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Sample records for acid synthase ahas

  1. Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides

    PubMed Central

    Lee, Hyejin; Rustgi, Sachin; Kumar, Neeraj; Burke, Ian; Yenish, Joseph P.; Gill, Kulvinder S.; von Wettstein, Diter; Ullrich, Steven E.

    2011-01-01

    Induced mutagenesis can be an effective way to increase variability in self-pollinated crops for a wide variety of agronomically important traits. Crop resistance to a given herbicide can be of practical value to control weeds with efficient chemical use. In some crops (for example, wheat, maize, and canola), resistance to imidazolinone herbicides (IMIs) has been introduced through mutation breeding and is extensively used commercially. However, this production system imposes plant-back restrictions on rotational crops because of herbicide residuals in the soil. In the case of barley, a preferred rotational crop after wheat, a period of 9–18 mo is required. Thus, introduction of barley varieties showing resistance to IMIs will provide greater flexibility as a rotational crop. The objective of the research reported was to identify resistance in barley for IMIs through induced mutagenesis. To achieve this objective, a sodium azide-treated M2/M3 population of barley cultivar Bob was screened for resistance against acetohydroxy acid synthase (AHAS)-inhibiting herbicides. The phenotypic screening allowed identification of a mutant line showing resistance against IMIs. Molecular analysis identified a single-point mutation leading to a serine 653 to asparagine amino acid substitution in the herbicide-binding site of the barley AHAS gene. The transcription pattern of the AHAS gene in the mutant (Ser653Asn) and WT has been analyzed, and greater than fourfold difference in transcript abundance was observed. Phenotypic characteristics of the mutant line are promising and provide the base for the release of IMI-resistant barley cultivar(s). PMID:21551103

  2. Acetohydroxyacid synthase activity and transcripts profiling reveal tissue-specific regulation of ahas genes in sunflower.

    PubMed

    Ochogavía, Ana C; Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana A; Nestares, Graciela

    2014-07-01

    Acetohydroxyacid synthase (AHAS) is the target site of several herbicides and catalyses the first step in the biosynthesis of branched chain amino acid. Three genes coding for AHAS catalytic subunit (ahas1, ahas2 and ahas3) have been reported for sunflower. The aim of this work was to study the expression pattern of ahas genes family and AHAS activity in sunflower (Helianthus annuus L.). Different organs (leaves, hypocotyls, roots, flowers and embryos) were evaluated at several developmental stages. The transcriptional profile was studied through RT-qPCR. The highest expression for ahas1 was shown in leaves, where all the induced and natural gene mutations conferring herbicide resistance were found. The maximal expression of ahas2 and ahas3 occurred in immature flowers and embryos. The highest AHAS activity was found in leaves and immature embryos. Correlation analysis among ahas gene expression and AHAS activity was discussed. Our results show that differences in ahas genes expression are tissue-specific and temporally regulated. Moreover, the conservation of multiple AHAS isoforms in sunflower seems to result from different expression requirements controlled by tissue-specific regulatory mechanisms at different developmental stages. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.).

    PubMed

    Thompson, Courtney; Tar'an, Bunyamin

    2014-07-01

    A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed. Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F2 population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.

  4. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    PubMed

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower.

  5. Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide.

    PubMed

    Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana; Nestares, Graciela

    2013-07-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first reaction in branch chain amino acids biosynthesis. This enzyme is the target of several herbicides, including all members of the imidazolinone family. Little is known about the expression of the three acetohydroxyacid synthase genes (ahas1, ahas2 and ahas3) in sunflower. The aim of this work was to evaluate ahas gene expression and AHAS activity in different tissues of sunflower plantlets. Three genotypes differing in imidazolinone resistance were evaluated, two of which carry an herbicide resistant-endowing mutation known as Ahasl1-1 allele. In vivo and in vitro AHAS activity and transcript levels were higher in leaves than in roots. The ahas3 transcript was the less abundant in both tissues. No significant difference was observed between ahas1 and ahas2 transcript levels of the susceptible genotype but a higher ahas1 transcript level was observed in leaves of genotypes carrying Ahasl1-1 allele. Similar transcript levels were found for ahas1 and ahas2 in roots of genotypes carrying Ahasl1-1 allele whereas higher ahas2 abundance was found in the susceptible genotype. Herbicide treatment triggered tissue-specific, gene and genotype-dependent changes in ahas gene expression. AHAS activity was highly inhibited in the susceptible genotype. Differential responses were observed between in vitro and in vivo AHAS inhibition assays. These findings enhance our understanding of AHAS expression in sunflower genotypes differing for herbicide resistance and its response to herbicide treatment.

  6. Lipoic Acid Synthase (LASY)

    PubMed Central

    Padmalayam, Indira; Hasham, Sumera; Saxena, Uday; Pillarisetti, Sivaram

    2009-01-01

    OBJECTIVE—Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes. RESEARCH DESIGN AND METHODS—We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake. RESULTS—LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with age- and sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-α and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulin-stimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-α–induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response. CONCLUSIONS—Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction. PMID:19074983

  7. Photoluminescence studies on the complexation of Eu(III) and Tb(III) with acetohydroxamic acid (AHA) in nitrate medium

    NASA Astrophysics Data System (ADS)

    Pathak, P. N.; Mohapatra, M.; Godbole, S. V.

    2013-11-01

    UREX process has been proposed for selective extraction of U(VI) and Tc(VII) from nitric acid medium (∼1 M HNO3) using tri-n-butyl phosphate (TBP) as extractant and retaining Pu, Np and fission products in the aqueous phase. The feasibility of the use of luminescence spectroscopy as a technique to understand the complexation of trivalent f-elements cations viz. Eu(III) and Tb(III) with acetohydroxamic acid (AHA) in nitric acid medium has been examined. The luminescence lifetimes for the 1 × 10-3 M Eu(III) and AHA complex system decreased with increased AHA concentration from 116 ± 0.2 μs (no AHA) to 1.6 ± 0.1 μs (0.1 M AHA) which was attributed to dynamic quenching. The corrected fluorescence intensities were used to calculate the stability constant (log K) for the formation of 1:1 Eu3+-AHA complex as 1.42 ± 0.64 under the conditions of this study. By contrast, the Tb(III)-AHA system at pH 3 (HNO3) did not show any significant variation in the life times of the excited state (364 ± 9 μs) suggesting the absence of dynamic quenching. The spectral changes in Tb(III)-AHA system showed the formation of 1:1 complex (log K: 1.72 ± 0.21). These studies suggest that the extent of AHA complexation with the rare earth elements will be insignificant as compared to tetravalent metal ions Pu(IV) and Np(IV) under UREX process conditions.

  8. Phosphanilic Acid Inhibits Dihydropteroate Synthase

    DTIC Science & Technology

    1989-11-01

    dihydropteroate synthases of P. aeruginosa and E . coli were about equally susceptible to inhibition by PA. These results suggest that cells of P. aeruginosa...are more permeable to PA than cells of E . coli . Although a weak inhibitor, PA acted on dihydropteroate synthase in the same manner as the sulfonamides...with which PA is structurally related. Inhibition of E . coli by PA in a basal salts-glucose medium was prevented by p-aminobenzoic acid (pABA). However

  9. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    PubMed

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  10. Producing dicarboxylic acids using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-10-29

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  11. Producing dicarboxylic acids using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-05-26

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

  12. Resistance to AHAS inhibitor herbicides: current understanding.

    PubMed

    Yu, Qin; Powles, Stephen B

    2014-09-01

    Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined. © 2013 Society of Chemical Industry.

  13. Diversity of the Sediment Microbial Community in the Aha Watershed (Southwest China) in Response to Acid Mine Drainage Pollution Gradients

    PubMed Central

    Sun, Weimin; Sun, Min; Dong, Yiran; Ning, Zengping; Xiao, Enzong; Tang, Song; Li, Jiwei

    2015-01-01

    Located in southwest China, the Aha watershed is continually contaminated by acid mine drainage (AMD) produced from upstream abandoned coal mines. The watershed is fed by creeks with elevated concentrations of aqueous Fe (total Fe > 1 g/liter) and SO42− (>6 g/liter). AMD contamination gradually decreases throughout downstream rivers and reservoirs, creating an AMD pollution gradient which has led to a suite of biogeochemical processes along the watershed. In this study, sediment samples were collected along the AMD pollution sites for geochemical and microbial community analyses. High-throughput sequencing found various bacteria associated with microbial Fe and S cycling within the watershed and AMD-impacted creek. A large proportion of Fe- and S-metabolizing bacteria were detected in this watershed. The dominant Fe- and S-metabolizing bacteria were identified as microorganisms belonging to the genera Metallibacterium, Aciditerrimonas, Halomonas, Shewanella, Ferrovum, Alicyclobacillus, and Syntrophobacter. Among them, Halomonas, Aciditerrimonas, Metallibacterium, and Shewanella have previously only rarely been detected in AMD-contaminated environments. In addition, the microbial community structures changed along the watershed with different magnitudes of AMD pollution. Moreover, the canonical correspondence analysis suggested that temperature, pH, total Fe, sulfate, and redox potentials (Eh) were significant factors that structured the microbial community compositions along the Aha watershed. PMID:25979900

  14. Diversity of the Sediment Microbial Community in the Aha Watershed (Southwest China) in Response to Acid Mine Drainage Pollution Gradients.

    PubMed

    Sun, Weimin; Xiao, Tangfu; Sun, Min; Dong, Yiran; Ning, Zengping; Xiao, Enzong; Tang, Song; Li, Jiwei

    2015-08-01

    Located in southwest China, the Aha watershed is continually contaminated by acid mine drainage (AMD) produced from upstream abandoned coal mines. The watershed is fed by creeks with elevated concentrations of aqueous Fe (total Fe > 1 g/liter) and SO4 (2-) (>6 g/liter). AMD contamination gradually decreases throughout downstream rivers and reservoirs, creating an AMD pollution gradient which has led to a suite of biogeochemical processes along the watershed. In this study, sediment samples were collected along the AMD pollution sites for geochemical and microbial community analyses. High-throughput sequencing found various bacteria associated with microbial Fe and S cycling within the watershed and AMD-impacted creek. A large proportion of Fe- and S-metabolizing bacteria were detected in this watershed. The dominant Fe- and S-metabolizing bacteria were identified as microorganisms belonging to the genera Metallibacterium, Aciditerrimonas, Halomonas, Shewanella, Ferrovum, Alicyclobacillus, and Syntrophobacter. Among them, Halomonas, Aciditerrimonas, Metallibacterium, and Shewanella have previously only rarely been detected in AMD-contaminated environments. In addition, the microbial community structures changed along the watershed with different magnitudes of AMD pollution. Moreover, the canonical correspondence analysis suggested that temperature, pH, total Fe, sulfate, and redox potentials (Eh) were significant factors that structured the microbial community compositions along the Aha watershed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  16. Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells.

    PubMed

    Aprile, Alessio; Federici, Claire; Close, Timothy J; De Bellis, Luigi; Cattivelli, Luigi; Roose, Mikeal L

    2011-12-01

    The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a "sweet" lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNA-specific, presumably pH-related genes, the homolog of Arabidopsis H(+)-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradation-related genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon.

  17. Reduction of Pertechnetate By Acetohydroxamic Acid: Formation of [tc**II(NO)(AHA)(2)(H(2)O)]**+ And Implications for the UREX Process

    SciTech Connect

    Gong, C.-M.S.; Lukens, W.W.; Poineau, F.; Czerwinski, K.R.

    2009-05-18

    Reductive nitrosylation and complexation of ammonium pertechnetate by acetohydroxamic acid has been achieved in aqueous nitric and perchloric acid solutions. The kinetics of the reaction depend on the relative concentrations of the reaction components and are accelerated at higher temperatures. The reaction does not occur unless conditions are acidic. Analysis of the X-ray absorption fine structure spectroscopic data is consistent with a pseudo-octahedral geometry and the linear Tc-N-O bond typical of technetium nitrosyl compounds, and electron spin resonance spectroscopy is consistent with a d{sup 5} Tc(II) nitrosyl complex. The nitrosyl source is generally AHA, but it may be augmented by some products of the reaction with nitric acid. The resulting low-valency trans-aquonitrosyl(diacetohydroxamic)-technetium(II) complex ([Tc{sup II}(NO)(AHA){sub 2}H{sub 2}O]{sup +}, 1) is highly soluble in water, extremely hydrophilic, and is not extracted by tri-n-butylphosphate in a dodecane diluent. Its extraction properties are not pH-dependent: potentiometric-spectrophotometric titration studies indicate a single species from pH 4 down to -0.6 (calculated). This molecule is resistant to oxidation by H{sub 2}O{sub 2}, even at high pH, and can undergo substitution to form other technetium nitrosyl complexes. The potential formation of 1 during reprocessing may strongly impact the fate of technetium in the nuclear fuel cycle.

  18. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    DTIC Science & Technology

    2010-05-31

    targeting. Ursolic acid , a pentacyclic triterpenoid acid , as well as the tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate...2007,  6(7), 2120‐2126.  73.  Liu, Y., Tian, W., Ma, X., and Ding, W. Evaluation of  inhibition of  fatty  acid  synthase by  ursolic   acid : positive...AD_________________ Award Number: W81XWH-09-1-0204 TITLE: Inhibitors of Fatty Acid Synthase for

  19. The impact of acid mine drainage on the methylmercury cycling at the sediment-water interface in Aha Reservoir, Guizhou, China.

