyamines (PAs) in plants are growth substrates with functions similar to phytohormones. Although they contribute to diverse processes, little is known about their role in stress responses, especially for perennial woody plants. We conducted a genome-wide investigation of 18 sequences involved in PA biosynthesis in the genome of apple (Malus domestica). Further analysis was performed to construct a phylogenetic tree, analyze their protein motifs and gene structures. In addition, we developed their expression profiles in response to stressed conditions. Both MDP0000171041 (MdSAMDC1) and MDP0000198590 (MdSPDS1) were induced by alkaline, salt, ABA, cold, and dehydration stress treatments, suggesting that these genes are the main contributors to activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermidine synthase (EC 2.5.1.16) in apple. Changes in PA biosynthesis under stress conditions indicated that spermidine and spermine are more essential than putrescine for apple, especially when responding to alkaline or salt stress. When seedlings of M. hupehensis Rehd. were supplied with exogenous PAs, their leaves showed less chlorosis under alkaline stress when compared with untreated plants. This application also inhibited the decline in SPAD levels and reduced relative electrolyte leakage in those stressed seedlings, while increasing their concentration of active iron. These results suggest that the alteration in PA biosynthesis confers enhanced tolerance to alkaline stress in M. hupehensis Rehd. Copyright © 2018. Published by Elsevier B.V.

  20. Occurrence and biosynthesis of carotenoids in phytoplankton.

    Science.gov (United States)

    Huang, Jim Junhui; Lin, Shaoling; Xu, Wenwen; Cheung, Peter Chi Keung

    2017-09-01

    Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Intracellular Biosynthesis and Antibacterial Activity of Silver Nanoparticles Using Edible Mushrooms

    Directory of Open Access Journals (Sweden)

    Sankaran MIRUNALINI

    2012-11-01

    Full Text Available The process of biosynthesis of silver nanoparticles is a simple, cost effective and eco-friendly approach. Biosynthesis of silver nanoparticles using some commonly available edible mushroom extracts and their antimicrobial activity was demonstrated in the current study. The formation of silver nanoparticles was confirmed by UV, FTIR and SEM and antibacterial activity was tested using disc diffusion method. From the results it is confirmed the successful formation of silver nanoparticles using mushroom extracts; they performed their role as a reducing and capping agent and also exhibited a potent antibacterial activity against S. aureus (gram positive bacteria. Thus the biosynthesis of silver nanoparticles using edible mushroom extract will deserve to be a good candidate as an antibacterial agent.

  2. Comparison of Effect of Brassinosteroid and Gibberellin Biosynthesis Inhibitors on Growth of Rice Seedlings

    Directory of Open Access Journals (Sweden)

    Tadashi Matusmoto

    2016-01-01

    Full Text Available Brassinosteroid (BR and gibberellin (GA are two predominant plant hormones that regulate plant cell elongation. Mutants disrupt the biosynthesis of these hormones and display different degrees of dwarf phenotypes in rice. Although the role of each plant hormone in promoting the longitudinal growth of plants has been extensively studied using genetic methods, their relationship is still poorly understood. In this study, we used two specific inhibitors targeting BR and GA biosynthesis to investigate the roles of BR and GA in growth of rice seedlings. Yucaizol, a specific inhibitor of BR biosynthesis, and Trinexapac-ethyl, a commercially available inhibitor of GA biosynthesis, were used. The effect of Yucaizol on rice seedlings indicated that Yucaizol significantly retarded stem elongation. The IC50 value was found to be approximately 0.8 μmol/L. Yucaizol also induced small leaf angle phenocopy in rice seedlings, similarly to BR-deficient rice, while Trinexapac-ethyl did not. When Yucaizol combined with Trinexapac-ethyl was applied to the rice plants, the mixture of these two inhibitors retarded stem elongation of rice at lower doses. Our results suggest that the use of a BR biosynthesis inhibitor combined with a GA biosynthesis inhibitor may be useful in the development of new technologies for controlling rice plant height.

  3. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

    2013-01-22

    Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  4. Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium.

    Science.gov (United States)

    Wu, Dan; Li, Ang; Ma, Fang; Yang, Jixian; Xie, Yutong

    2016-07-01

    Agrobacterium is a genus of gram-negative bacteria that can produce several typical exopolysaccharides with commercial uses in the food and pharmaceutical fields. In particular, succinoglycan and curdlan, due to their good quality in high yield, have been employed on an industrial scale comparatively early. Exopolysaccharide biosynthesis is a multiple-step process controlled by different functional genes, and various environmental factors cause changes in exopolysaccharide biosynthesis through regulatory mechanisms. In this mini-review, we focus on the genetic control and regulatory mechanisms of succinoglycan and curdlan produced by Agrobacterium. Some key functional genes and regulatory mechanisms for exopolysaccharide biosynthesis are described, possessing a high potential for application in metabolic engineering to modify exopolysaccharide production and physicochemical properties. This review may contribute to the understanding of exopolysaccharide biosynthesis and exopolysaccharide modification by metabolic engineering methods in Agrobacterium.

  5. Bioregulation of aflatoxin biosynthesis by unirradiated and irradiated conidia of Aspergillus flavus

    International Nuclear Information System (INIS)

    Aziz, N.H.; Abu-Shady, M.R.; El-Fouly, M.Z.; Moussa, L.A.

    1996-01-01

    A sequential technique involving the transfer of mycelia from peptone-based, aflatoxin-non-supporting medium to glucose based, aflatoxin-supporting medium was used to study the effect of γ-irradiation on the regulation of aflatoxin biosynthesis by Aspergillus flavus. Analysis indicated that irradiation at a dose of 1.00 kGy produced enhancement of aflatoxin biosynthesis in peptone-glucose mineral salt cultures with an increase of adenine nucleotide levels and fatty acid patterns of microsomes and mitochondria. The results suggest that aflatoxin synthesis is not regulated by the overall energy status of the fungal cell but that lipoperoxidation by γ-irradiation plays a role in aflatoxin biosynthesis

  6. The influence of auxins on the biosynthesis of isoprene derivatives in callus cultures of Vaccinium corymbosum var. bluecrop.

    Science.gov (United States)

    Migas, Piotr; Luczkiewicz, Maria; Cisowski, Wojciech

    2006-01-01

    Callus cultures of Vaccinium corymbosum var. bluecrop were optimized for their isoprene derivatives production by supplementing Schenk-Hildebrandt (SH) medium with constant concentration of kinetin (2.32 microM) and two different amounts of selected auxins. Every auxin, except for IBA, used in 10-time higher concentration (2,4D, NAA, IAA, NOA) stimulated biosynthesis of beta-sitosterol and inhibited triterpene synthesis. Quantitative analysis of isoprene derivatives in callus biomass collected on the 25th day of the experiment proved that the analyzed callus of Vaccinium corymbosum var. bluecrop synthesized the highest amount of isoprene derivatives after subculturing on SH medium modified with 22.6 microM of 2,4D and 2.32 microM of kinetin.

  7. Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

    Science.gov (United States)

    Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing

    2012-01-01

    Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425

  8. Fungal biosynthesis of gold nanoparticles: mechanism and scale up.

    Science.gov (United States)

    Kitching, Michael; Ramani, Meghana; Marsili, Enrico

    2015-11-01

    Gold nanoparticles (AuNPs) are a widespread research tool because of their oxidation resistance, biocompatibility and stability. Chemical methods for AuNP synthesis often produce toxic residues that raise environmental concern. On the other hand, the biological synthesis of AuNPs in viable microorganisms and their cell-free extracts is an environmentally friendly and low-cost process. In general, fungi tolerate higher metal concentrations than bacteria and secrete abundant extracellular redox proteins to reduce soluble metal ions to their insoluble form and eventually to nanocrystals. Fungi harbour untapped biological diversity and may provide novel metal reductases for metal detoxification and bioreduction. A thorough understanding of the biosynthetic mechanism of AuNPs in fungi is needed to reduce the time of biosynthesis and to scale up the AuNP production process. In this review, we describe the known mechanisms for AuNP biosynthesis in viable fungi and fungal protein extracts and discuss the most suitable bioreactors for industrial AuNP biosynthesis. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  9. Microbial Biosynthesis of Silver Nanoparticles in Different Culture Media.

    Science.gov (United States)

    Luo, Ke; Jung, Samuel; Park, Kyu-Hwan; Kim, Young-Rok

    2018-01-31

    Microbial biosynthesis of metal nanoparticles has been extensively studied for the applications in biomedical sciences and engineering. However, the mechanism for their synthesis through microorganism is not completely understood. In this study, several culture media were investigated for their roles in the microbial biosynthesis of silver nanoparticles (AgNPs). The size and morphology of the synthesized AgNPs were analyzed by UV-vis spectroscopy, Fourier-transform-infrared (FT-IR), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The results demonstrated that nutrient broth (NB) and Mueller-Hinton broth (MHB) among tested media effectively reduced silver ions to form AgNPs with different particle size and shape. Although the involved microorganism enhanced the reduction of silver ions, the size and shape of the particles were shown to mainly depend on the culture media. Our findings suggest that the growth media of bacterial culture play an important role in the synthesis of metallic nanoparticles with regard to their size and shape. We believe our findings would provide useful information for further exploration of microbial biosynthesis of AgNPs and their biomedical applications.