    PubMed

    He, Tianrong; Zhu, Yuzhen; Yin, Deliang; Luo, Guangjun; An, Yanlin; Yan, HaiYu; Qian, Xiaoli

    2015-04-01

    The methylmercury (MeHg) cycling at water-sediment interface in an acid mine drainage (AMD)-polluted reservoir (Aha Reservoir) and a reference site (Hongfeng Reservoir) were investigated and compared. Both reservoirs are seasonal anoxic and alkaline. The concentrations of sulfate, sulfide, iron, and manganese in Aha Reservoir were enriched compared to the reference levels in Hongfeng reservoir due to the AMD input. It was found that the MeHg accumulation layer in Aha Reservoir transitioned from the top sediment layer in winter to the water-sediment interface in spring and then to the overlying water above sediment in summer. It supported the assumption that spring methylation activity may start in sediments and migrate into the water column with seasonal variation. The weaker methylation in sediment during spring and summer was caused by the excessive sulfide (∼15-20 μM) that reduced the bioavailability of mercury, while sulfate reduction potential was in the optimal range for the methylation in the overlying water. This led to a transport flux of MeHg from water to sediment in spring and summer. In contrast, such inversion of MeHg accumulation layer did not occur in Hongfeng Reservoir. The sulfate reduction potential was in the optimal range for the methylation in top sediment, and dissolved MeHg was positively related to sulfide in pore water of Hongfeng Reservoir (r = 0.67, p < 0.001). This result suggested that accumulation of MeHg in lake water and cycling of MeHg at sediment-water interface associate with some sensitive environmental factors, such as sulfur.

  20. Producing a trimethylpentanoic acid using hybrid polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2014-10-07

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

  1. Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

    PubMed

    Hayashi, H; Huang, P; Inoue, K; Hiraoka, N; Ikeshiro, Y; Yazaki, K; Tanaka, S; Kushiro, T; Shibuya, M; Ebizuka, Y

    2001-12-01

    An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

  2. Mammalian fatty acid synthase: closure on a textbook mechanism?

    PubMed

    Leadlay, Peter; Baerga-Ortiz, Abel

    2003-02-01

    Mammalian fatty acid synthase is a classic example of a chain-building multienzyme. A cornerstone of its mechanism has been the obligatory collaboration of two identical subunits, with fatty acyl intermediates transferring between them. Now, fresh evidence has upset this view.

  3. Reduction of pertechnetate by acetohydroxamic acid: Formation of [TcNO(AHA)2(H2O)]+ and implications for the UREX process.

    SciTech Connect

    1Harry Reid Center for Environmental Studies, Nuclear Science and Technology Division, University of Nevada, Las Vegas, Las Vegas, NV, 89154-4006; Gong, Cynthia-May S; Poineau, Frederic; Lukens, Wayne W; Czerwinski, Kenneth R.

    2008-02-26

    Reductive nitrosylation and complexation of ammonium pertechnetate by acetohydroxamic acid has been achieved in aqueous nitric and perchloric acid solutions. The kinetics of the reaction depend on the relative concentrations of the reaction components and are accelerated at higher temperatures. The reaction does not occur unless conditions are acidic. Analysis of the x-ray absorption fine structure spectroscopic data is consistent with a pseudo-octahedral geometry with the linear Tc-N-O bond typical of technetium nitrosyl compounds, and electron spin resonance spectroscopy is consistent with a the d{sup 5} Tc(II) nitrosyl complex. The nitrosyl source is generally AHA, but may be augmented by products of reaction with nitric acid. The resulting low-valency trans-aquonitrosyl(diacetohydroxamic)-technetium(II) complex (1) is highly soluble in water, extremely hydrophilic, and is not extracted by tri-n-butylphosphate in a dodecane diluent. Its extraction properties are not pH-dependent; titration studies indicate a single species from pH 4.5 down to -0.6 (calculated). This molecule is resistant to oxidation by H{sub 2}O{sub 2}, even at high pH, and can undergo substitution to form other technetium nitrosyl complexes. The formation of 1 may strongly impact the fate of technetium in the nuclear fuel cycle.

  4. Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase.

    PubMed

    Lee, Yu-Ting; Cui, Chang-Jun; Chow, Eve W L; Pue, Nason; Lonhienne, Thierry; Wang, Jian-Guo; Fraser, James A; Guddat, Luke W

    2013-01-10

    The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

  5. How important is the synclinal conformation of sulfonylureas to explain the inhibition of AHAS: a theoretical study.

    PubMed

    Jaña, Gonzalo A; Delgado, Eduardo J; Medina, Fabiola E

    2014-03-24

    The inhibitory activity of 15 sulfonylureas on acetohydroxyacid synthase (AHAS) is addressed theoretically in order to stress how important the conformation is to explain their differences as AHAS inhibitors. The study includes calculations in gas phase, solution, and in the enzymatic environment. The results suggest that both the activation Gibbs free energy and Gibbs free energy change associated with the conformational change in solution allow for determining if sulfonylureas should have high or low inhibition activity. QM/MM calculations were also carried out in order to identify the role of the amino acid residues and the effects involved in the stabilization of the active conformation in the binding pocket. On the other hand, the analysis of the frontier molecular orbitals of the sulfonylureas in the binding pocket allowed us to explain the inhibitory activity in terms of the reactivity of the carbonyl carbon.

  6. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa.

    PubMed

    Taura, Futoshi; Sirikantaramas, Supaart; Shoyama, Yoshinari; Yoshikai, Kazuyoshi; Shoyama, Yukihiro; Morimoto, Satoshi

    2007-06-26

    Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants.

  7. Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

    PubMed

    Tittmann, Kai; Schröder, Kathrin; Golbik, Ralph; McCourt, Jennifer; Kaplun, Alexander; Duggleby, Ronald G; Barak, Ze'ev; Chipman, David M; Hübner, Gerhard

    2004-07-13

    Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

  8. Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase.

    PubMed

    Liu, Chia-I; Jeng, Wen-Yih; Chang, Wei-Jung; Ko, Tzu-Ping; Wang, Andrew H-J

    2012-05-25

    Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.

  9. The type I fatty acid and polyketide synthases: a tale of two megasynthases

    PubMed Central

    Tsai, Shiou-Chuan

    2008-01-01

    This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

  10. Purification and functional characterization of thermostable 5-aminolevulinic acid synthases.

    PubMed

    Meng, Qinglong; Zhang, Yanfei; Ma, Chunling; Ma, Hongwu; Zhao, Xueming; Chen, Tao

    2015-11-01

    As 5-aminolevulinic acid synthase (ALAS), the key enzyme for 5-aminolevulinic acid (ALA) synthesis, is unstable, we have sought to find thermostable ALASs from thermophilic organisms. Three ALASs from thermophiles Geobacillus thermoglucosidasius (GT-ALAS), Laceyella sacchari (LS-ALAS) and Pseudomonas alcaliphila (PA-ALAS) were purified and characterized. All enzymes were more stable than two previously studied ALASs from Rhodopseudomonas palustris and Rhodobacter sphaeroides. There was almost no activity change after 60 h at 37 °C for the three thermostable enzymes. This contrasts with the other two enzymes which lost over 90 % activities in just 1 h. Furthermore, the specific activity of LS-ALAS (7.8 U mg(-1)) was also higher than any previously studied ALASs. Thermostable ALASs were found in thermophilic organisms and this paves the way for developing cell free processes for enzymatic production of ALA from bulk chemicals succinate and glycine.

  11. Crystal structure of plant acetohydroxyacid synthase, the target for several commercial herbicides.

    PubMed

    Garcia, Mario Daniel; Wang, Jian-Guo; Lonhienne, Thierry; Guddat, Luke William

    2017-07-01

    Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plant AHAS in the absence of any inhibitor (2.9 Å resolution) and it shows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide binding in uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered, or adopt a different fold when the herbicide is not present. In addition, the structure provides an explanation as to why some herbicides are more potent inhibitors of Arabidopsis thaliana AHAS compared to AHASs from other species (e.g. S. cerevisiae). The elucidation of the native structure of plant AHAS provides a new platform for future rational structure-based herbicide design efforts. The coordinates and structure factors for uninhibited AtAHAS have been deposited in the Protein Data Bank (www.pdb.org) with the PDB ID code 5K6Q. © 2017 Federation of European Biochemical Societies.

  12. Tomato linalool synthase is induced in trichomes by jasmonic acid

    PubMed Central

    van Schie, Chris C. N.; Haring, Michel A.

    2007-01-01

    Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato. PMID:17440821

  13. Structure and function of eukaryotic fatty acid synthases.

    PubMed

    Maier, Timm; Leibundgut, Marc; Boehringer, Daniel; Ban, Nenad

    2010-08-01

    In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2·6 MDa α₆β₆ heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficiency.

  14. Novel Nuclear Localization of Fatty Acid Synthase Correlates with Prostate Cancer Aggressiveness

    PubMed Central

    Madigan, Allison A.; Rycyna, Kevin J.; Parwani, Anil V.; Datiri, Yeipyeng J.; Basudan, Ahmed M.; Sobek, Kathryn M.; Cummings, Jessica L.; Basse, Per H.; Bacich, Dean J.; O'Keefe, Denise S.

    2015-01-01

    Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is expressed at low levels in normal cells, it is currently a major target for anticancer drug design. Fatty acid synthase is normally found in the cytosol; however, we have discovered that it also localizes to the nucleus in a subset of prostate cancer cells. Analysis of the fatty acid synthase protein sequence indicated the presence of a nuclear localization signal, and subcellular fractionation of LNCaP prostate cancer cells, as well as immunofluorescent confocal microscopy of patient prostate tumor tissue and LNCaPs confirmed nuclear localization of this protein. Finally, immunohistochemical analysis of prostate cancer tissue indicated that nuclear localization of fatty acid synthase correlates with Gleason grade, implicating a potentially novel role in prostate cancer progression. Possible clinical implications include improving the accuracy of prostate biopsies in the diagnosis of low- versus intermediate-risk prostate cancer and the uncovering of novel metabolic pathways for the therapeutic targeting of androgen-independent prostate cancer. PMID:24907642

  15. Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.

    PubMed

    Zhao, Yuefang; Wen, Xin; Niu, Congwei; Xi, Zhen

    2012-11-05

    Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Expression of fatty acid synthase in nonalcoholic fatty liver disease.

    PubMed

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-03-25

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD.

  17. Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

    PubMed Central

    Lee, Yu-Ting; Duggleby, Ronald G.

    2006-01-01

    Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic subunit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs from that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required MgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator. PMID:16390333

  18. Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP.

    PubMed

    Lee, Yu-Ting; Duggleby, Ronald G

    2006-04-15

    Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic subunit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs from that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required MgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

  19. Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors.

    PubMed

    Birck, M R; Husain, A; Sheflyan, G Y; Ganem, B; Woodard, R W

    2001-11-05

    The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.

  20. Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

    PubMed Central

    Nouwens, Amanda; Lonhienne, Thierry G.; Guddat, Luke W.

    2017-01-01

    Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors. PMID:28137884

  1. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    PubMed

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

  2. Studies on tetrahydrocannabinolic acid synthase that produces the acidic precursor of tetrahydrocannabinol, the pharmacologically active cannabinoid in marijuana.

    PubMed

    Taura, F

    2009-06-01

    Tetrahydrocannabinol (THC), the psychoactive component of marijuana, is now regarded as a promising medicine because this cannabinoid has been shown to exert a variety of therapeutic activities. It has been demonstrated that THC is generated from the acidic precursor, tetrahydrocannabinolic acid (THCA) by nonenzymatic decarboxylation, and that THCA is biosynthesized by THCA synthase, which catalyzes a unique biosynthetic reaction, the stereospecific oxidative cyclization of the geranyl group of the substrate cannabigerolic acid. Molecular characterization of THCA synthase has revealed its structural characteristics and reaction mechanism. THCA synthase is the first cannabinoid synthase to be studied and is potentially attractive target for various biotechnological applications as it produces the direct precursor of THC. This review describes the research history of this enzyme, i.e., purification, molecular cloning, biochemical characterization, and possible biotechnological application of THCA synthase.

  3. Fatty acid synthase as a novel target for meningioma therapy

    PubMed Central

    Haase, Daniela; Schmidl, Stefan; Ewald, Christian; Kalff, Rolf; Huebner, Christian; Firsching, Raimund; Keilhoff, Gerburg; Evert, Matthias; Paulus, Werner; Gutmann, David H.; Lal, Anita; Mawrin, Christian

    2010-01-01

    High levels of fatty acid synthase (FAS) expression have been reported in hormone receptor-positive tumors, including prostate, breast, and ovarian cancers, and its inhibition reduces tumor growth in vitro and in vivo. Similar to other hormone receptor-positive tumor types, meningiomas are progesterone receptor- and estrogen receptor-immunoreactive brain tumors. To define the role of FAS in human meningioma growth control, we first analyzed the FAS expression using a tissue microarray containing 38 meningiomas and showed increased FAS expression in 70% of atypical WHO grade II and anaplastic WHO grade III meningiomas compared with 10% of benign WHO grade I tumors. We next confirmed this finding by real-time PCR and Western blotting. Second, we demonstrated that treatment with the FAS inhibitor, cerulenin (Cer), significantly decreased meningioma cell survival in vitro. Third, we showed that Cer treatment reduced FAS expression by modulating Akt phosphorylation (activation). Fourth, we demonstrated that Cer treatment of mice bearing meningioma xenografts resulted in significantly reduced tumor volumes associated with increased meningioma cell death. Collectively, our data suggest that the increased FAS expression in human meningiomas represents a novel therapeutic target for the treatment of unresectable or malignant meningioma. PMID:20511185

  4. Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase.

    PubMed

    Huang, W Y; Stoops, J K; Wakil, S J

    1989-04-01

    The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.

  5. The leaf extract of Siberian Crabapple (Malus baccata (Linn.) Borkh) contains potential fatty acid synthase inhibitors.

    PubMed

    Wei, Xiang; Zhao, Ran; Sun, Ying-Hui; Cong, Jian-Ping; Meng, Fan-Guo; Zhou, Hai-Meng

    2009-02-01

    The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver fatty acid synthase. The results showed that this extract had much stronger inhibitory ability on fatty acid synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver fatty acid synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of fatty acid synthase.

  6. Acetohydroxyacid Synthase, a Novel Target for Improvement of l-Lysine Production by Corynebacterium glutamicum▿ †

    PubMed Central

    Blombach, Bastian; Hans, Stephan; Bathe, Brigitte; Eikmanns, Bernhard J.