  10. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

    Energy Technology Data Exchange (ETDEWEB)

    Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

    2010-01-07

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

  11. Analyzing the structural aspects of Isoprenoid biosynthesis pathway proteins in Ocimum species

    Directory of Open Access Journals (Sweden)

    Muktesh Chandra

    2017-10-01

    Full Text Available Generally thought that the extremely diverse array of secondary metabolites observed within Ocimum species defends against a comparable diverse array of biotic pests, pathogens and herbivores encountered around its natural range. Along with defense the diverse array of secondary metabolite also leads to the therapeutic and remedial property which justifies Ocimum as natural medicinal and aromatic casket. Many of the defense compounds, aroma compounds and medicinal derivatives are secondary metabolites isolated from trichome glands, mainly consist of terpenoids as well as phenylpropanoids. Various pathways fabricating these compounds are known viz. mevalonate pathway (MVA, phenylpropanoid pathway and MEP pathways. The enzyme cascade responsible for various secondary metabolites, need to be explored in various aspects. Here we had studied the MVA pathway enzymes in O. basilicum and O. gratissimum to figure out variations in enzyme structures due to speciation. Hence, in depth analysis of the transcriptome of O. basilicum and O. gratissimum, varrying in qualitative and quantitative aspects of essential oil were carried out. The transcriptome data from NCBI server was assembled using bioinformatic approaches. nr database at NCBI repository used for annotation, which assigned 60% contigs to known functions. Contigs corresponding to Mevalonate pathway enzymes are isolated using perl pipelines developed in our lab, which were further assembled using CLC workbench to remove redundancy and make larger stretch of sequence. Blastx of these larger sequences assigned them function and they are mapped to validated sequences to make full length. Data from both species led us to overall seven enzymes (total 14 of MVA pathway. These enzymes are studied in detail for various physio-chemical properties, steriochemical properties and motif/domain for protein-protein interaction (PPI study. Homolog models of all enzymes were predicted, against templates from RCSB

  12. Oleic acid biosynthesis in cyanobacteria

    International Nuclear Information System (INIS)

    VanDusen, W.J.; Jaworski, J.G.

    1986-01-01

    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 14 CO 2 . None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating 14 CO 2 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

  13. Jasmonic Acid Enhances Al-Induced Root Growth Inhibition.

    Science.gov (United States)

    Yang, Zhong-Bao; He, Chunmei; Ma, Yanqi; Herde, Marco; Ding, Zhaojun

    2017-02-01

    Phytohormones such as ethylene and auxin are involved in the regulation of the aluminum (Al)-induced root growth inhibition. Although jasmonate (JA) has been reported to play a crucial role in the regulation of root growth and development in response to environmental stresses through interplay with ethylene and auxin, its role in the regulation of root growth response to Al stress is not yet known. In an attempt to elucidate the role of JA, we found that exogenous application of JA enhanced the Al-induced root growth inhibition. Furthermore, phenotype analysis with mutants defective in either JA biosynthesis or signaling suggests that JA is involved in the regulation of Al-induced root growth inhibition. The expression of the JA receptor CORONATINE INSENSITIVE1 (COI1) and the key JA signaling regulator MYC2 was up-regulated in response to Al stress in the root tips. This process together with COI1-mediated Al-induced root growth inhibition under Al stress was controlled by ethylene but not auxin. Transcriptomic analysis revealed that many responsive genes under Al stress were regulated by JA signaling. The differential responsive of microtubule organization-related genes between the wild-type and coi1-2 mutant is consistent with the changed depolymerization of cortical microtubules in coi1 under Al stress. In addition, ALMT-mediated malate exudation and thus Al exclusion from roots in response to Al stress was also regulated by COI1-mediated JA signaling. Together, this study suggests that root growth inhibition is regulated by COI1-mediated JA signaling independent from auxin signaling and provides novel insights into the phytohormone-mediated root growth inhibition in response to Al stress. © 2017 American Society of Plant Biologists. All Rights Reserved.

  14. Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials.

    Science.gov (United States)

    Lamsa, Anne; Lopez-Garrido, Javier; Quach, Diana; Riley, Eammon P; Pogliano, Joe; Pogliano, Kit

    2016-08-19

    Increasing antimicrobial resistance has become a major public health crisis. New antimicrobials with novel mechanisms of action (MOA) are desperately needed. We previously developed a method, bacterial cytological profiling (BCP), which utilizes fluorescence microscopy to rapidly identify the MOA of antimicrobial compounds. BCP is based upon our discovery that cells treated with antibiotics affecting different metabolic pathways generate different cytological signatures, providing quantitative information that can be used to determine a compound's MOA. Here, we describe a system, rapid inhibition profiling (RIP), for creating cytological profiles of new antibiotic targets for which there are currently no chemical inhibitors. RIP consists of the fast, inducible degradation of a target protein followed by BCP. We demonstrate that degrading essential proteins in the major metabolic pathways for DNA replication, transcription, fatty acid biosynthesis, and peptidoglycan biogenesis in Bacillus subtilis rapidly produces cytological profiles closely matching that of antimicrobials targeting the same pathways. Additionally, RIP and antibiotics targeting different steps in fatty acid biosynthesis can be differentiated from each other. We utilize RIP and BCP to show that the antibacterial MOA of four nonsteroidal anti-inflammatory antibiotics differs from that proposed based on in vitro data. RIP is a versatile method that will extend our knowledge of phenotypes associated with inactivating essential bacterial enzymes and thereby allow for screening for molecules that inhibit novel essential targets.

  15. Oleic Acid and Hydroxytyrosol Inhibit Cholesterol and Fatty Acid Synthesis in C6 Glioma Cells

    Directory of Open Access Journals (Sweden)

    Paola Priore

    2017-01-01

    Full Text Available Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA and hydroxytyrosol (HTyr, two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 μM OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.

  16. Biosynthesis of collagen by fibroblasts kept in culture

    International Nuclear Information System (INIS)

    Machado-Santelli, G.M.

    1978-01-01

    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.) [pt

  17. Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

    Science.gov (United States)

    Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

    2011-01-01

    Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

  18. Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Qingbo; Man, Cong; Li, Danning; Tan, Huijuan; Xie, Ye; Huang, Jirong

    2016-12-05

    Anthocyanins, a group of L-phenylalanine (Phe)-derived flavonoids, have been demonstrated to play important roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases (ADTs), the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adt1 adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression of ADT2, hyperaccumulate Phe and produce elevated level of anthocyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sustaining anthocyanin biosynthesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  19. Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    2013-01-01

    Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  20. Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE.

    Science.gov (United States)

    Huang, Xin; Li, Hao-ming

    2009-08-05

    Lovastatin is an effective drug for treatment of hyperlipidemia. This study aimed to clone lovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein. According to the lovastatin synthase gene sequence from genebank, primers were designed to amplify and clone the lovastatin biosynthesis regulatory gene lovE from Aspergillus terrus genomic DNA. Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through internet resources and software like DNAMAN. Target fragment lovE, almost 1500 bp in length, was amplified from Aspergillus terrus genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted. In the lovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-like transcriptional factor.

  1. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Gao-Yi Tan

    2016-02-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  2. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Tan Gao-Yi

    2015-12-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  3. Effects of obesity and exercise on testicular leptin signal transduction and testosterone biosynthesis in male mice.

    Science.gov (United States)

    Yi, Xuejie; Gao, Haining; Chen, Dequan; Tang, Donghui; Huang, Wanting; Li, Tao; Ma, Tie; Chang, Bo

    2017-04-01

    To explore the role of the testicular leptin and JAK-STAT[leptin (LEP)-JAK-STAT] pathway in testosterone biosynthesis during juvenile stages and exercise for weight loss, male C57BL/6J mice were randomly divided into normal-diet and high-fat diet groups. After 10 wk, mice in the high-fat diet-fed group were further divided randomly into obese control, obese moderate-volume exercise, and obese high-volume exercise groups. Mice in the obese moderate-volume exercise group were provided with 2 h/day, 6 days/wk swimming exercise for 8 wk, and mice in the obese high-volume exercise group underwent twice the amount of daily exercise intervention as the obese moderate-volume exercise group. The results showed that a high-fat diet causes obesity, leptin resistance, inhibition of the testicular LEP-JAK-STAT pathway, decreased mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and the P -450 side-chain cleavage enzyme, a decrease in the serum testosterone-to-estradiol ratio, and declines in sperm quality parameters. Both moderate and high-volume exercise were able to reduce body fat and increase the mRNA and protein expression of LEP-JAK-STAT, but only moderate exercise significantly increased the mRNA and protein expression of steroidogenic factor-1, steroidogenic acute regulatory protein, and P -450 side-chain cleavage enzyme and significantly reversed the serum testosterone-to-estradiol ratio and sperm quality parameters. These findings suggest that by impairing the testicular LEP-JAK-STAT pathway, early-stage obesity inhibits the biosynthesis of testosterone and sexual development and reduces male reproductive potential. Long-term moderate and high-volume exercise can effectively reduce body fat and improve obesity-induced abnormalities in testicular leptin signal transduction, whereas only moderate-volume exercise can reverse the negative impacts of obesity on male reproductive function. Copyright © 2017 the American

  4. Topical problems in the biosynthesis of red blood pigment

    International Nuclear Information System (INIS)

    Franck, B.