    2009-01-01

    The influence of acetohydroxy acid synthase (AHAS) on l-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower Km for the substrate pyruvate and an about fourfold-lower Vmax; (ii) a slightly increased Km for the substrate α-ketobutyrate with an about twofold-lower Vmax; and (iii) insensitivity against the inhibitors l-valine, l-isoleucine, and l-leucine (10 mM each). Introduction of the modified AHAS into the l-lysine producers C. glutamicum DM1729 and DM1933 increased l-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased l-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to l-valine, l-isoleucine, and l-leucine auxotrophy and to further-improved l-lysine production. In batch fermentations, C. glutamicum DM1729 ΔilvB produced about 85% more l-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 ΔilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the l-lysine biosynthetic pathway. PMID:19047397

  7. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    PubMed Central

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  8. Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A Randomized Controlled Trial

    DTIC Science & Technology

    2007-03-01

    14. ABSTRACT: See next page. 15. SUBJECT TERMS Prostate Cancer; Lipid Medtabolism, Clinical Trial; Omega -3 Fatty Acids 16. SECURITY...AD_________________ Award Number: W81XWH-04-1-0296 TITLE: Fish Oil Supplementation and Fatty Acid ...SUBTITLE Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A 5a. CONTRACT NUMBER Randomized

  9. Acyl-carrier protein - Phosphopantetheinyltransferase partnerships in fungal fatty acid synthases

    USDA-ARS?s Scientific Manuscript database

    The synthesis of fatty acids is an essential primary metabolic process for energy storage and cellular structural integrity. Assembly of saturated fatty acids is achieved by fatty acid synthases (FASs) that combine acetyl- and malonyl-CoAs by repetitive decarboxylative Claisen condensations with su...

  10. Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase

    SciTech Connect

    Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

    1986-05-01

    Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

  11. Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I.

    PubMed

    Boshoff, Helena I; Mizrahi, Valerie; Barry, Clifton E

    2002-04-01

    The effects of low extracellular pH and intracellular accumulation of weak organic acids were compared with respect to fatty acid synthesis by whole cells of Mycobacterium tuberculosis and Mycobacterium smegmatis. The profile of fatty acids synthesized during exposure to benzoic, nicotinic, or pyrazinoic acids, as well as that observed during intracellular hydrolysis of the corresponding amides, was not a direct consequence of modulation of fatty acid synthesis by these compounds but reflected the response to inorganic acid stress. Analysis of fatty acid synthesis in crude mycobacterial cell extracts demonstrated that pyrazinoic acid failed to directly modulate the fatty acid synthase activity catalyzed by fatty acid synthase I (FAS-I). However, fatty acid synthesis was irreversibly inhibited by 5-chloro-pyrazinamide in a time-dependent fashion. Moreover, we demonstrate that pyrazinoic acid does not inhibit purified mycobacterial FAS-I, suggesting that this enzyme is not the immediate target of pyrazinamide.

  12. Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples 1

    PubMed Central

    Bufler, Gebhard

    1984-01-01

    Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO2 production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO2; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene. Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO2, but not by 1.25% CO2. However, each of these CO2 concentrations reduced the rate of increase of ACC synthase activity. PMID:16663569

  13. The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species.

    PubMed

    Zhao, Yuefang; Niu, Congwei; Wen, Xin; Xi, Zhen

    2013-04-15

    Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte.

    PubMed

    Fan, Huijin; Wu, Dan; Tian, Weixi; Ma, Xiaofeng

    2013-07-01

    Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC50) of 0.14 microM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARgamma during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.

  15. Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae.

    PubMed Central

    Carabaza, A; Arino, J; Fox, J W; Villar-Palasi, C; Guinovart, J J

    1990-01-01

    Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected. Images Fig. 1. Fig. 2. Fig. 3. PMID:2114092

  16. Screening for latent acute intermittent porphyria: the value of measuring both leucocyte delta-aminolaevulinic acid synthase and erythrocyte uroporphyrinogen-1-synthase activities.

    PubMed Central

    McColl, K E; Moore, M R; Thompson, G G; Goldberg, A

    1982-01-01

    Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder of haem biosynthesis characterised by reduced activity of the enzyme uroporphyrinogen-1-(URO) synthase and compensatory increased activity of the rate controlling enzyme delta-aminolaevulinic acid (ALA) synthase. Subjects with the disorder should be identified as they are at risk of developing severe porphyric attacks if exposed to a variety of drugs or chemicals. We have assessed the value of measuring the activities of ALA synthase and URO synthase in peripheral blood cells as a means of identifying latent cases in affected families. In AIP subjects, ALA synthase activity was increased and URO synthase decreased compared to controls, through there was considerable overlap between the two groups when either enzyme was examined alone. When both enzymes were examined together, all but one of the 19 AIP patients had both increased ALA synthase activity (greater than 250 nmol ALA/g protein/h) and reduced URO synthase activity (less than 25.1 nmol URO/l RBC/h), whereas none of the 62 controls showed this enzyme pattern. Examination of 35 asymptomatic first degree blood relatives of AIP patients showed that 17 (49%) had the porphyric enzyme pattern with no sex bias. The combined study of these two enzymes permits accurate detection of latent cases of AIP and confirms its autosomal dominant inheritance. PMID:7120315

  17. Expanding the product portfolio of fungal type I fatty acid synthases.

    PubMed

    Zhu, Zhiwei; Zhou, Yongjin J; Krivoruchko, Anastasia; Grininger, Martin; Zhao, Zongbao K; Nielsen, Jens

    2017-04-01

    Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.

  18. Polyunsaturated fatty acid inhibition of fatty acid synthase transcription is independent of PPAR activation.

    PubMed

    Clarke, S D; Turini, M; Jump, D B; Abraham, S; Reedy, M

    1998-01-01

    Polyunsaturated fatty acids (PUFA) of the (n-6) and (n-3) families inhibit the rate of gene transcription for a number of hepatic lipogenic and glycolytic genes, e.g., fatty acid synthase (FAS). In contrast, saturated and monounsaturated fatty acids have no inhibitory capability. The suppression of gene transcription resulting from the addition of PUFA to a high carbohydrate diet: occurs quickly (< 3 h) after its addition to a high glucose diet; can be recreated with hepatocytes cultured in a serum-free medium containing insulin and glucocorticoids; can be demonstrated in diabetic rats fed fructose; and is independent of glucagon. While the nature of the intracellular PUFA inhibitor is unclear, it appears that delta-6 desaturation is a required step in the process. Recently, the fatty acid activated nuclear factor, peroxisome-proliferator activated receptor (PPAR) was suggested to be the PUFA-response factor. However, the potent PPAR activators ETYA and Wy-14643 did not suppress hepatic expression of FAS, but did induce the PPAR-responsive gene, acyl-CoA oxidase (AOX). Similarly, treating rat hepatocytes with 20:4 (n-6) suppressed FAS expression but had no effect on AOX. Thus, it appears that the PUFA regulation of gene transcription involves a PUFA-response factor that is independent from PPAR.

  19. Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L.. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid.

    PubMed

    Taura, F; Morimoto, S; Shoyama, Y

    1996-07-19

    We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.

  20. Pyrazinoic acid and its n-propyl ester inhibit fatty acid synthase type I in replicating tubercle bacilli.

    PubMed

    Zimhony, Oren; Vilchèze, Catherine; Arai, Masayoshi; Welch, John T; Jacobs, William R

    2007-02-01

    The activity of different analogs of pyrazinamide on Mycobacterium tuberculosis fatty acid synthase type I (FASI) in replicating bacilli was studied. Palmitic acid biosynthesis was diminished by 96% in bacilli treated with n-propyl pyrazinoate, 94% in bacilli treated with 5-chloro-pyrazinamide, and 97% in bacilli treated with pyrazinoic acid, the pharmacologically active agent of pyrazinamide. We conclude that the minimal structure of pyrazine ring with an acyl group is sufficient for FASI inhibition and antimycobacterial activity.

  1. Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75

    PubMed Central

    Babich, John W.; Mairs, Robert J.

    2016-01-01

    Abstract The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer‐killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase‐1. C75 is administered in the form of a racemic mixture of (−) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase‐1. Therefore, we assessed the relative cancer‐killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (−)‐C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)‐C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor‐killing activity of ionizing radiation, while minimizing weight loss in cancer patients. PMID:27901292

  2. Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75.

    PubMed

    Rae, Colin; Babich, John W; Mairs, Robert J

    2017-01-01

    The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer-killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase-1. C75 is administered in the form of a racemic mixture of (-) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase-1. Therefore, we assessed the relative cancer-killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (-)-C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)-C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor-killing activity of ionizing radiation, while minimizing weight loss in cancer patients.

  3. Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

    PubMed

    Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

    2016-01-01

    Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  4. Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase.

    PubMed

    Wang, Jian-Guo; Lee, Patrick K-M; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G; Li, Zheng-Ming; Guddat, Luke W

    2009-03-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg(2+) and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 A, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

  5. Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase

    SciTech Connect

    Wang, Jian-Guo; Lee, Patrick K.-M.; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G.; Li, Zheng-Ming; Guddat, Luke W.

    2009-08-17

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg{sup 2+} and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 {angstrom}, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

  6. Apoptotic effect of tannic acid on fatty acid synthase over-expressed human breast cancer cells.

    PubMed

    Nie, Fangyuan; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-02-01

    Breast cancer is one of the most common cancers and is the second leading cause of cancer mortality in women worldwide. Novel therapies and chemo-therapeutic drugs are urgently needed to be developed for the treatment of breast cancer. Increasing evidence suggests that fatty acid synthase (FAS) plays an important role in breast cancer, for the expression of FAS is significantly higher in human breast cancer cells than in normal cells. Tannic acid (TA), a natural polyphenol, possesses significant biological functions, including bacteriostasis, hemostasis, and anti-oxidant. Our previous studies demonstrated that TA is a natural FAS inhibitor whose inhibitory activity is stronger than that of classical FAS inhibitors, such as C75 and cerulenin. This study further assessed the effect and therapeutic potential of TA on FAS over-expressed breast cancer cells, and as a result, TA had been proven to possess the functions of inhibiting intracellular FAS activity, down-regulating FAS expression in human breast cancer MDA-MB-231 and MCF-7 cells, and inducing cancer cell apoptosis. Since high-expressed FAS is recognized as a molecular marker for breast cancer and plays an important role in cancer prognosis, these findings suggest that TA is a potential drug candidate for treatment of breast cancer.

  7. Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

    SciTech Connect

    Dotson, G.D.; Woodard, R.W.

    1994-12-01

    The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3{sup 2}H)PEP, (2-{sup 13}C)PEP, and (2-{sup 13}C,{sup 18}O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our {sup 1}H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3-{sup 2}H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3-{sup 2}H)PEP gave predominantly (3S)-(3{sup 2}H)KDO 8-P and (E)-(3-{sup 2}H)PEP gave predominantly (3R)-(3{sup 2}H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2-{sup 13}C, {sup 18}O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both {sup 13}C- and {sup 31}P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the {sup 18}O.

  8. Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops.

    PubMed

    Tan, S; Evans, R; Singh, B

    2006-03-01

    Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes.

  9. Crystallization of Δ{sup 1}-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    SciTech Connect

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Shoyama, Yukihiro; Morimoto, Satoshi

    2005-08-01

    Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å{sup 3} Da{sup −1} assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

  10. Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida Lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation.

    PubMed

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.

  11. Recent advances in targeting the fatty acid biosynthetic pathway using fatty acid synthase inhibitors.

    PubMed

    Angeles, Thelma S; Hudkins, Robert L

    2016-12-01

    Elevated lipogenesis has been associated with a variety of diseases including obesity, cancer and nonalcoholic fatty liver disease (NAFLD). Fatty acid synthase (FASN) plays a pivotal role in de novo lipogenesis, making this multi-catalytic protein an attractive target for therapeutic intervention. Recently, the first FASN inhibitor successfully advanced through the drug development process and entered clinical evaluation in oncology. Areas covered: This review discusses the biological roles of FASN in three prominent disease areas: cancer, obesity-related disorders and NAFLD. Recent advances in drug discovery strategies and design of newer FASN inhibitors are also highlighted. Expert opinion: Despite the abundance of evidence linking the lipogenic pathway to cancer, progression of FASN-targeted molecules has been rather slow and challenging and no compounds have moved past the preclinical phase. The landscape has recently changed with the recent advancement of the first FASN inhibitor into clinical evaluation for solid tumors. Needless to say, the successful translation into the clinical setting will open opportunities for expanding the therapeutic utility of FASN inhibitors not just in oncology but in other diseases associated with elevated lipogenesis such as obesity, type 2 diabetes, and NAFLD.

  12. Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding.

    PubMed

    Kim, Joungmok; Beak, Dong-Gil; Kim, Young-Tae; Choi, Jung-Do; Yoon, Moon-Young

    2004-11-15

    AHAS (acetohydroxyacid synthase) catalyses the first committed step in the biosynthesis of branched-chain amino acids, such as valine, leucine and isoleucine. Owing to the unique presence of these biosynthetic pathways in plants and micro-organisms, AHAS has been widely investigated as an attractive target of several classes of herbicides. Recently, the crystal structure of the catalytic subunit of yeast AHAS has been resolved at 2.8 A (1 A=0.1 nm), showing that the active site is located at the dimer interface and is near the herbicide-binding site. In this structure, the existence of two disordered regions, a 'mobile loop' and a C-terminal 'lid', is worth notice. Although these regions contain the residues that are known to be important in substrate specificity and in herbicide resistance, they are poorly folded into any distinct secondary structure and are not within contact distance of the cofactors. In the present study, we have tried to demonstrate the role of these regions of tobacco AHAS by constructing variants with serial deletions, based on the structure of yeast AHAS. In contrast with the wild-type AHAS, the truncated mutant which removes the C-terminal lid, Delta630, and the internal deletion mutant without the mobile loop, Delta567-582, impaired the binding affinity for ThDP (thiamine diphosphate), and showed different elution profiles representing a monomeric form in gel-filtration chromatography. Our results suggest that these regions are involved in the binding/stabilization of the active dimer and ThDP binding.

  13. Enrichment and identification of Δ(9)-Tetrahydrocannabinolic acid synthase from Pichia pastoris culture supernatants.

    PubMed

    Lange, Kerstin; Poetsch, Ansgar; Schmid, Andreas; Julsing, Mattijs K

    2015-09-01

    This data article refers to the report Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) production in Pichia pastoris enables chemical synthesis of cannabinoids (Lange et. al. 2015) [2]. THCAS was produced on a 2 L lab scale using recombinant P. pastoris KM71 KE1. Enrichment of THCAS as a technically pure enzyme was realized using dialysis and cationic exchange chromatography. nLC-ESI-MS/MS analysis identified THCAS in different fractions obtained by cationic exchange chromatography.

  14. Activation of beta-glucan synthases by wall-bound purple acid phosphatase in tobacco cells.

    PubMed

    Kaida, Rumi; Satoh, Yumi; Bulone, Vincent; Yamada, Yohko; Kaku, Tomomi; Hayashi, Takahisa; Kaneko, Takako S

    2009-08-01

    Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

  15. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    PubMed

    Sirikantaramas, Supaart; Morimoto, Satoshi; Shoyama, Yoshinari; Ishikawa, Yu; Wada, Yoshiko; Shoyama, Yukihiro; Taura, Futoshi

    2004-09-17

    Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

  16. Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

    PubMed

    Rigouin, Coraline; Gueroult, Marc; Croux, Christian; Dubois, Gwendoline; Borsenberger, Vinciane; Barbe, Sophie; Marty, Alain; Daboussi, Fayza; André, Isabelle; Bordes, Florence

    2017-06-21

    Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS. This enabled to point out the key role of an isoleucine residue, I1220, from the fatty acid binding site, which could be targeted by mutagenesis. To address this challenge, TALEN (transcription activator-like effector nucleases)-based genome editing technology was applied for the first time to Yarrowia lipolytica and proved to be very efficient for inducing targeted genome modifications. Among the generated FAS mutants, those having a bulky aromatic amino acid residue in place of the native isoleucine at position 1220 led to a significant increase of myristic acid (C14) production compared to parental wild-type KS. Particularly, the best performing mutant, I1220W, accumulates C14 at a level of 11.6% total fatty acids. Overall, this work illustrates how a combination of molecular modeling and genome-editing technology can offer novel opportunities to rationally engineer complex systems for synthetic biology.

  17. myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d-glucuronic Acid Biosynthesis in Plants 12

    PubMed Central

    Loewus, Mary W.; Loewus, Frank

    1974-01-01

    Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all. PMID:16658890

  18. Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines.

    PubMed

    Li, N; Parsons, B L; Liu, D R; Mattoo, A K

    1992-02-01

    Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

  19. The very-long-chain fatty acid synthase is inhibited by chloroacetamides.

    PubMed

    Götz, Thomas; Böger, Peter

    2004-01-01

    The first elongation step to form very-long-chain fatty acids (VLCFAs) is catalyzed by the VLCFA-synthase. CoA-activated fatty acids react with malonyl-CoA to condense a C2-unit. As shown with recombinant enzyme this reaction is specifically inhibited by chloroacetamide herbicides. The inhibition is alleviated when the inhibitor (e.g. metazachlor) is incubated together with adequate concentrations of the substrate (e.g. oleoyl-CoA). Malonyl-CoA has no influence. However, once a chloroacetamide has been tightly bound to the synthase after an appropriate time it cannot be displaced anymore by the substrate. In contrast, oleoyl-CoA, is easily removed from the synthase by metazachlor. The irreversible binding of the chloroacetamides and their competition with the substrate explains the very low half-inhibition values of 10(-8) M and below. Chiral chloroacetamides like metolachlor or dimethenamid give identical results. However, only the (S)-enantiomers are active.

  20. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  1. Exploration of Learning Strategies Associated With Aha Learning Moments.

    PubMed

    Pilcher, Jobeth W

    2016-01-01

    Educators recognize aha moments as powerful aspects of learning. Yet limited research has been performed regarding how to promote these learning moments. This article describes an exploratory study of aha learning moments as experienced and described by participants. Findings showed use of visuals, scenarios, storytelling, Socratic questions, and expert explanation led to aha learning moments. The findings provide guidance regarding the types of learning strategies that can be used to promote aha moments.

  2. Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

    PubMed Central

    Petkowski, Janusz J.; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Zheng, Heping; Skarina, Tatiana; Onopriyenko, Olena; Cymborowski, Marcin T.; Koclega, Katarzyna D.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2007-01-01

    Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice. PMID:17586771

  3. REACTION MECHANISMS OF 15-HYDROPEROXYEICOSATETRAENOIC ACID CATALYZED BY HUMAN PROSTACYCLIN AND THROMBOXANE SYNTHASES

    PubMed Central

    Yeh, Hui-Chun; Tsai, Ah-Lim; Wang, Lee-Ho

    2007-01-01

    Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H2 (PGH2) to produce prostacyclin (PGI2) and thromboxane A2 (TXA2). PGI2 and TXA2 have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-OH-14,15-EET), like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O–O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The Vmax of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min−1 vs. 320 min−1) and 1.4-fold faster for TXAS (170 min−1 vs. 120 min−1). Similar KM values for homolytic and heterolytic cleavages were found for PGIS (∼60 μM 15-HPETE) and TXAS (∼80 μM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction. PMID:17459323

  4. Triazolopyrimidines as a New Herbicidal Lead for Combating Weed Resistance Associated with Acetohydroxyacid Synthase Mutation.

    PubMed

    Liu, Yu-Chao; Qu, Ren-Yu; Chen, Qiong; Yang, Jing-Fang; Cong-Wei, Niu; Zhen, Xi; Yang, Guang-Fu

    2016-06-22

    Acetohydroxyacid synthase (AHAS; also known as acetolactate synthase; EC 2.2.1.6, formerly EC 4.1.3.18) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. Weed resistance to AHAS-inhibiting herbicides, increasing at an exponential rate, is becoming a global problem and leading to an urgent demand of developing novel compounds against both resistant and wild AHAS. In the present work, a series of novel 2-aroxyl-1,2,4-triazolopyrimidine derivatives (a total of 55) were designed and synthesized with the aim to discover an antiresistant lead compound. Fortunately, the screening results indicated that many of the newly synthesized compounds showed a better, even excellent, inhibition effect against both the wild-type Arabidopsis thaliana AHAS and P197L mutants. Among them, compounds 5-3 to 5-17, compounds 5-19 to 5-26, compounds 5-28 to 5-45, and compound 5-48 have the lower values of resistance factor (RF) and display a potential power to overcome resistance associated with the P197L mutation in the enzyme levels. Further greenhouse in vivo assay showed that compounds 5-15 and 5-20 displayed "moderate" to "good" herbicidal activity against both the wild type-and the resistant (P197L mutation) Descurainia sophia, even at a rate as low as 0.9375 (g of ai/ha). The above results indicated that these two compounds could be used as new leads for the future development of antiresistance herbicides.

  5. Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production.

    PubMed

    Ruijter, G J; Panneman, H; Xu, D; Visser, J

    2000-03-01

    Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated M(r) of A. niger citrate synthase is 48501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain.

  6. Crystallization of Delta1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa.

    PubMed

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Shoyama, Yukihiro; Morimoto, Satoshi

    2005-08-01

    Delta1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure-function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A. The calculated Matthews coefficient was approximately 4.1 or 2.0 A3 Da(-1) assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

  7. Using Aha! Moments to Understand Leadership Theory

    ERIC Educational Resources Information Center

    Moore, Lori L.; Lewis, Lauren J.

    2012-01-01

    As Huber (2002) noted, striving to understand how leadership is taught and learned is both a challenge and an opportunity facing leadership educators. This article describes the "Leadership Aha! Moment" assignment used in a leadership theory course to help students recognize the intersection of leadership theories and their daily lives while…

  8. Using Aha! Moments to Understand Leadership Theory

    ERIC Educational Resources Information Center

    Moore, Lori L.; Lewis, Lauren J.

    2012-01-01

    As Huber (2002) noted, striving to understand how leadership is taught and learned is both a challenge and an opportunity facing leadership educators. This article describes the "Leadership Aha! Moment" assignment used in a leadership theory course to help students recognize the intersection of leadership theories and their daily lives while…

  9. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1.

    PubMed

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-07-01

    Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

  10. Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger.

    PubMed

    Lv, Yangyong; Xiao, Jing; Pan, Li

    2014-11-01

    Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H](-)), was detected by ESI-MS in the negative ionization mode, and (1)H and (13)C NMR data confirmed its structure.

  11. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination.

    PubMed

    Pracharoenwattana, Itsara; Cornah, Johanna E; Smith, Steven M

    2005-07-01

    We tested the hypothesis that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of triacylglycerol in Arabidopsis thaliana seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings, and seeds from plants with both CSY genes disrupted were dormant and did not metabolize triacylglycerol. Germination was achieved by removing the seed coat and supplying sucrose, but the seedlings still did not use triacylglycerol. The mutant seedlings were resistant to 2,4-dichlorophenoxybutyric acid, indicating a block in peroxisomal beta-oxidation, and were unable to develop further after transfer to soil. The mutant phenotype was complemented with a cDNA encoding CSY with either its native peroxisomal targeting sequence (PTS2) or a heterologous PTS1 sequence from pumpkin (Cucurbita pepo) malate synthase. These results suggest that peroxisomal CSY in Arabidopsis is not only a key enzyme of the glyoxylate cycle but also catalyzes an essential step in the respiration of fatty acids. We conclude that citrate is exported from the peroxisome during fatty acid respiration, whereas in yeast, acetylcarnitine is exported.

  12. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    PubMed

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors.

  13. Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase.

    PubMed

    Pecchia, Paola; Cammareri, Maria; Malafronte, Nicola; Consiglio, M Federica; Gualtieri, Maria Josefina; Conicella, Clara

    2011-11-01

    Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus.

  14. Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

    PubMed Central

    Nguyen, Long Nam; Trofa, David; Nosanchuk, Joshua D.

    2009-01-01

    Cytosolic fungal fatty acid synthase is composed of two subunits α and β, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14∶0), palmitic acid (16∶0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target. PMID:20027295

  15. The Aeromonas caviae AHA0618 gene modulates cell length and influences swimming and swarming motility.

    PubMed

    Lowry, Rebecca C; Parker, Jennifer L; Kumbhar, Ramhari; Mesnage, Stephane; Shaw, Jonathan G; Stafford, Graham P

    2014-12-17

    Aeromonas caviae is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular adherence and biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O-linked glycosylation with pseudaminic acid on 6-8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum. Aeromonas caviae possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the AHA0618 gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen Helicobacter pylori, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an AHA0618 A. caviae mutant revealed increased swimming and swarming motility compared to the wild-type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of l,d-transpeptidases involved in cell wall biology and peptidoglycan cross-linking (YkuD-like). Scanning electron microscopy (SEM) and fluorescence microscopy analysis of the wild-type and AHA0618-mutant A. caviae strains revealed the mutant to be subtly but significantly shorter than wild-type cells; a phenomenon that could be recovered when either AHA0618 or H. pylori HP0518 were introduced. We can therefore conclude that AHA0618 does not affect A. caviae behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering

  16. The Aeromonas caviae AHA0618 gene modulates cell length and influences swimming and swarming motility

    PubMed Central

    Lowry, Rebecca C; Parker, Jennifer L; Kumbhar, Ramhari; Mesnage, Stephane; Shaw, Jonathan G; Stafford, Graham P

    2015-01-01

    Aeromonas caviae is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular adherence and biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O-linked glycosylation with pseudaminic acid on 6–8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum. Aeromonas caviae possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the AHA0618 gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen Helicobacter pylori, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an AHA0618 A. caviae mutant revealed increased swimming and swarming motility compared to the wild-type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of l,d-transpeptidases involved in cell wall biology and peptidoglycan cross-linking (YkuD-like). Scanning electron microscopy (SEM) and fluorescence microscopy analysis of the wild-type and AHA0618-mutant A. caviae strains revealed the mutant to be subtly but significantly shorter than wild-type cells; a phenomenon that could be recovered when either AHA0618 or H. pylori HP0518 were introduced. We can therefore conclude that AHA0618 does not affect A. caviae behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering

  17. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    PubMed

    Salim, Vonny; Wiens, Brent; Masada-Atsumi, Sayaka; Yu, Fang; De Luca, Vincenzo

    2014-05-01

    Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. 3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

    PubMed Central

    Tai, H; Jaworski, J G

    1993-01-01

    A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals. PMID:8290632

  19. Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes.

    PubMed

    Sirikantaramas, Supaart; Taura, Futoshi; Tanaka, Yumi; Ishikawa, Yu; Morimoto, Satoshi; Shoyama, Yukihiro

    2005-09-01

    Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.

  20. Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events.

    PubMed

    McCarthy, A D; Goldring, J P; Hardie, D G

    1983-10-17

    The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium [3H]borohydride. Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein. The results show that the enoyl reductase lies between the 3-oxoacylsynthase and the acyl carrier. This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events.

  1. A single amino acid change in acetolactate synthase confers resistance to valine in tobacco.

    PubMed

    Hervieu, F; Vaucheret, H

    1996-05-23

    The metabolic control of branches chain amino acid (BCAA) biosynthesis involves allosteric regulation of acetolactate synthase (ALS) by the end-products of the pathway, valine, leucine and isoleucine. We describe here the molecular basis of valine resistance. We cloned and sequenced an ALS gene from the tobacco mutant Valr-1 and found a single basepair substitution relative to the wild-type allele. This mutation causes a serine to leucine change in the amino acid sequence of ALS at position 214. We then mutagenized the wild-type allele of the ALS gene of Arabidopsis and found that it confers valine resistance when introduced into tobacco plants. Taken together, these results suggest that the serine to leucine change at position 214 of ALS is responsible for valine resistance in tobacco.