    1982-01-01

    Uroporphyrinogen III plays a key role in the biosynthesis of heme, the red pigment of blood. In vivo studies with specifically 14 C- and 3 H-labeled precursors have revealed that the formation of uroporphyrinogen III in the organism follows several primary and subsidiary pathways. Model experiments on the pattern of biosynthesis have led to simple and effective methods of synthesizing uroporphyrin analogs and have shwon that their production is strongly favored thermodynamically, The biologically important porphyrins thus available permit a mechanistic explanantion of the light-induced dermatoses in porphyria diseases and suggest promising medical applications in diagnosis and therapy. (orig.)

  5. 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

  6. Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated Gentiana macrophylla Seedlings.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Cao

    Full Text Available Gentiana macrophylla, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in G. macrophylla. Our HPLC results showed that Gentiana macrophylla seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 μmol L-1 MeJA (C or 250 μmol L-1 MeJA (M5 and detected differentially expressed genes (DEGs. In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46% sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in G. macrophylla responds to MeJA.

  7. Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

    Science.gov (United States)

    Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A.; Wilensky, Robert L.; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A.

    2012-01-01

    The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy. PMID:22406532

  8. Improvement of Folate Biosynthesis by Lactic Acid Bacteria Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Norfarina Muhamad Nor

    2010-01-01

    Full Text Available Lactic acid bacteria (Lactococcus lactis NZ9000, Lactococcus lactis MG1363, Lactobacillus plantarum I-UL4 and Lactobacillus johnsonii DSM 20553 have been screened for their ability to produce folate intracellularly and/or extracellularly. L. plantarum I-UL4 was shown to be superior producer of folate compared to other strains. Statistically based experimental designs were used to optimize the medium formulation for the growth of L. plantarum I-UL4 and folate biosynthesis. The optimal values of important factors were determined by response surface methodology (RSM. The effects of carbon sources, nitrogen sources and para-aminobenzoic acid (PABA concentrations on folate biosynthesis were determined prior to RSM study. The biosynthesis of folate by L. plantarum I-UL4 increased from 36.36 to 60.39 µg/L using the optimized medium formulation compared to the selective Man de Rogosa Sharpe (MRS medium. Conditions for the optimal growth of L. plantarum I-UL4 and folate biosynthesis as suggested by RSM were as follows: lactose 20 g/L, meat extract 16.57 g/L and PABA 10 µM.

  9. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-01

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  10. Genes encoding enzymes of the lignin biosynthesis pathway in Eucalyptus

    Directory of Open Access Journals (Sweden)

    Ricardo Harakava

    2005-01-01

    Full Text Available Eucalyptus ESTs libraries were screened for genes involved in lignin biosynthesis. This search was performed under the perspective of recent revisions on the monolignols biosynthetic pathway. Eucalyptus orthologues of all genes of the phenylpropanoid pathway leading to lignin biosynthesis reported in other plant species were identified. A library made with mRNAs extracted from wood was enriched for genes involved in lignin biosynthesis and allowed to infer the isoforms of each gene family that play a major role in wood lignin formation. Analysis of the wood library suggests that, besides the enzymes of the phenylpropanoids pathway, chitinases, laccases, and dirigent proteins are also important for lignification. Colocalization of several enzymes on the endoplasmic reticulum membrane, as predicted by amino acid sequence analysis, supports the existence of metabolic channeling in the phenylpropanoid pathway. This study establishes a framework for future investigations on gene expression level, protein expression and enzymatic assays, sequence polymorphisms, and genetic engineering.

  11. Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

    Science.gov (United States)

    Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

    2015-01-14

    Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

  12. The mechanism of ethylene signaling induced by endophytic fungus Gilmaniella sp. AL12 mediating sesquiterpenoids biosynthesis in Atractylodes lancea

    Directory of Open Access Journals (Sweden)

    Jie eYuan

    2016-03-01

    Full Text Available Ethylene, the first known gaseous phytohormone, is involved in plant growth, development as well as responses to environmental signals. However, limited information is available on the role of ethylene in endophytic fungi induced secondary metabolites biosynthesis. Atractylodes lancea is a traditional Chinese herb, and its quality depends on the main active compounds sesquiterpenoids. This work showed that the endophytic fungus Gilmaniella sp. AL12 induced ethylene production in Atractylodes lancea. Pre-treatment of plantlets with ethylene inhibiter aminooxyacetic acid (AOA suppressed endophytic fungi induced accumulation of ethylene and sesquiterpenoids. Plantlets were further treated with AOA, salicylic acid (SA biosynthesis inhibitor paclobutrazol (PAC, jasmonic acid inhibitor ibuprofen (IBU, hydrogen peroxide (H2O2 scavenger catalase (CAT, nitric oxide (NO-specific scavenger 2-(4-Carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO. With endophytic fungi inoculation, IBU or PAC did not inhibit ethylene production, and JA and SA generation were suppressed by AOA, showing that ethylene may act as an upstream signal of JA and SA pathway. With endophytic fungi inoculation, CAT or cPTIO suppressed ethylene production, and H2O2 or NO generation was not affected by 1-aminocyclopropane-1-carboxylic acid (ACC, showing that ethylene may act as a downstream signal of H2O2 and NO pathway. Then, plantlets were treated with ethylene donor ACC, JA, SA, H2O2, NO donor sodium nitroprusside (SNP. Exogenous ACC could trigger JA and SA generation, whereas exogenous JA or SA did not affect ethylene production, and the induced sesquiterpenoids accumulation triggered by ACC was partly suppressed by IBU and PAC, showing that ethylene acted as an upstream signal of JA and SA pathway. Exogenous ACC did not affect H2O2 or NO generation, whereas exogenous H2O2 and SNP induced ethylene production, and the induced sesquiterpenoids

  13. Identification and biosynthesis of a novel xanthomonadin-dialkylresorcinol-hybrid from Azoarcus sp. BH72.

    Directory of Open Access Journals (Sweden)

    Tim A Schöner

    Full Text Available A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.

  14. Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

    Science.gov (United States)

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

  15. Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis

    Science.gov (United States)

    Kelly, Christine; Jones, Opal; Barnhart, Christopher; Lajoie, Curtis

    Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/1 or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

  16. Biosynthesis of anatoxin-a and analogues (anatoxins) in cyanobacteria.

    Science.gov (United States)

    Méjean, Annick; Paci, Guillaume; Gautier, Valérie; Ploux, Olivier

    2014-12-01

    Freshwater cyanobacteria produce secondary metabolites that are toxic to humans and animals, the so-called cyanotoxins. Among them, anatoxin-a and homoanatoxin-a are potent neurotoxins that are agonists of the nicotinic acetylcholine receptor. These alkaloids provoke a rapid death if ingested at low doses. Recently, the cluster of genes responsible for the biosynthesis of these toxins, the ana cluster, has been identified in Oscillatoria sp. PCC 6506, and a biosynthetic pathway was proposed. This biosynthesis was reconstituted in vitro using purified enzymes confirming the predicted pathway. One of the enzymes, AnaB a prolyl-acyl carrier protein oxidase, was crystallized and its three dimensional structure solved confirming its reaction mechanism. Three other ana clusters have now been identified and sequenced in other cyanobacteria. These clusters show similarities and some differences suggesting a common evolutionary origin. In particular, the cluster from Cylindrospermum stagnale PCC 7417, possesses an extra gene coding for an F420-dependent oxidoreductase that is likely involved in the biosynthesis of dihydroanatoxin-a. This review summarizes all these new data and discusses them in relation to the production of anatoxins in the environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Biosynthesis of silver nanoparticles synthesized by Aspergillus

    Indian Academy of Sciences (India)

    In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour ...

  18. Observational Study of a French and Belgian Multicenter Cohort of 23 Patients Diagnosed in Adulthood With Mevalonate Kinase Deficiency.