  2. Para-aminobenzoic acid (PABA) synthase enhances thermotolerance of mushroom Agaricus bisporus.

    PubMed

    Lu, Zhonglei; Kong, Xiangxiang; Lu, Zhaoming; Xiao, Meixiang; Chen, Meiyuan; Zhu, Liang; Shen, Yuemao; Hu, Xiangyang; Song, Siyang

    2014-01-01

    Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom's innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins.

  3. A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

    PubMed

    Cabruja, Matías; Mondino, Sonia; Tsai, Yi Ting; Lara, Julia; Gramajo, Hugo; Gago, Gabriela

    2017-02-01

    Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

  4. Structure and conformational variability of the mycobacterium tuberculosis fatty acid synthase multienzyme complex.

    PubMed

    Ciccarelli, Luciano; Connell, Sean R; Enderle, Mathias; Mills, Deryck J; Vonck, Janet; Grininger, Martin

    2013-07-02

    Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis.

  5. Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L.

    PubMed

    Weitzel, Corinna; Petersen, Maike

    2011-05-01

    Lemon balm (Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene (MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2011-08-15

    Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.

  7. Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis

    PubMed Central

    Heaton, Nicholas S.; Perera, Rushika; Berger, Kristi L.; Khadka, Sudip; LaCount, Douglas J.; Kuhn, Richard J.; Randall, Glenn

    2010-01-01

    Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway. PMID:20855599

  8. Fatty acid synthase is a metabolic oncogene targetable in malignant peripheral nerve sheath tumors

    PubMed Central

    Patel, Ami V.; Johansson, Gunnar; Colbert, Melissa C.; Dasgupta, Biplab; Ratner, Nancy

    2015-01-01

    Background Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas with minimal therapeutic opportunities. We observed that lipid droplets (LDs) accumulate in human MPNST cell lines and in primary human tumor samples. The goal of this study was to investigate the relevance of lipid metabolism to MPNST survival and as a possible therapeutic target. Methods Based on preliminary findings that MPNSTs accumulate LDs, we hypothesized that a deregulated lipid metabolism supports MPNST cell survival/proliferation rate. To test this, we examined respiration, role of fatty acid oxidation (FAO), and the enzyme fatty acid synthase involved in de novo fatty acid synthesis in MPNSTs using both genetic and pharmacological tools. Results We demonstrate that LDs accumulate in MPNST cell lines, primary human and mouse MPNST tumors, and neural crest cells. LDs from MPNST cells disappear on lipid deprivation, indicating that LDs can be oxidized as a source of energy. Inhibition of FAO decreased oxygen consumption and reduced MPNST survival, indicating that MPNST cells likely metabolize LDs through active FAO. FAO inhibition reduced oxygen consumption and survival even in the absence of exogenous lipids, indicating that lipids synthesized de novo can also be oxidized. Consequently, inhibition of de novo fatty acid synthesis, which is overexpressed in human MPNST cell lines, effectively reduced MPNST survival and delayed induction of tumor growth in vivo. Conclusion Our results show that MPNSTs depend on lipid metabolic pathways and suggest that disrupting lipid metabolism could be a potential new strategy for the development of MPNST therapeutics. PMID:26116612

  9. Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes.

    PubMed Central

    Stuible, H P; Wagner, C; Andreou, I; Huter, G; Haselmann, J; Schweizer, E

    1996-01-01

    The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid. PMID:8759839

  10. Presence of fatty acid synthase inhibitors in the rhizome of Alpinia officinarum hance.

    PubMed

    Li, Bing-Hui; Tian, Wei-Xi

    2003-08-01

    The galangal (the rhizome of Alpinia officinarum, Hance) is popular in Asia as a traditional herbal medicine. The present study reports that the galangal extract (GE) can potently inhibit fatty-acid synthase (FAS, E.C.2.3.1.85). The inhibition consists of both reversible inhibition with an IC50 value of 1.73 microg dried GE/ml, and biphasic slow-binding inactivation. Subsequently the reversible inhibition and slow-binding inactivation to FAS were further studied. The inhibition of FAS by galangin, quercetin and kaempferol, which are the main flavonoids existing in the galangal, showed that quercetin and kaempferol had potent reversible inhibitory activity, but all three flavonoids had no obvious slow-binding inactivation. Analysis of the kinetic results led to the conclusion that the inhibitory mechanism of GE is totally different from that of some other previously reported inhibitors of FAS, such as cerulenin, EGCG (epigallocatechin gallate) and C75.

  11. Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana.

    PubMed

    Jiang, He Zhong; Quan, Xiao Fang; Tian, Wei Xi; Hu, Jiang Miao; Wang, Peng Cheng; Huang, Sheng Zhuo; Cheng, Zhong Quan; Liang, Wen Juan; Zhou, Jun; Ma, Xiao Feng; Zhao, You Xing

    2010-10-15

    Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1-13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC(50) values ranging from 1.24 to 91.07 μM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by orlistat

    SciTech Connect

    Pemble,C.; Johnson, L.; Kridel, S.; Lowther, W.

    2007-01-01

    Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-{angstrom}-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

  13. Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

    PubMed

    Shoyama, Yoshinari; Tamada, Taro; Kurihara, Kazuo; Takeuchi, Ayako; Taura, Futoshi; Arai, Shigeki; Blaber, Michael; Shoyama, Yukihiro; Morimoto, Satoshi; Kuroki, Ryota

    2012-10-12

    ∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

  14. The 2.0 Å X-ray structure for yeast acetohydroxyacid synthase provides new insights into its cofactor and quaternary structure requirements

    PubMed Central

    Lonhienne, Thierry; Garcia, Mario D.; Fraser, James A.; Williams, Craig M.; Guddat, Luke W.

    2017-01-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin adenine dinucleotide (FAD) as indispensable co-factors. A new, high resolution, 2.0 Å crystal structure of Saccharomyces cerevisiae AHAS reveals that the dimer is asymmetric, with the catalytic centres having distinct structures where FAD is trapped in two different conformations indicative of different redox states. Two molecules of oxygen (O2) are bound on the surface of each active site and a tunnel in the polypeptide appears to passage O2 to the active site independently of the substrate. Thus, O2 appears to play a novel “co-factor” role in this enzyme. We discuss the functional implications of these features of the enzyme that have not previously been described. PMID:28178302

  15. The 2.0 Å X-ray structure for yeast acetohydroxyacid synthase provides new insights into its cofactor and quaternary structure requirements.

    PubMed

    Lonhienne, Thierry; Garcia, Mario D; Fraser, James A; Williams, Craig M; Guddat, Luke W

    2017-01-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin adenine dinucleotide (FAD) as indispensable co-factors. A new, high resolution, 2.0 Å crystal structure of Saccharomyces cerevisiae AHAS reveals that the dimer is asymmetric, with the catalytic centres having distinct structures where FAD is trapped in two different conformations indicative of different redox states. Two molecules of oxygen (O2) are bound on the surface of each active site and a tunnel in the polypeptide appears to passage O2 to the active site independently of the substrate. Thus, O2 appears to play a novel "co-factor" role in this enzyme. We discuss the functional implications of these features of the enzyme that have not previously been described.

  16. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

    PubMed

    Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-02-01

    Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  18. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    PubMed

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  19. Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation

    PubMed Central

    2012-01-01

    Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo. PMID:24900571

  20. Low-temperature Storage of Cucumbers Induces Changes in the Organic Acid Content and in Citrate Synthase Activity

    USDA-ARS?s Scientific Manuscript database

    To elucidate the cause of reported pyruvate accumulation in chilled stored cucumbers (Cucumis sativus L.) cv. ‘Toppugurin’, we have examined differences in the extent of incorporation of acetate-1,2-14C into the tricarboxylic acid (TCA) cycle and the specific activity of the enzyme citrate synthase ...

  1. Cooperative functioning between phenylalanine ammonia lyase and isochorishmate synthase activities contributes to salicylic acid biosynthesis in soybean

    USDA-ARS?s Scientific Manuscript database

    Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL), or the isochorishmate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We...

  2. Dietary Soy Protein Inhibits DNA Damage and Cell Survival of Colon Epithelial Cells through Attenuated Expression of Fatty Acid Synthase

    USDA-ARS?s Scientific Manuscript database

    Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of Fatty Acid Synthase (FASN) underlies, in part, the tumor preventive effects of soy protein, since FASN over-expression characterizes early tumorigene...

  3. Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene from Chromobacterium violaceum

    PubMed Central

    Kolibachuk, Dana; Miller, Andrea; Dennis, Douglas

    1999-01-01

    The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaCCv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaCCv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaACv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaCCv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaCCv accumulated significant levels of PHA. Sequence analysis of the phaCCv product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274). PMID:10427049

  4. Leucine deprivation inhibits proliferation and induces apoptosis of human breast cancer cells via fatty acid synthase

    PubMed Central

    Xiao, Fei; Wang, Chunxia; Yin, Hongkun; Yu, Junjie; Chen, Shanghai; Fang, Jing; Guo, Feifan

    2016-01-01

    Substantial studies on fatty acid synthase (FASN) have focused on its role in regulating lipid metabolism and researchers have a great interest in treating cancer with dietary manipulation of amino acids. In the current study, we found that leucine deprivation caused the FASN-dependent anticancer effect. Here we showed that leucine deprivation inhibited cell proliferation and induced apoptosis of MDA-MB-231 and MCF-7 breast cancer cells. In an in vivo tumor xenograft model, the leucine-free diet suppressed the growth of human breast cancer tumors and triggered widespread apoptosis of the cancer cells. Further study indicated that leucine deprivation decreased expression of lipogenic gene FASN in vitro and in vivo. Over-expression of FASN or supplementation of palmitic acid (the product of FASN action) blocked the effects of leucine deprivation on cell proliferation and apoptosis in vitro and in vivo. Moreover, leucine deprivation suppressed the FASN expression via regulating general control non-derepressible (GCN)2 and sterol regulatory element-binding protein 1C (SREBP1C). Taken together, our study represents proof of principle that anticancer effects can be obtained with strategies to deprive tumors of leucine via suppressing FASN expression, which provides important insights in prevention of breast cancer via metabolic intervention. PMID:27579768

  5. Using modern tools to probe the structure-function relationship of fatty acid synthases

    PubMed Central

    Burkart, Michael D.

    2015-01-01

    Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory. PMID:25676190

  6. Δ(9)-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids.

    PubMed

    Lange, Kerstin; Schmid, Andreas; Julsing, Mattijs K

    2015-10-10

    Δ(9)-Tetrahydrocannabinol (THC) is of increasing interest as a pharmaceutical and bioactive compound. Chemical synthesis of THC uses a laborious procedure and does not satisfy the market demand. The implementation of biocatalysts for specific synthesis steps might be beneficial for making natural product availability independent from the plant. Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from C. sativa L. catalyzes the cyclization of cannabigerolic acid (CBGA) to Δ(9)-tetrahydrocannabinolic acid (THCA), which is non-enzymatically decarboxylated to THC. We report the preparation of THCAS in amounts sufficient for the biocatalytic production of THC(A). Active THCAS was most efficiently obtained from Pichia pastoris. THCAS was produced on a 2L bioreactor scale and the enzyme was isolated by single-step chromatography with a specific activity of 73Ug(-1)total protein. An organic/aqueous two-liquid phase setup for continuous substrate delivery facilitated in situ product removal. In addition, THCAS activity in aqueous environments lasted for only 20min whereas the presence of hexane stabilized the activity over 3h. In conclusion, production of THCAS in P. pastoris Mut(S) KM71 KE1, subsequent isolation, and its application in a two-liquid phase setup enables the synthesis of THCA on a mg scale.

  7. Inhibition of Fatty Acid Synthase Reduces Blastocyst Hatching through Regulation of the AKT Pathway in Pigs

    PubMed Central

    Guo, Jing; Kim, Nam-Hyung; Cui, Xiang-Shun

    2017-01-01

    Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs. PMID:28107461

  8. A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

    PubMed Central

    Taura, Futoshi; Iijima, Miu; Yamanaka, Eriko; Takahashi, Hironobu; Kenmoku, Hiromichi; Saeki, Haruna; Morimoto, Satoshi; Asakawa, Yoshinori; Kurosaki, Fumiya; Morita, Hiroyuki

    2016-01-01

    Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum. PMID:27729920

  9. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  10. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    PubMed

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  11. Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration.

    PubMed

    Chang, Z; Vining, L C

    2002-07-01

    A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase

  12. A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

    PubMed Central

    Cabruja, Matías; Mondino, Sonia; Tsai, Yi Ting; Lara, Julia; Gramajo, Hugo

    2017-01-01

    Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria. PMID:28228470

  13. [Correlation analysis between single nucleotide polymorphism of beta-amyrin synthase and content of glycyrrhizic acid in Glycyrrhiza uralensis].

    PubMed

    Shen, Zhanyun; Liu, Chunsheng; Wang, Xueyong; Guo, Wei; Li, Beining

    2010-04-01

    To analyze the correlation between content of glycyrrhizic acid and the single nucleotide polymorphism of beta-amyrin synthase (bAS) in Glycyrrhiza uralensis. glycyrrhizic acid content in 80 samples of the cultivated G. uralensis were determined by HPLC; According to the very significant level (P < 0.000 1), 80 samples in accordance with glycyrrhizic acid will be grouped by SAS 9.0; Using RT-PCR strategy to amplification the Open Reading Frame of beta-amyrin synthase with the template of total RNA extracted from roots of G. uralensis and then using DNAman to analyze the relationship between glycyrrhizic acid content and the single nucleotide polymorphism of beta-amyrin synthase (bAS). There exited two mutation sites 94 bp and 254 bp, G/A conversion occurred at 94 bp site, which belonged to a missense mutation. G/A conversion led to the corresponding amino acid conversion (Gly --> Asp); C/T conversion occurred at 254 bp site, which belonged to a synonymous mutation. According to sequence variation, the samples were divided into four genotypes: G-T genotype, A-T genotype, G/A-C genotype and G-T genotype. A-T genotype, G/A-C genotype and G-T genotype are correlated with the high content of glycyrrhizic acid.

  14. Enzymatic formation of a resorcylic acid by creating a structure-guided single-point mutation in stilbene synthase.

    PubMed

    Bhan, Namita; Li, Lingyun; Cai, Chao; Xu, Peng; Linhardt, Robert J; Koffas, Mattheos A G

    2015-02-01

    A novel C17 resorcylic acid was synthesized by a structure-guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl-CoA, to p-coumaroyl-CoA. The resulting novel pentaketide can be produced strictly by STS-like enzymes and not by Chalcone synthase-like type III polyketide synthases; due to the unique thioesterase like activity of STS-like enzymes. We utilized a liquid chromatography mass spectrometry-based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor-directed biosynthesis and identification of unnatural polyketides by structure-guided mutation of STS-like enzymes.

  15. Fatty acid synthase - Modern tumor cell biology insights into a classical oncology target.

    PubMed

    Buckley, Douglas; Duke, Gregory; Heuer, Timothy S; O'Farrell, Marie; Wagman, Allan S; McCulloch, William; Kemble, George

    2017-02-12

    Decades of preclinical and natural history studies have highlighted the potential of fatty acid synthase (FASN) as a bona fide drug target for oncology. This review will highlight the foundational concepts upon which this perspective is built. Published studies have shown that high levels of FASN in patient tumor tissues are present at later stages of disease and this overexpression predicts poor prognosis. Preclinical studies have shown that experimental overexpression of FASN in previously normal cells leads to changes that are critical for establishing a tumor phenotype. Once the tumor phenotype is established, FASN elicits several changes to the tumor cell and becomes intertwined with its survival. The product of FASN, palmitate, changes the biophysical nature of the tumor cell membrane; membrane microdomains enable the efficient assembly of signaling complexes required for continued tumor cell proliferation and survival. Membranes densely packed with phospholipids containing saturated fatty acids become resistant to the action of other chemotherapeutic agents. Inhibiting FASN leads to tumor cell death while sparing normal cells, which do not have the dependence of this enzyme for normal functions, and restores membrane architecture to more normal properties thereby resensitizing tumors to killing by chemotherapies. One compound has recently reached clinical studies in solid tumor patients and highlights the need for continued evaluation of the role of FASN in tumor cell biology. Significant advances have been made and much remains to be done to optimally apply this class of pharmacological agents for the treatment of specific cancers.

  16. Clinical and therapeutic relevance of the metabolic oncogene fatty acid synthase in HER2+ breast cancer.

    PubMed

    Corominas-Faja, Bruna; Vellon, Luciano; Cuyàs, Elisabet; Buxó, Maria; Martin-Castillo, Begoña; Serra, Dolors; García, Jordi; Lupu, Ruth; Menendez, Javier A

    2017-07-01

    Fatty acid synthase (FASN) is a key lipogenic enzyme for de novo fatty acid biosynthesis and a druggable metabolic oncoprotein that is activated in most human cancers. We evaluated whether the HER2-driven lipogenic phenotype might represent a biomarker for sensitivity to pharmacological FASN blockade. A majority of clinically HER2-positive tumors were scored as FASN overexpressors in a series of almost 200 patients with invasive breast carcinoma. Re-classification of HER2-positive breast tumors based on FASN gene expression predicted a significantly inferior relapse-free and distant metastasis-free survival in HER2+/FASN+ patients. Notably, non-tumorigenic MCF10A breast epithelial cells engineered to overexpress HER2 upregulated FASN gene expression, and the FASN inhibitor C75 abolished HER2-induced anchorage-independent growth and survival. Furthermore, in the presence of high concentrations of C75, HER2-negative MCF-7 breast cancer cells overexpressing HER2 (MCF-7/HER2) had significantly higher levels of apoptosis than HER2-negative cells. Finally, C75 at non-cytotoxic concentrations significantly reduced the capacity of MCF-7/HER2 cells to form mammospheres, an in vitro indicator of cancer stem-like cells. Collectively, our findings strongly suggest that the HER2-FASN lipogenic axis delineates a group of breast cancer patients that might benefit from treatment with therapeutic regimens containing FASN inhibitors.

  17. Identification of amino acid residues essential for onion lachrymatory factor synthase activity.

    PubMed

    Masamura, Noriya; Ohashi, Wakana; Tsuge, Nobuaki; Imai, Shinsuke; Ishii-Nakamura, Anri; Hirota, Hiroshi; Nagata, Toshiyuki; Kumagai, Hidehiko

    2012-01-01

    Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H(+) substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23-169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23-169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket.

  18. Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

    PubMed

    Krishnamoorthy, Ezhilarasi; Hassan, Sameer; Hanna, Luke Elizabeth; Padmalayam, Indira; Rajaram, Rama; Viswanathan, Vijay

    2017-05-07

    Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

    PubMed Central

    2015-01-01

    Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

  20. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

    PubMed

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L; Katz, Leonard; Petzold, Christopher J; Keasling, Jay D

    2016-01-15

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  1. Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit.

    PubMed

    Aluru, Maneesha R; Mazourek, Michael; Landry, Laurie G; Curry, Jeanne; Jahn, Molly; O'Connell, Mary A

    2003-07-01

    The biosynthesis of capsaicinoids in the placenta of chilli fruit is modelled to require components of the fatty acid synthase (FAS) complex. Three candidate genes for subunits in this complex, Kas, Acl, and Fat, isolated based on differential expression, were characterized. Transcription of these three genes was placental-specific and RNA abundance was positively correlated with degree of pungency. Kas and Acl were mapped to linkage group 1 and Fat to linkage group 6. None of the genes is linked to the pungency locus, C, on linkage group 2. KAS accumulation was positively correlated with pungency. Western blots of placental extracts and histological sections both demonstrated that the accumulation of this enzyme was correlated with fruit pungency and KAS was immunolocalized to the expected cell layer, the placental epidermis. Enzyme activity of the recombinant form of the placental-specific KAS was confirmed using crude cell extracts. These FAS components are fruit-specific members of their respective gene families. These genes are predicted to be associated with Capsicum fruit traits, for example, capsaicinoid biosynthesis or fatty acid biosynthesis necessary for placental development.

  2. Structural and mechanistic comparison of the Cyclopropane Mycolic Acid Synthases (CMAS) protein family of Mycobacterium tuberculosis.

    PubMed

    Defelipe, Lucas A; Osman, Federico; Marti, Marcelo A; Turjanski, Adrián G

    2017-08-30

    Tuberculosis (TB) is a chronic disease caused by the bacillus Mycobacterium tuberculosis(Mtb) and remains a leading cause of mortality worldwide. The bacteria has an external wall which protects it from being killed, and the enzymes involved in the biosynthesis of the cell wall components have been proposed as promising targets for future drug development efforts. Cyclopropane Mycolic Acid Synthases (CMAS) constitute a group of ten homologous enzymes which belong to the mycolic acid biosynthesis pathway. These enzymes have S-adenosyl-l-methionine (SAM) dependent methyltransferase activity with a peculiarity, each one of them has strong substrate selectivity and reaction specificity, being able to produce among other things cyclopropanes or methyl-alcohol groups from the lipid olefin group. How each CMAS processes its substrate and how the specificity and selectivity are encoded in the protein sequence and structure, is still unclear. In this work, by using a combination of modeling tools, including comparative modeling, docking, all-atom MD and QM/MM methodologies we studied in detail the reaction mechanism of cmaA2, mmaA4, and mmaA1 CMAS and described the molecular determinants that lead to different products. We have modeled the protein-substrate complex structure and determined the free energy pathway for the reaction. The combination of modeling tools at different levels of complexity allows having a complete picture of the CMAS structure-activity relationship. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Suppression of rat hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fat.

    PubMed

    Blake, W L; Clarke, S D

    1990-12-01

    The objective of this research was to determine whether dietary polyunsaturated fatty acids suppress hepatic fatty acid synthase (FAS) mRNA levels by altering FAS gene transcription. Male Sprague-Dawley rats were meal-fed for 10 d a high glucose diet supplemented with 20% digestible energy as menhaden oil or tripalmitin. The transcription rate for FAS was determined by nuclear run-on analysis in hepatic nuclei isolated from rats 2 h postmeal. The values for transcription rates of FAS and S14 (a putative lipogenic protein) in rats fed menhaden oil were only 6 and 21%, respectively, of the rates in rats fed the tripalmitin diet (p less than 0.02). Gene transcription for beta-actin and phosphoenolpyruvate carboxykinase did not differ between treatments. The reduction in hepatic FAS mRNA levels caused by dietary polyunsaturated fats appears to be caused primarily by an inhibition of FAS transcription. The control of transcription by polyunsaturated fats appears not to be mediated by cAMP because the transcription rate for phosphoenolpyruvate carboxykinase (whose gene is very sensitive to cAMP stimulation) was unaffected by the source of dietary fat.

  4. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid

    SciTech Connect

    Hagen, Andrew; Poust, Sean; Rond, Tristan de; Fortman, Jeffrey L.; Katz, Leonard; Petzold, Christopher J.; Keasling, Jay D.

    2015-10-26

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  5. Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines.

    PubMed

    Niranjan, S K; Goyal, S; Dubey, P K; Kumari, N; Mishra, S K; Mukesh, M; Kataria, R S

    2016-01-10

    Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines

  6. The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis

    PubMed Central

    Sacco, Emmanuelle; Covarrubias, Adrian Suarez; O'Hare, Helen M.; Carroll, Paul; Eynard, Nathalie; Jones, T. Alwyn; Parish, Tanya; Daffé, Mamadou; Bäckbro, Kristina; Quémard, Annaïk

    2007-01-01

    The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (≥C12) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs. PMID:17804795

  7. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

    PubMed Central

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M.; Baerga-Ortiz, Abel

    2014-01-01

    Increasing the production of fatty acids by microbial fermentation remains an important step towards the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations towards accessible biodiesel precursors. PMID:24411456

  8. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli.

    PubMed

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M; Baerga-Ortiz, Abel

    2014-02-05

    Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.

  9. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis

    SciTech Connect

    Guerinot, M.L.; Chelm, B.K.

    1986-03-01

    Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. The authors now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolated the wild-type hemA gene encoding delta-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizobium meliloti hemA gene as a hybridization probe. A deletion of the hemA gene region, generated in vitro, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no delta-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless delta-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial delta-aminolevulinic acid synthase activity as the exclusive source of delta-aminolevulinic acid for heme formation in soybean nodules.

  10. Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis

    SciTech Connect

    Yu X. H.; Shanklin J.; Rawat, R.

    2011-05-01

    Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory. Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate gene for

  11. DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in "drug-type" and "fiber-type" Cannabis sativa L.

    PubMed

    Kojoma, Mareshige; Seki, Hikaru; Yoshida, Shigeo; Muranaka, Toshiya

    2006-06-02

    The cannabinoid content of 13 different strains of cannabis plant (Cannabis sativa L.) was analyzed. Six strains fell into the "drug-type" class, with high Delta-9-tetrahydrocannabinolic acid (THCA) content, and seven strains into the "fiber-type" class, with low THCA using HPLC analysis. Genomic DNA sequence polymorphisms in the THCA synthase gene from each strain were studied. A single PCR fragment of the THCA synthase gene was detected from six strains of "drug-type" plants. We could also detect the fragment from seven strains of "fiber-type" plants, although no or very low content of THCA were detected in these samples. These were 1638 bp from all 13 strains and no intron among the sequences obtained. There were two variants of the THCA synthase gene in the "drug-type" and "fiber-type" cannabis plants, respectively. Thirty-seven major substitutions were detected in the alignment of the deduced amino acid sequences from these variants. Furthermore, we identified a specific PCR marker for the THCA synthase gene for the "drug-type" strains. This PCR marker was not detected in the "fiber-type" strains.

  12. Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II of Mycobacterium tuberculosis▿

    PubMed Central

    Sacco, Emmanuelle; Slama, Nawel; Bäckbro, Kristina; Parish, Tanya; Laval, Françoise; Daffé, Mamadou; Eynard, Nathalie; Quémard, Annaık̈

    2010-01-01

    The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-IIMt) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-IIMt cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-IIMt enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-IIMt. The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-IIMt. The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned. PMID:20511508

  13. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.

    PubMed

    Yu, Xingye; Liu, Tiangang; Zhu, Fayin; Khosla, Chaitan

    2011-11-15

    Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.

  14. Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication.

    PubMed

    Wilsky, Steffi; Sobotta, Katharina; Wiesener, Nadine; Pilas, Johanna; Althof, Nadine; Munder, Thomas; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Coxsackievirus B3 (CVB3) is a human pathogen that causes acute and chronic infections, but an antiviral drug to treat these diseases has not yet been developed for clinical use. Several intracellular pathways are altered to assist viral transcription, RNA replication, and progeny release. Among these, fatty acid synthase (FAS) expression is increased. In order to test the potential of FAS inhibition as an anti-CVB3 strategy, several experiments were performed, including studies on the correlation of CVB3 replication and FAS expression in human Raji cells and an analysis of the time and dose dependence of the antiviral effect of FAS inhibition due to treatment with amentoflavone. The results demonstrate that CVB3 infection induces an up-regulation of FAS expression already at 1 h postinfection (p.i.). Incubation with increasing concentrations of amentoflavone inhibited CVB3 replication significantly up to 8 h p.i. In addition, suppression of p38 MAP kinase activity by treatment with SB239063 decreased FAS expression as well as viral replication. These data provide evidence that FAS inhibition via amentoflavone administration might present a target for anti-CVB3 therapy.

  15. Biphasic modulation of fatty acid synthase by hydrogen peroxide in Saccharomyces cerevisiae.

    PubMed

    Matias, Ana C; Marinho, H Susana; Cyrne, Luísa; Herrero, Enrique; Antunes, Fernando

    2011-11-01

    Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150μM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit β. However, the exposure to a steady-state 50μM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.