    Science.gov (United States)

    Durel, Cécile-Audrey; Aouba, Achille; Bienvenu, Boris; Deshayes, Samuel; Coppéré, Brigitte; Gombert, Bruno; Acquaviva-Bourdain, Cécile; Hachulla, Eric; Lecomte, Frédéric; Touitou, Isabelle; Ninet, Jacques; Philit, Jean-Baptiste; Messer, Laurent; Brouillard, Marc; Girard-Madoux, Marie-Hélène; Moutschen, Michel; Raison-Peyron, Nadia; Hutin, Pascal; Duffau, Pierre; Trolliet, Pierre; Hatron, Pierre-Yves; Heudier, Philippe; Cevallos, Ramiro; Lequerré, Thierry; Brousse, Valentine; Lesire, Vincent; Audia, Sylvain; Maucort-Boulch, Delphine; Cuisset, Laurence; Hot, Arnaud

    2016-03-01

    The aim of this study was to describe the clinical and biological features of Mevalonate kinase deficiency (MKD) in patients diagnosed in adulthood. This is a French and Belgian observational retrospective study from 2000 to 2014. To constitute the cohort, we cross-check the genetic and biochemical databases. The clinical, enzymatic, and genetic data were gathered from medical records. Twenty-three patients were analyzed. The mean age at diagnosis was 40 years, with a mean age at onset of symptoms of 3 years. All symptomatic patients had fever. Febrile attacks were mostly associated with arthralgia (90.9%); lymphadenopathy, abdominal pain, and skin lesions (86.4%); pharyngitis (63.6%); cough (59.1%); diarrhea, and hepatosplenomegaly (50.0%). Seven patients had psychiatric symptoms (31.8%). One patient developed recurrent seizures. Three patients experienced renal involvement (13.6%). Two patients had angiomyolipoma (9.1%). All but one tested patients had elevated serum immunoglobulin (Ig) D level. Twenty-one patients had genetic diagnosis; most of them were compound heterozygote (76.2%). p.Val377Ile was the most prevalent mutation. Structural articular damages and systemic AA amyloidosis were the 2 most serious complications. More than 65% of patients displayed decrease in severity and frequency of attacks with increasing age, but only 35% achieved remission. MKD diagnosed in adulthood shared clinical and genetic features with classical pediatric disease. An elevated IgD concentration is a good marker for MKD in adults. Despite a decrease of severity and frequency of attacks with age, only one-third of patients achieved spontaneous remission.

  19. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    International Nuclear Information System (INIS)

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-01-01

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  20. Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

    Directory of Open Access Journals (Sweden)

    Antonia Y. Tetteh

    2014-01-01

    Full Text Available Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0, but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3 treatment and then used quantitative real-time PCR (qRT-PCR to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

  1. In Vitro Biosynthesis of Unnatural Enterocin and Wailupemycin Polyketides¥

    Science.gov (United States)

    Kalaitzis, John A.; Cheng, Qian; Thomas, Paul M.; Kelleher, Neil L.; Moore, Bradley S.

    2009-01-01

    Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways. PMID:19215142

  2. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  3. Structure and Biosynthesis of Branched Wax Compounds on Wild Type and Wax Biosynthesis Mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Busta, Lucas; Jetter, Reinhard

    2017-06-01

    The cuticle is a waxy composite that protects the aerial organs of land plans from non-stomatal water loss. The chemical make-up of the cuticular wax mixture plays a central role in defining the water barrier, but structure-function relationships have not been established so far, in part due to gaps in our understanding of wax structures and biosynthesis. While wax compounds with saturated, linear hydrocarbon tails have been investigated in detail, very little is known about compounds with modified aliphatic tails, which comprise substantial portions of some plant wax mixtures. This study aimed to investigate the structures, abundances and biosynthesis of branched compounds on the species for which wax biosynthesis is best understood: Arabidopsis thaliana. Microscale derivatization, mass spectral interpretation and organic synthesis identified homologous series of iso-alkanes and iso-alcohols on flowers and leaves, respectively. These comprised approximately 10-15% of wild type wax mixtures. The abundances of both branched wax constituents and accompanying unbranched compounds were reduced on the cer6, cer3 and cer1 mutants but not cer4, indicating that branched compounds are in part synthesized by the same machinery as unbranched compounds. In contrast, the abundances of unbranched, but not branched, wax constituents were reduced on the cer2 and cer26 mutants, suggesting that the pathways to both types of compounds deviate in later steps of chain elongation. Finally, the abundances of branched, but not unbranched, wax compounds were reduced on the cer16 mutant, and the (uncharacterized) CER16 protein may therefore be controlling the relative abundances of iso-alkanes and iso-alcohols on Arabidopsis surfaces. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Biosynthesis and metabolic fate of phenylalanine in conifers

    Directory of Open Access Journals (Sweden)

    María Belén Pascual

    2016-07-01

    Full Text Available The amino acid phenylalanine (Phe is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

  5. Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

    Science.gov (United States)

    Pascual, María B; El-Azaz, Jorge; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cánovas, Francisco M

    2016-01-01

    The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

  6. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

    Science.gov (United States)

    Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

    2010-02-01

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

  7. Citrate coated silver nanoparticles with modulatory effects on aflatoxin biosynthesis in Aspergillus parasiticus

    Science.gov (United States)

    Mitra, Chandrani

    fungus successfully reverts its aflatoxin biosynthesis to normal levels once the level of AgNp-cit decreased significantly in the growth medium. A stability study of AgNp-cit in the fungal growth medium, along with mycelia, was conducted using UV-vis spectroscopy. The result showed that the distinctive peak (at 395nm wavelength) of silver nanoparticles, size of 20nm, shifted to a higher wavelength (400nm-500nm), broadened, and decreased over time. At 30-hour post inoculation the UV-vis peak at 395 nm wavelength was not observed at all. The peak shifts may occur due to organic molecules from the medium replacing the citrate surface coating. Another possible explanation for the peak shift are the interactions between the surface coating and other inorganic components in the medium. Peak broadening may suggest possible aggregation or formation of corona on the surface of AgNp due to particle-protein interactions (leading to AgNp aggregation in the growth medium). Reduction of peak height may suggest nanoparticle uptake by the mycelia, dissolution of nanoparticles into charged ions as well as possible interaction with other ions in the growth medium or the formation of precipitate of silver salt. We have investigated effects of different sizes (15 nm, 20 nm, and 30 nm) of AgNp-cit and pvp coated silver nanoparticles (AgNp-pvp (20 nm)) on growth and aflatoxin B1 biosynthesis in A. parasiticus. AgNp-cit size of 15nm showed maximum aflatoxin inhibition at 25ng/mL. For 20nm and 30nm AgNp-cit the strongest aflatoxin inhibition was observed at 50ng/mL concentration. The aflatoxin inhibitory effect was also found to be AgNp coating dependent. For 20nm AgNp-cit the strongest aflatoxin inhibition was seen at 50ng/mL (calculated) while for 20nm AgNp-pvp, the maximum aflatoxin inhibition was observed at 60ng/mL (calculated) concentration. Acute toxicity of silver nanoparticles on various organisms are well-studied but large knowledge gap still exist on the assessment of its chronic

  8. 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

  9. Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine.

    Science.gov (United States)

    Nagarajah, James; Le, Mina; Knauf, Jeffrey A; Ferrandino, Giuseppe; Montero-Conde, Cristina; Pillarsetty, Nagavarakishore; Bolaender, Alexander; Irwin, Christopher; Krishnamoorthy, Gnana Prakasam; Saqcena, Mahesh; Larson, Steven M; Ho, Alan L; Seshan, Venkatraman; Ishii, Nobuya; Carrasco, Nancy; Rosen, Neal; Weber, Wolfgang A; Fagin, James A

    2016-11-01

    Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.

  10. DGAT enzymes and triacylglycerol biosynthesis

    OpenAIRE

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, ...

  11. Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis.

    Science.gov (United States)

    Wang, Zhong-Wei; Jiang, Cong; Wen, Qiang; Wang, Na; Tao, Yuan-Yuan; Xu, Li-An

    2014-03-15

    Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1).

    Science.gov (United States)

    Demidenko, Aleksandr; Akberdin, Ilya R; Allemann, Marco; Allen, Eric E; Kalyuzhnaya, Marina G

    2016-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1) . Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE , was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE -knockout mutants and farE -overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE -strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

  13. Study on the secondary metabolic regulation of callus of Panax quinquefolium L

    International Nuclear Information System (INIS)

    Zhang Meiping; Wang Yi; Sun Chunyu; Li Xianggao

    2003-01-01

    The effects of precursors with different kind, concentration, growth phase, time and different ATP, nicotinic acid concentration on the callus growth and saponin formation with the buds of Panax quinquefolium L. has been studied. The results showed that 0.1% mevalonic acid inhibited the callus growth but promoted the saponin formation; L-leucine of 100 mg/L had no obvious effects on callus growth, but promoted the saponin formation; Mg(Ac) 2 of 444 mg/L promoted both the callus growth and saponin content. The optimum time for precursor feeding is at the beginning of regeneration. The optimum time for mevalonic acid is 6h; ATP of 160 mg/L promoted both the callus growth and saponin formation. Nicotinic acid of 1.0 mg/L promoted callus growth and saponin formation. With 4000 Gy of 60 Co γ-rays, the saponin content has been increased 1 time compared with nonirradiation

  14. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

    International Nuclear Information System (INIS)

    Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1986-01-01

    Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32 Pi and L-[U- 14 C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells

  15. Metabolic engineering for improved heterologous terpenoid biosynthesis

    NARCIS (Netherlands)

    Ryden, A.; Melillo, E.; Czepnik, M.; Kayser, O.

    Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood

  16. Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

    Science.gov (United States)

    Sheridan, Kevin J; Lechner, Beatrix Elisabeth; Keeffe, Grainne O'; Keller, Markus A; Werner, Ernst R; Lindner, Herbert; Jones, Gary W; Haas, Hubertus; Doyle, Sean

    2016-10-17

    Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H 2 O 2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H 2 O 2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H 2 O 2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

  17. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  18. 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

  19. Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

    Science.gov (United States)

    Shrivastava-Ranjan, Punya; Flint, Mike; Bergeron, Éric; McElroy, Anita K; Chatterjee, Payel; Albariño, César G; Nichol, Stuart T; Spiropoulou, Christina F

    2018-05-01

    Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD. IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune

  20. Biosynthesis of two dihydropyrrole-polyketides from a marine-derived Penicillium citrinum

    Energy Technology Data Exchange (ETDEWEB)

    Romminger, Stelamar; Pimenta, Eli F.; Berlinck, Roberto G.S., E-mail: rgsberlinck@iqsc.usp.br [Universidade de Sao Paulo (USP), Sao Carlos, SP (Brazil). Inst. de Quimica; Nascimento, Eduardo S.; Ferreira, Antonio G. [Universidade Federal de Sao Carlos (UFSCAR), SP (Brazil). Dept. de Quimica

    2012-10-15

    Feeding experiments with {sup 13}C-labeled precursors were performed in order to establish the biosynthesis of two N-acylated dihydro pyrroles, (8E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldec- 8-ene-1,3-dione (1) and 1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldecane-1,3-dione (2), isolated from the cultures of a marine-derived Penicillium citrinum. The biosynthesis of both, 1 and 2, involves the incorporation of acetate, methionine and ornithine. (author)

  1. Biosynthesis of two dihydropyrrole-polyketides from a marine-derived Penicillium citrinum

    International Nuclear Information System (INIS)

    Romminger, Stelamar; Pimenta, Eli F.; Berlinck, Roberto G.S.; Nascimento, Eduardo S.; Ferreira, Antonio G.

    2012-01-01

    Feeding experiments with 13 C-labeled precursors were performed in order to establish the biosynthesis of two N-acylated dihydro pyrroles, (8E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldec- 8-ene-1,3-dione (1) and 1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldecane-1,3-dione (2), isolated from the cultures of a marine-derived Penicillium citrinum. The biosynthesis of both, 1 and 2, involves the incorporation of acetate, methionine and ornithine. (author)

  2. epsilon-N-trimethyllysine availability regulates the rate of carnitine biosynthesis in the growing rat

    International Nuclear Information System (INIS)

    Rebouche, C.J.; Lehman, L.J.; Olson, L.

    1986-01-01

    Rates of carnitine biosynthesis in mammals depend on the availability of substrates and the activity of enzymes subserving the pathway. This study was undertaken to test the hypothesis that the availability of epsilon-N-trimethyllysine is rate-limiting for synthesis of carnitine in the growing rat and to evaluate diet as a source of this precursor for carnitine biosynthesis. Rats apparently absorbed greater than 90% of a tracer dose of [methyl- 3 H]epsilon-N-trimethyllysine, and approximately 30% of that was incorporated into tissues as [ 3 H]carnitine. Rats given oral supplements of epsilon-N-trimethyllysine (0.5-20 mg/d), but no dietary carnitine, excreted more carnitine than control animals receiving no dietary epsilon-N-trimethyllysine or carnitine. Rates of carnitine excretion increased in a dose-dependent manner. Tissue and serum levels of carnitine also increased with dietary epsilon-N-trimethyllysine supplementation. There was no evidence that the capacity for carnitine biosynthesis was saturated even at the highest level of oral epsilon-N-trimethyllysine supplementation. Common dietary proteins (casein, soy protein and wheat gluten) were found to be poor sources of epsilon-N-trimethyllysine for carnitine biosynthesis. The results of this study indicate that the availability of epsilon-N-trimethyllysine limits the rate of carnitine biosynthesis in the growing rat

  3. Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes.

    Science.gov (United States)

    Argyris, Jason; Dahal, Peetambar; Hayashi, Eiji; Still, David W; Bradford, Kent J

    2008-10-01

    Lettuce (Lactuca sativa 'Salinas') seeds fail to germinate when imbibed at temperatures above 25 degrees C to 30 degrees C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 degrees C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

  4. Fusarium proliferatum strains change fumonisin biosynthesis and accumulation when exposed to host plant extracts.

    Science.gov (United States)

    Górna, Karolina; Pawłowicz, Izabela; Waśkiewicz, Agnieszka; Stępień, Łukasz

    2016-01-01

    Fumonisin concentrations in mycelia and media were studied in liquid Fusarium proliferatum cultures supplemented with host plant extracts. Furthermore, the kinetics of fumonisin accumulation in media and mycelia collected before and after extract addition was analysed as well as the changes in the expression of the FUM1 gene. Fumonisin content in culture media increased in almost all F. proliferatum strains shortly after plant extracts were added. The asparagus extract induced the highest FB level increase and the garlic extract was the second most effective inducer. Fumonisin level decreased constantly until 14th day of culturing, though for some strains also at day 8th an elevated FB level was observed. Pineapple extract induced the highest increase of fum1 transcript levels as well as fumonisin synthesis in many strains, and the peas extract inhibited fungal growth and fumonisin biosynthesis. Moreover, fumonisins were accumulated in mycelia of studied strains and in the respective media. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  5. Biosynthesis of polyhydroxyalkanotes in wildtype yeasts | Desuoky ...

    African Journals Online (AJOL)

    Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different ...

  6. Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

    Science.gov (United States)

    Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

    2011-09-18

    Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

  7. Omethoate treatment mitigates high salt stress inhibited maize seed germination.

    Science.gov (United States)

    Yang, Kejun; Zhang, Yifei; Zhu, Lianhua; Li, Zuotong; Deng, Benliang

    2018-01-01

    Omethoate (OM) is a highly toxic organophophate insecticide, which is resistant to biodegradation in the environment and is widely used for pest control in agriculture. The effect of OM on maize seed germination was evaluated under salt stress. Salt (800mM) greatly reduced germination of maize seed and this could be reversed by OM. Additionally, H 2 O 2 treatment further improved the effect of OM on seed germination. Higher H 2 O 2 content was measured in OM treated seed compared to those with salt stress alone. Dimethylthiourea (DTMU), a specific scavenger of reactive oxygen species (ROS), inhibited the effect of OM on seed germination, as did IMZ (imidazole), an inhibitor of NADPH oxidase. Abscisic acid (ABA) inhibited the effect of OM on seed germination, whereas fluridone, a specific inhibitor of ABA biosynthesis, enhanced the effect of OM. Taken together, these findings suggest a role of ROS and ABA in the promotion of maize seed germination by OM under salt stress. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. 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.

  9. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  10. Simvastatin attenuates acrolein-induced mucin production in rats: involvement of the Ras/extracellular signal-regulated kinase pathway.

    Science.gov (United States)

    Chen, Ya-Juan; Chen, Peng; Wang, Hai-Xia; Wang, Tao; Chen, Lei; Wang, Xun; Sun, Bei-Bei; Liu, Dai-Shun; Xu, Dan; An, Jing; Wen, Fu-Qiang

    2010-06-01

    Airway mucus overproduction is a cardinal feature of airway inflammatory diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. Since the small G-protein Ras is known to modulate cellular functions in the lung, we sought to investigate whether the Ras inhibitor simvastatin could attenuate acrolein-induced mucin production in rat airways. Rats were exposed to acrolein for 12 days, after first being pretreated intragastrically for 24 h with either simvastatin alone or simvastatin in combination with mevalonate, which prevents the isoprenylation needed for Ras activation. Lung tissue was analyzed for extracellular signal-regulated kinase (ERK) activity, goblet cell metaplasia and mucin production. To analyze the effect of simvastatin on mucin production in more detail, acrolein-exposed human airway epithelial NCI-H292 cells were pretreated with simvastatin alone or together with mevalonate. Culture medium was collected to detect mucin secretion, and cell lysates were examined for Ras-GTPase activity and epidermal growth factor receptor (EGFR)/ERK phosphorylation. In vivo, simvastatin treatment dose-dependently suppressed acrolein-induced goblet cell hyperplasia and metaplasia in bronchial epithelium and inhibited ERK phosphorylation in rat lung homogenates. Moreover, simvastatin inhibited Muc5AC mucin synthesis at both the mRNA and protein levels in the lung. In vitro, simvastatin pretreatment attenuated the acrolein-induced significant increase in MUC5AC mucin expression, Ras-GTPase activity and EGFR/ERK phosphorylation. These inhibitory effects of simvastatin were neutralized by mevalonate administration both in vitro and in vivo. Our results suggest that simvastatin may attenuate acrolein-induced mucin protein synthesis in the airway and airway inflammation, possibly by blocking ERK activation mediated by Ras protein isoprenylation. Thus, the evidence from the experiment suggests that human trials are warranted to determine the potential

  11. Taurine biosynthesis in frog retina: effects of light and dark adaptations

    International Nuclear Information System (INIS)

    Nishimura, C.; Ida, S.; Kuriyama, K.