  16. Potent Inhibitory Effect of Chinese Dietary Spices on Fatty Acid Synthase.

    PubMed

    Jiang, Bing; Liang, Yan; Sun, Xuebing; Liu, Xiaoxin; Tian, Weixi; Ma, Xiaofeng

    2015-09-01

    Dietary spices have been adopted in cooking since ancient times to enhance flavor and also as food preservatives and disease remedies. In China, the use of spices and other aromatic plants as food flavoring is an integral part of dietary behavior, but relatively little is known about their functions. Fatty acid synthase (FAS) has been recognized as a remedy target, and its inhibitors might be applied in disease treatment. The present work was designed to assess the inhibitory activities on FAS of spices extracts in Chinese menu. The in vitro inhibitory activities on FAS of 22 extracts of spices were assessed by spectrophotometrically monitoring oxidation of NADPH at 340 nm. Results showed that 20 spices extracts (90.9 %) exhibited inhibitory activities on FAS, with half inhibition concentration (IC(50)) values ranging from 1.72 to 810.7 μg/ml. Among them, seven spices showed strong inhibitory effect with IC(50) values lower than 10 μg/ml. These findings suggest that a large proportion of the dietary spices studied possess promising inhibitory activities on FAS, and subsequently might be applied in the treatment of obesity and obesity-related human diseases.

  17. (-)-UB006: A new fatty acid synthase inhibitor and cytotoxic agent without anorexic side effects.

    PubMed

    Makowski, Kamil; Mir, Joan Francesc; Mera, Paula; Ariza, Xavier; Asins, Guillermina; Hegardt, Fausto G; Herrero, Laura; García, Jordi; Serra, Dolors

    2017-05-05

    C75 is a synthetic anticancer drug that inhibits fatty acid synthase (FAS) and shows a potent anorexigenic side effect. In order to find new cytotoxic compounds that do not impact food intake, we synthesized a new family of C75 derivatives. The most promising anticancer compound among them was UB006 ((4SR,5SR)-4-(hydroxymethyl)-3-methylene-5-octyldihydrofuran-2(3H)-one). The effects of this compound on cytotoxicity, food intake and body weight were studied in UB006 racemic mixture and in both its enantiomers separately. The results showed that both enantiomers inhibit FAS activity and have potent cytotoxic effects in several tumour cell lines, such as the ovarian cell cancer line OVCAR-3. The (-)-UB006 enantiomer's cytotoxic effect on OVCAR-3 was 40-fold higher than that of racemic C75, and 2- and 38-fold higher than that of the racemic mixture and its opposite enantiomer, respectively. This cytotoxic effect on the OVCAR-3 cell line involves mechanisms that reduce mitochondrial respiratory capacity and ATP production, DDIT4/REDD1 upregulation, mTOR activity inhibition, and caspase-3 activation, resulting in apoptosis. In addition, central and peripheral administration of (+)-UB006 or (-)-UB006 into rats and mice did not affect food intake or body weight. Altogether, our data support the discovery of a new potential anticancer compound (-)-UB006 that has no anorexigenic side effects.

  18. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes.

    PubMed

    Yang, Jeong-Yeh; Koo, Bon-Sun; Kang, Mi-Kyung; Rho, Hye-Won; Sohn, Hee-Sook; Jhee, Eun-Chung; Park, Jin-Woo

    2002-11-30

    The present study was undertaken to explore whether retinoids, which are known to have immunomodulatory actions, could attenuate tumor necrosis factor-alpha (TNF)-stimulated inducible nitric oxide synthase (iNOS) expression in 3T3-L1 adipocytes. Adipocytes incubated with TNF induced dose- and time-dependent accumulation of nitrite in the culture medium through the iNOS induction as confirmed by Western blotting. Treatment of cells with TNF in the presence of all-trans-retinoic acid (RA) significantly decreased their ability to produce nitrite and iNOS induction. Both 13-cis- and all- trans-RA-induced suppression was dose-dependent, and all-trans-RA was somewhat potent than 13-cis-RA. The inhibitory effect of RA on TNF-induced iNOS induction was reversible, completely recovered after 2 days, and was exerted through the inhibition of NF-kappaB activation. TNF also suppressed the lipoprotein lipase (LPL) activity of 3T3-L1 adipocytes. RA could not reverse the TNF- induced LPL suppression at RA levels causing near complete inhibition of the TNF-induced NO production. These results indicate that RAs attenuate iNOS expression reversibly in TNF-stimulated 3T3-L1 adipocytes, and that the TNF-induced LPL suppression is not the result of NO overproduction.

  19. Inhibitory effects of sea buckthorn procyanidins on fatty acid synthase and MDA-MB-231 cells.

    PubMed

    Wang, Yi; Nie, Fangyuan; Ouyang, Jian; Wang, Xiaoyan; Ma, Xiaofeng

    2014-10-01

    Fatty acid synthase (FAS) is overexpressed in many human cancers including breast cancer and is considered to be a promising target for therapy. Sea buckthorn has long been used to treat a variety of maladies. Here, we investigated the inhibitory effect of sea buckthorn procyanidins (SBPs) isolated from the seeds of sea buckthorn on FAS and FAS overexpressed human breast cancer MDA-MB-231 cells. The FAS activity and FAS inhibition were measured by a spectrophotometer at 340 nm of nicotinamide adenine dinucleotide phosphate (NADPH) absorption. We found that SBP potently inhibited the activity of FAS with a half-inhibitory concentration (IC50) value of 0.087 μg/ml. 3-4,5-Dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide (MTT) assay was used to test the cell viability. SBP reduced MDA-MB-231 cell viability with an IC50 value of 37.5 μg/ml. Hoechst 33258/propidium iodide dual staining and flow cytometric analysis showed that SBP induced MDA-MB-231 cell apoptosis. SBP inhibited intracellular FAS activity with a dose-dependent manner. In addition, sodium palmitate could rescue the cell apoptosis induced by SBP. These results showed that SBP was a promising FAS inhibitor which could induce the apoptosis of MDA-MB-231 cells via inhibiting FAS. These findings suggested that SBP might be useful for preventing or treating breast cancer.

  20. Circulating Fatty Acid Synthase in pregnant women: Relationship to blood pressure, maternal metabolism and newborn parameters

    PubMed Central

    Carreras-Badosa, Gemma; Prats-Puig, Anna; Puig, Teresa; Vázquez-Ruíz, Montserrat; Bruel, Monserrat; Mendoza, Ericka; de Zegher, Francis; Ibáñez, Lourdes; López-Bermejo, Abel; Bassols, Judit

    2016-01-01

    The enzyme FASN (fatty acid synthase) is potentially related with hypertension and metabolic dysfunction. FASN is highly expressed in the human placenta. We aimed to investigate the relationship circulating FASN has with blood pressure, maternal metabolism and newborn parameters in healthy pregnant women. Circulating FASN was assessed in 115 asymptomatic pregnant women in the second trimester of gestation along with C-peptide, fasting glucose and insulin, post-load glucose lipids, HMW-adiponectin and blood pressure (the latter was assessed in each trimester of gestation). At birth, newborns and placentas were weighed. FASN expression was also able to be assessed in 80 placentas. Higher circulating FASN was associated with lower systolic blood pressure (SBP), with a more favourable metabolic phenotype (lower fasting glucose and insulin, post load glucose, HbAc1, HOMA-IR and C-peptide), and with lower placental and birth weight (all p < 0.05 to p < 0.001). Placental FASN expression related positively to circulating FASN (p < 0.005) and negatively to placental weight (p < 0.05). Our observations suggest a physiological role of placental FASN in human pregnancy. Future studies will clarify whether circulating FASN of placental origin does actually regulate placental and fetal growth, and (thereby) has a favourable influence on the pregnant mother’s insulin sensitivity and blood pressure. PMID:27090298

  1. Circulating Fatty Acid Synthase in pregnant women: Relationship to blood pressure, maternal metabolism and newborn parameters.

    PubMed

    Carreras-Badosa, Gemma; Prats-Puig, Anna; Puig, Teresa; Vázquez-Ruíz, Montserrat; Bruel, Monserrat; Mendoza, Ericka; de Zegher, Francis; Ibáñez, Lourdes; López-Bermejo, Abel; Bassols, Judit

    2016-04-19

    The enzyme FASN (fatty acid synthase) is potentially related with hypertension and metabolic dysfunction. FASN is highly expressed in the human placenta. We aimed to investigate the relationship circulating FASN has with blood pressure, maternal metabolism and newborn parameters in healthy pregnant women. Circulating FASN was assessed in 115 asymptomatic pregnant women in the second trimester of gestation along with C-peptide, fasting glucose and insulin, post-load glucose lipids, HMW-adiponectin and blood pressure (the latter was assessed in each trimester of gestation). At birth, newborns and placentas were weighed. FASN expression was also able to be assessed in 80 placentas. Higher circulating FASN was associated with lower systolic blood pressure (SBP), with a more favourable metabolic phenotype (lower fasting glucose and insulin, post load glucose, HbAc1, HOMA-IR and C-peptide), and with lower placental and birth weight (all p < 0.05 to p < 0.001). Placental FASN expression related positively to circulating FASN (p < 0.005) and negatively to placental weight (p < 0.05). Our observations suggest a physiological role of placental FASN in human pregnancy. Future studies will clarify whether circulating FASN of placental origin does actually regulate placental and fetal growth, and (thereby) has a favourable influence on the pregnant mother's insulin sensitivity and blood pressure.

  2. {alpha}-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

    SciTech Connect

    Lee, Young; Naseem, R. Haris; Park, Byung-Hyun; Garry, Daniel J.; Richardson, James A.; Schaffer, Jean E.; Unger, Roger H. . E-mail: roger.unger@utsouthwestern.edu

    2006-05-26

    {alpha}-Lipoic acid ({alpha}-LA) mimics the hypothalamic actions of leptin on food intake, energy expenditure, and activation of AMP-activated protein kinase (AMPK). To determine if, like leptin, {alpha}-LA protects against cardiac lipotoxicity, {alpha}-LA was fed to transgenic mice with cardiomyocyte-specific overexpression of the acyl CoA synthase (ACS) gene. Untreated ACS-transgenic mice died prematurely with increased triacylglycerol content and dilated cardiomyopathy, impaired systolic function and myofiber disorganization, apoptosis, and interstitial fibrosis on microscopy. In {alpha}-LA-treated ACS-transgenic mice heart size, echocardiogram and TG content were normal. Plasma TG fell 50%, hepatic-activated phospho-AMPK rose 6-fold, sterol regulatory element-binding protein-1c declined 50%, and peroxisome proliferator-activated receptor-{gamma} cofactor-1{alpha} mRNA rose 4-fold. Since food restriction did not prevent lipotoxicity, we conclude that {alpha}-LA treatment, like hyperleptinemia, protects the heart of ACS-transgenic mice from lipotoxicity.

  3. Influence of Different Levels of Lipoic Acid Synthase Gene Expression on Diabetic Nephropathy

    PubMed Central

    Xu, Longquan; Hiller, Sylvia; Simington, Stephen; Nickeleit, Volker; Maeda, Nobuyo; James, Leighton R.; Yi, Xianwen

    2016-01-01

    Oxidative stress is implicated in the pathogenesis of diabetic nephropathy (DN) but outcomes of many clinical trials are controversial. To define the role of antioxidants in kidney protection during the development of diabetic nephropathy, we have generated a novel genetic antioxidant mouse model with over- or under-expression of lipoic acid synthase gene (Lias). These models have been mated with Ins2Akita/+ mice, a type I diabetic mouse model. We compare the major pathologic changes and oxidative stress status in two new strains of the mice with controls. Our results show that Ins2Akita/+ mice with under-expressed Lias gene, exhibit higher oxidative stress and more severe DN features (albuminuria, glomerular basement membrane thickening and mesangial matrix expansion). In contrast, Ins2Akita/+ mice with highly-expressed Lias gene display lower oxidative stress and less DN pathologic changes. Our study demonstrates that strengthening endogenous antioxidant capacity could be an effective strategy for prevention and treatment of DN. PMID:27706190

  4. Extracellular Fatty Acid Synthase: A Possible Surrogate Biomarker of Insulin Resistance

    PubMed Central

    Fernandez-Real, Jose Manuel; Menendez, Javier A.; Moreno-Navarrete, Jose Maria; Blüher, Matthias; Vazquez-Martin, Alejandro; Vázquez, María Jesús; Ortega, Francisco; Diéguez, Carlos; Frühbeck, Gema; Ricart, Wifredo; Vidal-Puig, Antonio

    2010-01-01

    CONTEXT Circulating fatty acid synthase (FASN) is a biomarker of metabolically demanding human diseases. The aim of this study was to determine whether circulating FASN could be a biomarker of overnutrition-induced metabolic stress and insulin resistance in common metabolic disorders. RESEARCH DESIGN AND METHODS Circulating FASN was evaluated in two cross-sectional studies in association with insulin sensitivity and in four longitudinal studies investigating the effect of diet- and surgery-induced weight loss, physical training, and adipose tissue expansion using peroxisome proliferator–activated receptor agonist rosiglitazone on circulating FASN. RESULTS Age- and BMI-adjusted FASN concentrations were significantly increased in association with obesity-induced insulin resistance in two independent cohorts. Both visceral and subcutaneous FASN expression and protein levels correlated inversely with extracellular circulating FASN (P = −0.63; P < 0.0001), suggesting that circulating FASN is linked to depletion of intracellular FASN. Improved insulin sensitivity induced by therapeutic strategies that decreased fat mass (diet induced, surgery induced, or physical training) all led to decreased FASN levels in blood (P values between 0.02 and 0.04). To discriminate whether this was an effect related to insulin sensitization, we also investigated the effects of rosiglitazone. Rosiglitazone did not lead to significant changes in circulating FASN concentration. CONCLUSIONS Our results suggest that circulating FASN is a biomarker of overnutrition-induced insulin resistance that could provide diagnostic and prognostic advantages by providing insights on the individualized metabolic stress. PMID:20299470

  5. Inhibitory effect of emodin on fatty acid synthase, colon cancer proliferation and apoptosis.

    PubMed

    Lee, Kyung Ha; Lee, Myung Sun; Cha, Eun Young; Sul, Ji Young; Lee, Jin Sun; Kim, Jin Su; Park, Jun Beom; Kim, Ji Yeon