    1983-01-01

    The retinal uptake and metabolism of cysteine, a precursor for taurine biosynthesis, were analysed using the bull frog. The [ 14 C] cysteine uptake into isolated retina had some specific properties: It was rather temperature independent, required Na ions, was inhibited by ouabain but not by dinitrophenol, and exhibited saturation kinetics composed of two components. When retinal homogenate was incubated with 12-30 microM of L-[U- 14 C]cysteine, the accumulation of labeled alanine, cysteine sulfinic acid (CSA), cysteic acid (CA), hypotaurine, and taurine was detected. The metabolic conversions of [ 14 C] cysteine to labeled alanine, hypotaurine, and taurine were linear over 90 minutes. Prolonged light adaptation (3 weeks) induced a significant reduction in the formation of labeled CA, CSA, hypotaurine, and taurine from [ 14 C] cysteine. On the other hand, it was found that in dark-adapted retinae, the formation of labeled taurine from [ 14 C] cysteine increased significantly in spite of the reduction in the formation of labeled CA. These results indicate that biosynthetic pathways exist for taurine from cysteine in frog retina, and that these metabolic pathways are involved in the regulation of retinal taurine content under continuous visual adaptation

  12. 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.

  13. Identification of Putative Genes Involved in Limonoids Biosynthesis in Citrus by Comparative Transcriptomic Analysis

    Directory of Open Access Journals (Sweden)

    Fusheng Wang

    2017-05-01

    Full Text Available Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L. Osbeck at different development stages with variances in limonoids contents were used for digital gene expression profiling analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

  14. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    Science.gov (United States)

    Villarreal, Fernando D; Kültz, Dietmar

    2015-01-01

    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.

  15. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  16. Role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by Serratia marcescens Db10.

    Science.gov (United States)

    Gerc, Amy J; Stanley-Wall, Nicola R; Coulthurst, Sarah J

    2014-08-01

    Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites. © 2014 The Authors.

  17. Modulation of biosynthesis and regulatory action of 24(S),25-epoxycholesterol (S-EC) in cultured cells by progesterone (PG)

    International Nuclear Information System (INIS)

    Panini, S.R.; Gupta, A.K.; Sexton, R.C.; Parish, E.J.; Rudney, H.

    1987-01-01

    Treatment of IEC-6 cells with PG caused a strong inhibition of cholesterol biosynthesis at the level of desmosterol reductase. In addition, two new products were observed in PG-treated cells. The first compound was designated as cholesta-5,7,24-trien-3β-ol based on its HPLC chromatographic properties. The second compound was identified as S-EC based on (1) a comparison of its chromatographic properties with those of authentic EC and (2) by its conversion to 25-hydroxycholesterol (HC) upon reduction with LiAlH 4 . In spite of cellular accumulation of S-EC in the presence of PG, the activity of HMG-CoA reductase (HMGR) which is known to be sensitive to oxysterols, was elevated rather than suppressed. On the other hand, when PG-treated cells were refed fresh medium without PG, HMGR activity was suppressed. Exogenous S-EC was a potent suppressor of HMGR in untreated IEC-6 cells. Suppression of HMGR by S-EC but not by HC could be prevented by progesterone. Exogenous [ 3 H]S-EC was not metabolized by IEC-6 cells. These results support the hypothesis that S-EC plays a normal regulatory role in sterol biosynthesis and indicate that enhanced S-EC synthesis observed in the presence of PG may be due to interference with this regulatory action

  18. Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

    NARCIS (Netherlands)

    Hogenboom, Sietske; Romeijn, Gerrit Jan; Houten, Sander M.; Baes, Myriam; Wanders, Ronald J. A.; Waterham, Hans R.

    2002-01-01

    To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger

  19. Dolichol related intermediates involved in N-linked gloycoprotein synthesis in Drosophila Kc cells

    International Nuclear Information System (INIS)

    Sagami, H.; Chap, R.; Rivero, M.; Lennarz, W.J.

    1986-01-01

    The purpose of this investigation is to study the regulation of the biosynthesis of dolichyl phosphate (Dol-P) in embryonic cells. Using embryonic Drosophila Kc cells which are known to lack the squalene-cholesterol branch of polyisoprenoid biosynthetic pathway, the authors labeled the cells in suspension culture for 4 hrs with [5- 3 H]mevalonic acid. After harvesting the cells, the incorporated radioactive lipids were extracted with CHCl 3 /CH 3 OH (2/1). Analysis of the extract by a combination of several methods consisting of ion exchange chromatography on DEAE-cellulose, gel filtration on Fractogel TSK-HW 40, and silica gel thin layer chromatography revealed that the extract contained coenzyme Q, farnesol, dolichol (Dol), Dol-P, and dolichol-linked sugar (Dol-P-Sugar). In a pulse-chase experiment the metabolism of these intermediates was measured for various times up to 30 hrs. The amount of radioactivity in Dol slightly increased with the chase time, while that in Dol-P decreased. The amount of radioactive Dol-P-Sugar remained unchanged. These results suggest that the de novo end product in dolichol biosynthesis is Dol-P and that the level of Dol-P can be controlled by varying the rate of de novo biosynthesis and by dephosphorylation. The authors are now investigating the effect of ecdysone, the molting hormone, on the level of these intermediates and on incorporation of sugars into glycoproteins

  20. 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.

  1. Studies on the biosynthesis of the modified-peptide antibiotic, nosiheptide

    International Nuclear Information System (INIS)

    Houck, D.R.

    1986-01-01

    The biosynthesis of nosiheptide, a highly modified-peptide antibiotic, was studied by feeding 14 C- and 13 C-labeled precursors to the producing organism, Streptomyces actuosus. To this end, methods were developed for the fermentation of S. Actuosus, and the isolation of nosiheptide. Nosiheptide was produced in a synthetic medium at levels of 175 to 200 mg/L. In addition, a HPLC method was developed for rapid and accurate assay of nosiheptide in fermentation broths. During the fermentation studies, nosiheptide production was found to be inhibited by tryptophan, cysteine, ammonia, and phosphate. Feeding experiments with DL-[3- 13 C]cysteine, L-[3- 13 C]serine, DL-[1- 13 C]serine, L-[CH 3 - 13 C]methionine, and L-[2,1'- 13 C 2 ]tryptophan uncovered the biosynthetic precursors of the residues in nosiheptide. The double-quantum NMR technique known as INADEQUATE was used to analyze nosiheptide biosynthesized from [U- 13 Cnumber]glycerol. This experiment permitted unambiguous 13 C-NMR assignments to be made, as well as elucidation of the biochemically maintained 13 C- 13 C connectivities in the conversion of the labeled glycerol to nosiheptide. Making some reasonable assumptions, a logical peptide precursor of nosiheptide is proposed: H 2 N-ser-cys-thr-thr-cys-glu-cys-cys-cys-ser-cys-ser-CO 2

  2. Comparative Transcriptome Analysis Identifies Putative Genes Involved in Steroid Biosynthesis in Euphorbia tirucalli

    Directory of Open Access Journals (Sweden)

    Weibo Qiao

    2018-01-01

    Full Text Available Phytochemical analysis of different Euphorbia tirucalli tissues revealed a contrasting tissue-specificity for the biosynthesis of euphol and β-sitosterol, which represent the two pharmaceutically active steroids in E. tirucalli. To uncover the molecular mechanism underlying this tissue-specificity for phytochemicals, a comprehensive E. tirucalli transcriptome derived from its root, stem, leaf and latex was constructed, and a total of 91,619 unigenes were generated with 51.08% being successfully annotated against the non-redundant (Nr protein database. A comparison of the transcriptome from different tissues discovered members of unigenes in the upstream steps of sterol backbone biosynthesis leading to this tissue-specific sterol biosynthesis. Among them, the putative oxidosqualene cyclase (OSC encoding genes involved in euphol synthesis were notably identified, and their expressions were significantly up-regulated in the latex. In addition, genome-wide differentially expressed genes (DEGs in the different E. tirucalli tissues were identified. The cluster analysis of those DEGs showed a unique expression pattern in the latex compared with other tissues. The DEGs identified in this study would enrich the insights of sterol biosynthesis and the regulation mechanism of this latex-specificity.

  3. Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

    Science.gov (United States)

    T, Vinutha; Bansal, Navita; Kumari, Khushboo; Prashat G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, S K; Sachdev, Archana; Praveen, Shelly

    2017-12-20

    Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

  4. Insect molting hormone and sterol biosynthesis in spinach

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  5. Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

    Science.gov (United States)

    Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

    2017-07-01

    NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

  6. Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5

    Science.gov (United States)

    Zhang, Duanwu; Tomisato, Wataru; Su, Lijing; Sun, Lei; Choi, Jin Huk; Zhang, Zhao; Wang, Kuan-wen; Zhan, Xiaoming; Choi, Mihwa; Li, Xiaohong; Tang, Miao; Castro-Perez, Jose M.; Hildebrand, Sara; Murray, Anne R.; Moresco, Eva Marie Y.; Beutler, Bruce

    2017-01-01

    The recessive N-ethyl-N-nitrosourea–induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body. PMID:28607088

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

    Directory of Open Access Journals (Sweden)

    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.

  8. ODORANT1 Regulates Fragrance Biosynthesis in Petunia FlowersW⃞

    Science.gov (United States)

    Verdonk, Julian C.; Haring, Michel A.; van Tunen, Arjen J.; Schuurink, Robert C.

    2005-01-01

    Floral scent is important to plant reproduction because it attracts pollinators to the sexual organs. Therefore, volatile emission is usually tuned to the foraging activity of the pollinators. In Petunia hybrida, volatile benzenoids determine the floral aroma. Although the pathways for benzenoid biosynthesis have been characterized, the enzymes involved are less well understood. How production and emission are regulated is unknown. By targeted transcriptome analyses, we identified ODORANT1 (ODO1), a member of the R2R3-type MYB family, as a candidate for the regulation of volatile benzenoids in Petunia hybrida cv W115 (Mitchell) flowers. These flowers are only fragrant in the evening and at night. Transcript levels of ODO1 increased before the onset of volatile emission and decreased when volatile emission declined. Downregulation of ODO1 in transgenic P. hybrida Mitchell plants strongly reduced volatile benzenoid levels through decreased synthesis of precursors from the shikimate pathway. The transcript levels of several genes in this pathway were reduced by suppression of ODO1 expression. Moreover, ODO1 could activate the promoter of the 5-enol-pyruvylshikimate-3-phosphate synthase gene. Flower pigmentation, which is furnished from the same shikimate precursors, was not influenced because color and scent biosynthesis occur at different developmental stages. Our studies identify ODO1 as a key regulator of floral scent biosynthesis. PMID:15805488

  9. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

    KAUST Repository

    Kuwahara, Hiroyuki; Alazmi, Meshari; Cui, Xuefeng; Gao, Xin

    2016-01-01

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/.

  10. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

    KAUST Repository

    Kuwahara, Hiroyuki

    2016-04-29

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/.

  11. Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    van der Graaff, Eric; Hooykaas, Paul; Lein, Wolfgang

    2004-01-01

    Purine nucleotides are essential components to sustain plant growth and development. In plants they are either synthesized "de novo" during the process of purine biosynthesis or are recycled from purine bases and purine nucleosides throughout the salvage pathway. Comparison between animals...... biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening...... strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role...

  12. BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

    Science.gov (United States)

    Creelman, Robert A.; Mullet, John E.

    1997-06-01

    Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

  13. CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa.

    Science.gov (United States)

    Landolfo, Sara; Ianiri, Giuseppe; Camiolo, Salvatore; Porceddu, Andrea; Mulas, Giuliana; Chessa, Rossella; Zara, Giacomo; Mannazzu, Ilaria

    2018-01-01

    A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

  14. Triterpene biosynthesis in plants.

    Science.gov (United States)

    Thimmappa, Ramesha; Geisler, Katrin; Louveau, Thomas; O'Maille, Paul; Osbourn, Anne

    2014-01-01

    The triterpenes are one of the most numerous and diverse groups of plant natural products. They are complex molecules that are, for the most part, beyond the reach of chemical synthesis. Simple triterpenes are components of surface waxes and specialized membranes and may potentially act as signaling molecules, whereas complex glycosylated triterpenes (saponins) provide protection against pathogens and pests. Simple and conjugated triterpenes have a wide range of applications in the food, health, and industrial biotechnology sectors. Here, we review recent developments in the field of triterpene biosynthesis, give an overview of the genes and enzymes that have been identified to date, and discuss strategies for discovering new triterpene biosynthetic pathways.

  15. A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis

    Science.gov (United States)

    Frelin, Océane; Huang, Lili; Hasnain, Ghulam; Jeffryes, James G.; Ziemak, Michael J.; Rocca, James R.; Wang, Bing; Rice, Jennifer; Roje, Sanja; Yurgel, Svetlana N.; Gregory, Jesse F.; Edison, Arthur S.; Henry, Christopher S.; deCrécy-Lagard, Valérie; Hanson, Andrew D.

    2015-01-01

    Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multistep pathway. The early intermediates of this pathway are notoriously reactive, and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. Here we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA, respectively). We present cheminformatic, biochemical, genetic, and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multienzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites. PMID:25431972

  16. Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10.

    Science.gov (United States)

    Balagurunathan, R; Radhakrishnan, M; Rajendran, R Babu; Velmurugan, D

    2011-10-01

    Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.

  17. Biosynthesis of membrane lipids of thermophilic archaebacteria and its implication to early evolution of life

    International Nuclear Information System (INIS)

    Oshima, Tairo

    1995-01-01

    The unit lipid of cell membranes of archaebacteria is unique ether lipids, O-dialkylated glycerol with a polar head group at sn-1 position. The chirality of glycerol moiety of the lipids is opposite to that of other kingdoms. The hydrophobic potion consists of saturated C 20 isoprenoid hydrocarbon backbone and is connected to glycerol by an ether linkage. In addition, cell membrane of some of thermophilic archaebacteria are monolayer (in stead of bilayer) of tetraether lipids in which both tails of hydrocarbon chains of two diether lipids are covalently connected in a tail-to-tail fashion. Although the host cell from which contemporary eukaryotes have been derived by endosymbiosis, is speculated to be an archaebacterium, the unique ether lipids raised a serious question to the idea of archabacterial origin of eukaryote cells; why the unique ether lipids are not used to construct cytoplasmic membranes of eukaryotes? The author and his colleagues have studied biosynthesis of membrane liquids of two thermo-acidophilic archaebacteria, Thermoplasma and Sulfolobus. It was found that origins of stereospecificity of glycerol moiety of archaebacterial ether lipids differs form species to species. In Sulfolobus sn-glycerol-1-phosphate (the abnormal isomer of glycerol phosphate) seems to be directly synthesized from glycerol, whereas in Halobacterium stereospecificity of glycerol phosphate is inverted during the lipid synthesis. Recently we found that specific inhibitors for eukaryotes squalene epoxidase inhibit the condensation of diether lipids to tetraether lipids in cell-free extracts of these thermophilic archaebacteria. The results suggest evolutionary implication of archaebacterial tetraether condensing enzyme to eukaryote sterol biosynthesis. Relationships between chemical structures of membrane lipids and early evolution of life will be discussed. (author). Abstract only

  18. Macromolecule biosynthesis assay and fluorescence spectroscopy methods to explore antimicrobial peptide mode(s) of action

    DEFF Research Database (Denmark)

    Jana, Bimal; Baker, Kristin Renee; Guardabassi, Luca

    2017-01-01

    the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods...

  19. Biosynthesis and therapeutic properties of Lavandula essential oil constituents.

    Science.gov (United States)

    Woronuk, Grant; Demissie, Zerihun; Rheault, Mark; Mahmoud, Soheil

    2011-01-01

    Lavenders and their essential oils have been used in alternative medicine for several centuries. The volatile compounds that comprise lavender essential oils, including linalool and linalyl acetate, have demonstrative therapeutic properties, and the relative abundance of these metabolites is greatly influenced by the genetics and environment of the developing plants. With the rapid progress of molecular biology and the genomic sciences, our understanding of essential oil biosynthesis has greatly improved over the past few decades. At the same time, there is a recent surge of interest in the use of natural remedies, including lavender essential oils, in alternative medicine and aromatherapy. This article provides a review of recent developments related to the biosynthesis and medicinal properties of lavender essential oils. © Georg Thieme Verlag KG Stuttgart · New York.

  20. [A systematic review of biosynthesis of poly (3-hydroxypropionate)].

    Science.gov (United States)

    Chang, Le; Zhan, Yuanlong; Liu, Changli

    2018-04-25

    Poly (3-hydroxypropionate) (P3HP), a new member of thermoplastic of family polyhydroxyalkanoates (PHAs), has excellent characteristics of biodegradability and biocompatibility. By now no reports can be found about wild-type bacteria that naturally synthesize P3HP, so the main way to produce P3HP is chemical and biological methods. Chemical method by adding high cost 3-HP monomers or their structural analogs as precursors, has the drawbacks of toxicity, low effectiveness and high cost. Biological method using engineered strain may utilize inexpensive and renewable carbon source to produce P3HP and has gradually become more and more popular. We systematically review here the biosynthesis of P3HP research progress. The advantages and disadvantages of biosynthesis pathways of glycerol pathway, malonyl-CoA pathway and β-alanine pathway were analyzed.

  1. Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans*

    Science.gov (United States)

    Wang, Wenqing; McReynolds, Melanie R.; Goncalves, Jimmy F.; Shu, Muya; Dhondt, Ineke; Braeckman, Bart P.; Lange, Stephanie E.; Kho, Kelvin; Detwiler, Ariana C.; Pacella, Marisa J.; Hanna-Rose, Wendy

    2015-01-01

    Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD+ consumers to resynthesize NAD+, resulting in a reduction in global NAD+ bioavailability. We manipulate NAD+ levels to demonstrate that a minor deficit in NAD+ availability is incompatible with a normal pace of gonad development. The NAD+ deficit compromises NAD+ consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD+ biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD+ salvage biosynthesis for the purposes of inhibiting tumor growth. PMID:26350462

  2. Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the Developing Embryo of Pecan (Carya illinoinensis).

    Science.gov (United States)

    Huang, Ruimin; Huang, Youjun; Sun, Zhichao; Huang, Jianqin; Wang, Zhengjia

    2017-05-24

    Pecan (Carya illinoinensis) is an important woody tree species because of the high content of healthy oil in its nut. Thus far, the pathways and key genes related to oil biosynthesis in developing pecan seeds remain largely unclear. Our analyses revealed that mature pecan embryo accumulated more than 80% oil, in which 90% was unsaturated fatty acids with abundant oleic acid. RNA sequencing generated 84,643 unigenes in three cDNA libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering (DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, 7 for oil bodies, and 5 for transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil began to increase while crude fat contents increased from 16.61 to 74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologues were not detected. Consequently, we inferred that the linolenic acid in developing pecan embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula. During pecan embryo development, different unigenes are expressed for plastidial and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well consistent with their expression in transcriptomic analyses. Our data would be important for the metabolic engineering of pecans to increase oil contents and modify fatty acid composition.

  3. Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

    Science.gov (United States)

    Matuschewski, Kai; Haussig, Joana M.

    2016-01-01

    Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

  4. A model for evolution and regulation of nicotine biosynthesis regulon in tobacco.

    Science.gov (United States)

    Kajikawa, Masataka; Sierro, Nicolas; Hashimoto, Takashi; Shoji, Tsubasa

    2017-06-03

    In tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.

  5. Bioactive Mushroom Polysaccharides: A Review on Monosaccharide Composition, Biosynthesis and Regulation.

    Science.gov (United States)

    Wang, Qiong; Wang, Feng; Xu, Zhenghong; Ding, Zhongyang

    2017-06-13

    Mushrooms are widely distributed around the world and are heavily consumed because of their nutritional value and medicinal properties. Polysaccharides (PSs) are an important component of mushrooms, a major factor in their bioactive properties, and have been intensively studied during the past two decades. Monosaccharide composition/combinations are important determinants of PS bioactivities. This review summarizes: (i) monosaccharide composition/combinations in various mushroom PSs, and their relationships with PS bioactivities; (ii) possible biosynthetic pathways of mushroom PSs and effects of key enzymes on monosaccharide composition; (iii) regulation strategies in PS biosynthesis, and prospects for controllable biosynthesis of PSs with enhanced bioactivities.

  6. Extracellular facile biosynthesis, characterization and stability of gold nanoparticles by Bacillus licheniformis.

    Science.gov (United States)

    Singh, Sneha; Vidyarthi, Ambarish Sharan; Nigam, Vinod Kumar; Dev, Abhimanyu

    2014-02-01

    The development of a reliable, eco-friendly process for synthesis of gold nanoparticles (AuNPs) has gained impetus in recent years to counter the drawbacks of chemical and physical methods. This study illustrates simple, green synthesis of AuNPs in vitro using cell lysate supernatant (CLS) of non-pathogenic bacteria and to investigate its potential antimicrobial activity. Gold nanoparticles were synthesized by the reduction of precursor AuCl4- ions using the CLS of Bacillus licheniformis at 37°C upon 24 h of incubation. The nanoparticles were characterized for their morphology, particle size, optical absorption, zeta potential, and stability. Further the antimicrobial activity was assayed using cup-plate method. The process of biosynthesis was extracellular and the gold ions were reduced to stable nanogold of average size 38 nm. However, upon storage of AuNPs for longer duration at room temperature stability was influenced in terms of increase in particle size and decrease in zeta potential with respect to as synthesized nanoparticles. SEM micrographs revealed the spherical shape of AuNPs and EDX analysis confirmed the presence of gold in the sample. Also clear zone of inhibition was observed against Bacilllus subtilis MTCC 8364, Pseudomonas aeruginosa MTCC 7925, and Escherichia coli MTCC 1698 confirming the antimicrobial activity of AuNPs. The bioprocess under study was simple and less time consuming as compared to other methods as the need for harvesting AuNPs from within the microbial cells via downstream process will be eliminated. Nanoparticles exhibited good stability even in absence of external stabilizing agents. AuNPs showed good antimicrobial activity against several Gram-negative and Gram-positive pathogenic bacteria. The extracellular biosynthesis from CLS may serve as a suitable alternative for large scale synthesis of gold nanoparticles in vitro. The synthesis from lysed bacterial cell strongly suggests that exposure of microbial whole cells to the

  7. Biosynthesis of silver nanoparticles and its antibacterial activity ...

    African Journals Online (AJOL)

    In the present research work, biosynthesis of silver nanoparticles and its activity on bacterial pathogens were investigated. Silver nanoparticles were rapidly synthesized using Urospora sp. and the formation of nanoparticles was observed within 30 min. The results recorded from UV–vis spectrum, Fourier Transform Infrared ...

  8. Biosynthesis of silver nanoparticles by Leishmania tropica | Rahi ...

    African Journals Online (AJOL)

    A novel biosynthesis route for Silver Nanoparticles (Ag-NPs) was attempted in the present study using Leishmania tropica the causative agent of cutaneous leishmaniasis in different countries, particularly in Mediterranean region in Iraq. Silver nanoparticles were successfully synthesized from AgNO3 by reduction of ...

  9. Biosynthesis of silver nanoparticles and its antibacterial activity ...

    African Journals Online (AJOL)

    Dr.Rajasekar

    2012-07-19

    Jul 19, 2012 ... Available online at http://www.academicjournals.org/AJB ... Transmission Electron Microscopy (HRTEM) support the biosynthesis and characterization of silver nanoparticles. ... nanoparticle from seaweed is a green chemical method ... operating at a voltage of 80 kV and a current of 30 mA (Chandran.

  10. Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

    Science.gov (United States)

    Lazar, Nathaniel; Fay, Allison; Nandakumar, Madhumitha; Boyle, Kerry E; Xavier, Joao; Rhee, Kyu; Glickman, Michael S

    2017-12-01

    Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism. © 2017 John Wiley & Sons Ltd.

  11. Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Bonde, Mette; Højland, Dorte Heidi; Kolmos, Hans Jørn

    2011-01-01

    have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis...... in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall...... reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis....

  12. Proanthocyanidins in seed coat tegmen and endospermic cap inhibit seed germination in Sapium sebiferum.

    Science.gov (United States)

    Shah, Faheem Afzal; Ni, Jun; Chen, Jing; Wang, Qiaojian; Liu, Wenbo; Chen, Xue; Tang, Caiguo; Fu, Songling; Wu, Lifang

    2018-01-01

    Sapium sebiferum , an ornamental and bio-energetic plant, is propagated by seed. Its seed coat contains germination inhibitors and takes a long time to stratify for germination. In this study, we discovered that the S. sebiferum seed coat (especially the tegmen) and endospermic cap (ESC) contained high levels of proanthocyanidins (PAs). Seed coat and ESC removal induced seed germination, whereas exogenous application with seed coat extract (SCE) or PAs significantly inhibited this process, suggesting that PAs in the seed coat played a major role in regulating seed germination in S. sebiferum . We further investigated how SCE affected the expression of the seed-germination-related genes. The results showed that treatment with SCE upregulated the transcription level of the dormancy-related gene, gibberellins (GAs) suppressing genes, abscisic acid (ABA) biosynthesis and signalling genes. SCE decreased the transcript levels of ABA catabolic genes, GAs biosynthesis genes, reactive oxygen species genes and nitrates-signalling genes. Exogenous application of nordihydroguaiaretic acid, gibberellic acid, hydrogen peroxide and potassium nitrate recovered seed germination in seed-coat-extract supplemented medium. In this study, we highlighted the role of PAs, and their interactions with the other germination regulators, in the regulation of seed dormancy in S. sebiferum .

  13. Pyridine-substituted thiazolylphenol derivatives: Synthesis, modeling studies, aromatase inhibition, and antiproliferative activity evaluation.

    Science.gov (United States)

    Ertas, Merve; Sahin, Zafer; Berk, Barkin; Yurttas, Leyla; Biltekin, Sevde N; Demirayak, Seref

    2018-04-01

    Drugs used in breast cancer treatments target the suppression of estrogen biosynthesis. During this suppression, the main goal is to inhibit the aromatase enzyme that is responsible for the cyclization and structuring of estrogens either with steroid or non-steroidal-type inhibitors. Non-steroidal derivatives generally have a planar aromatic structure attached to the triazole ring system in their structures, which inhibits hydroxylation reactions during aromatization by coordinating the heme group. Bioisosteric replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase the selectivity for aromatase enzyme inhibition. In this study, pyridine-substituted thiazolylphenol