    2017-04-01

    Fatty acid synthase (FASN) is a key anabolic enzyme for de novo fatty acid synthesis, which is important in the development of colon carcinoma. The high expression of FASN is considered a promising molecular target for colon cancer therapy. Emodin, a naturally occurring anthraquinone, exhibits an anticancer effect in various types of human cancer, including colon cancer; however, the molecular mechanisms remain to be fully elucidated. Cell viability was evaluated using a Cell Counting Kit‑8 assay. The apoptosis rate of cells was quantified via flow cytometry following Annexin V/propidium iodide staining. FASN activity was measured by monitoring oxidation of nicotinamide adenine dinucleotide phosphate at a wavelength of 340 nm, and intracellular free fatty acid levels were detected using a Free Fatty Acid Quantification kit. Western blot analysis and reverse transcription‑polymerase chain reaction were used to detect target gene and protein expression. The present study was performed to investigate whether the gene expression of FASN and its enzymatic activity are regulated by emodin in a human colon cancer cell line. Emodin markedly inhibited the proliferation of HCT116 cells and a higher protein level of FASN was expressed, compared with that in SW480, SNU-C2A or SNU‑C5 cells. Emodin significantly downregulated the protein expression of FASN in HCT116 cells, which was caused by protein degradation due to elevated protein ubiquitination. Emodin also inhibited intracellular FASN enzymatic activity and reduced the levels of intracellular free fatty acids. Emodin enhanced antiproliferation and apoptosis in a dose‑ and time‑dependent manner. The combined treatment of emodin and cerulenin, a commercial FASN inhibitor, had an additive effect on these activities. Palmitate, the final product of the FASN reaction, rescued emodin‑induced viability and apoptosis. In addition, emodin altered FASN‑involved signaling pathways, including phosphatidylinositol 3

  6. Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

    PubMed

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2016-02-01

    Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

  7. The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together.

    PubMed

    Lomakin, Ivan B; Xiong, Yong; Steitz, Thomas A

    2007-04-20

    In yeast, the whole metabolic pathway for making 16- and 18-carbon fatty acids is carried out by fatty acid synthase, a 2.6 megadalton molecular-weight macromolecular assembly containing six copies of all eight catalytic centers. We have determined its crystal structure, which illuminates how this enzyme is initially activated and then carries out multiple steps of synthesis in each of six sterically isolated reaction chambers. Six of the catalytic sites are in the wall of the assembly facing an acyl carrier protein (ACP) bound to the ketoacyl synthase domain. Two-dimensional diffusion of substrates to the catalytic sites may be achieved by the electrostatically negative ACP swinging to each of the six electrostatically positive catalytic sites. The phosphopantetheinyl transferase domain lies outside the shell of the assembly, inaccessible to ACP that lies inside, suggesting that the attachment of the pantetheine arm to ACP must occur before complete assembly of the complex.

  8. Biosynthesis of biphenyls and benzophenones--evolution of benzoic acid-specific type III polyketide synthases in plants.

    PubMed

    Beerhues, Ludger; Liu, Benye

    2009-01-01

    Type III polyketide synthases (PKSs) generate a diverse array of secondary metabolites by varying the starter substrate, the number of condensation reactions, and the mechanism of ring closure. Among the starter substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the formation of identical linear tetraketide intermediates from benzoyl-CoA and three molecules of malonyl-CoA but use alternative intramolecular cyclization reactions to form 3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS and BPS group together closely, indicating that they arise from a relatively recent functional diversification of a common ancestral gene. The functionally diverse PKSs, which include BIS and BPS, and the ubiquitously distributed chalcone synthases (CHSs) form separate clusters, which originate from a gene duplication event prior to the speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism. Biphenyls and the related dibenzofurans are the phytoalexins of the Maloideae. This subfamily of the Rosaceae includes a number of economically important fruit trees, such as apple and pear. When incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known anticoagulant derivative of this enzymatic product is dicoumarol. Elicitor-treated cell cultures of Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized benzophenone derivatives. BPS was converted into a functional phenylpyrone synthase (PPS) by a single amino acid substitution in the initiation/elongation cavity. The

  9. Fatty acid synthase regulates estrogen receptor-α signaling in breast cancer cells

    PubMed Central

    Menendez, J A; Lupu, R

    2017-01-01

    Fatty acid synthase (FASN), the key enzyme for endogenous synthesis of fatty acids, is overexpressed and hyperactivated in a biologically aggressive subset of sex steroid-related tumors, including breast carcinomas. Using pharmacological and genetic approaches, we assessed the molecular relationship between FASN signaling and estrogen receptor alpha (ERα) signaling in breast cancer. The small compound C75, a synthetic slow-binding inhibitor of FASN activity, induced a dramatic augmentation of estradiol (E2)-stimulated, ERα-driven transcription. FASN and ERα were both necessary for the synergistic activation of ERα transcriptional activity that occurred following co-exposure to C75 and E2: first, knockdown of FASN expression using RNAi (RNA interference) drastically lowered (>100 fold) the amount of E2 required for optimal activation of ERα-mediated transcriptional activity; second, FASN blockade synergistically increased E2-stimulated ERα-mediated transcriptional activity in ERα-negative breast cancer cells stably transfected with ERα, but not in ERα-negative parental cells. Non-genomic, E2-regulated cross-talk between the ERα and MAPK pathways participated in these phenomena. Thus, treatment with the pure antiestrogen ICI 182 780 or the potent and specific inhibitor of MEK/ERK, U0126, was sufficient to abolish the synergistic nature of the interaction between FASN blockade and E2-stimulated ERα transactivation. FASN inhibition suppressed E2-stimulated breast cancer cell proliferation and anchorage-independent colony formation while promoting the reduction of ERα protein. FASN blockade resulted in the increased expression and nuclear accumulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, two critical mediators of the therapeutic effects of antiestrogen in breast cancer, while inactivating AKT, a key mediator of E2-promoted anchorage-independent growth. The ability of FASN to regulate E2/ERα signaling may represent a

  10. Tissue Localization of a Submergence-Induced 1-Aminocyclopropane-1-Carboxylic Acid Synthase in Rice1

    PubMed Central

    Zhou, Zhongyi; de Almeida Engler, Janice; Rouan, Dominique; Michiels, Frank; Van Montagu, Marc; Van Der Straeten, Dominique

    2002-01-01

    At least two 1-aminocyclopropane-1-carboxylic acid synthase genes (ACS) are implicated in the submergence response of rice (Oryza sativa). Previously, the OS-ACS5 gene has been shown to be induced during short- as well as long-term complete submergence of seedlings and to be controlled by a balance of gibberellin and abscisic acid in both lowland and deepwater rice. This study demonstrates that OS-ACS5 mRNA is localized in specific tissues and cells both during normal development and in response to complete submergence. The temporal and spatial regulation of OS-ACS5 expression is presented by in situ hybridization and histochemical analysis of β-glucuronidase (GUS) activity in transgenic rice carrying an OS-ACS5-gus fusion. Whole-mount in situ hybridization revealed that in air-grown rice seedlings, OS-ACS5 was expressed at a low level in the shoot apex, meristems, leaf, and adventitious root primordia, and in vascular tissues of nonelongated stems and leaf sheaths. In response to complete submergence, the expression in vascular bundles of young stems and leaf sheaths was strongly induced. The results of histochemical GUS assays were consistent with those found by whole-mount in situ hybridization. Our findings suggest that OS-ACS5 plays a role in vegetative growth of rice under normal conditions and is also recruited for enhanced growth upon complete submergence. The possible implication of OS-ACS5 in root-shoot communication during submergence stress and its putative role in aerenchyma formation upon low-oxygen stress are discussed. PMID:12011339

  11. Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress.

    PubMed

    Groves, Jennifer A; Maduka, Austin O; O'Meally, Robert N; Cole, Robert N; Zachara, Natasha E

    2017-02-23

    The dynamic post-translational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins and this increase is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc respectively, are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc levels in U2OS cells, but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination with Stable Isotope Labeling of Amino Acids in Cell culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70 kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (≈85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation. While the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation.

  12. Diet-induced alteration of fatty acid synthase in prostate cancer progression

    PubMed Central

    Huang, M; Koizumi, A; Narita, S; Inoue, T; Tsuchiya, N; Nakanishi, H; Numakura, K; Tsuruta, H; Saito, M; Satoh, S; Nanjo, H; Sasaki, T; Habuchi, T

    2016-01-01

    Fatty acid synthase (FASN) is a cytosolic metabolic enzyme that catalyzes de novo fatty acid synthesis. A high-fat diet (HFD) is attributed to prostate cancer (PCa) progression, but the role FASN on HFD-mediated PCa progression remains unclear. We investigated the role of FASN on PCa progression in LNCaP xenograft mice fed with HFD or low-fat diet (LFD), in PCa cells, and in clinical PCa. The HFD promoted tumour growth and FASN expression in the LNCaP xenograft mice. HFD resulted in AKT and extracellular signal-regulated kinase (ERK) activation and 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation. Serum FASN levels were significantly lower in the HFD group (P=0.026) and correlated inversely with tumour volume (P=0.022). Extracellular FASN release was enhanced in the PCa cells with phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinase (MAPK) inhibition and AMPK signalling activation. FASN inhibition resulted in decrease of PCa cell proliferation through PI3K/MAPK downregulation and AMPK activation. Furthermore, AMPK activation was associated with FASN downregulation and PI3K/MAPK inactivation. Clinically, high FASN expression was significantly associated with high Gleason scores and advanced pathological T stage. Moreover, FASN expression was markedly decreased in the PCa response to androgen deprivation therapy and chemotherapy. HFD modulates FASN expression, which may be an important mechanism in HFD-associated PCa progression. Furthermore, a critical stimulatory loop exists between FASN and the PI3K/MAPK system, whereas AMPK signalling was associated with suppression. These may offer appropriate targets for chemoprevention and cancer therapy in HFD-induced PCa. PMID:26878389

  13. Inhibition of Mycobacterium tuberculosis dihydrodipicolinate synthase by alpha-ketopimelic acid and its other structural analogues

    PubMed Central

    Shrivastava, Priyanka; Navratna, Vikas; Silla, Yumnam; Dewangan, Rikeshwer P.; Pramanik, Atreyi; Chaudhary, Sarika; Rayasam, GeethaVani; Kumar, Anuradha; Gopal, Balasubramanian; Ramachandran, Srinivasan

    2016-01-01

    The Mycobacterium tuberculosis dihydrodipicolinate synthase (Mtb-dapA) is an essential gene. Mtb-DapA catalyzes the aldol condensation between pyruvate and L-aspartate-beta-semialdehyde (ASA) to yield dihydrodipicolinate. In this work we tested the inhibitory effects of structural analogues of pyruvate on recombinant Mtb-DapA (Mtb-rDapA) using a coupled assay with recombinant dihydrodipicolinate reductase (Mtb-rDapB). Alpha-ketopimelic acid (α-KPA) showed maximum inhibition of 88% and IC50 of 21 μM in the presence of pyruvate (500 μM) and ASA (400 μM). Competition experiments with pyruvate and ASA revealed competition of α-KPA with pyruvate. Liquid chromatography-mass spectrometry (LC-MS) data with multiple reaction monitoring (MRM) showed that the relative abundance peak of final product, 2,3,4,5-tetrahydrodipicolinate, was decreased by 50%. Thermal shift assays showed 1 °C Tm shift of Mtb-rDapA upon binding α-KPA. The 2.4 Å crystal structure of Mtb-rDapA-α-KPA complex showed the interaction of critical residues at the active site with α-KPA. Molecular dynamics simulations over 500 ns of pyruvate docked to Mtb-DapA and of α-KPA-bound Mtb-rDapA revealed formation of hydrogen bonds with pyruvate throughout in contrast to α-KPA. Molecular descriptors analysis showed that ligands with polar surface area of 91.7 Å2 are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA. PMID:27501775

  14. The 3-hydroxyacyl-ACP dehydratase component of the plant mitochondrial fatty acid synthase system.

    PubMed

    Guan, Xin; Okazaki, Yozo; Lithio, Andrew; Li, Ling; Zhao, Xuefeng; Jin, Huanan; Nettleton, Dan; Saito, Kazuki; Nikolau, Basil J

    2017-02-15

    We report the characterization of the Arabidopsis 3-hydroxyacyl-acyl carrier protein (ACP) dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein (GFP) transgenesis experiment, and by in vivo complementation and in vitro enzymatic assays. RNAi knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA-Seq analysis revealed that mthd-rnai and mtkas mutants are near equivalent to each other in altering transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system, but independent of photorespiratory deficiency. These data demonstrate the non-redundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism.

  15. Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation1[W][OPEN

    PubMed Central

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation. PMID:24204024

  16. Cloning and Functional Analysis of a beta-amyrin synthase gene associated with oleanolic acid biosynthesis in Gentiana straminea MAXIM.

    PubMed

    Liu, Yanling; Cai, Yunfei; Zhao, Zhongjuan; Wang, Junfeng; Li, Jing; Xin, Wei; Xia, Guangmin; Xiang, Fengning

    2009-05-01

    Phytosterols and triterpenes are synthesized by oxidosqualene cyclases (OSCs) via the isoprenoid pathway. Here, GsAS1--a full-length beta-amyrin synthase cDNA isolated from Gentiana straminea MAXIM.--was characterized. Its open reading frame consists of 2268 bp, predicted to encode a 756 residue protein containing four QW and one Asp-Cys-Thr-Ala-Glu (DCTAE) motifs, which are both well conserved among known triterpene synthases. The deduced GsAS1 peptide sequence shares 76.2% homology with that of Panax ginseng beta-amyrin synthase. A phylogenetic analysis showed that GsAS1 is closely related to other plant OSCs, and particularly to the beta-amyrin synthases. When the GsAS1 sequence was heterologously expressed in Escherichia coli, an 88 kDa gene product was produced, and this reacted with the appropriate antibody. The sequence was also heterologously expressed in the Pichia pastoris yeast. GsAS1 is expressed in a tissue-specific manner, with its expression in the leaf being ca. 4.5-fold than that in the root, and nearly three-fold than that in the stem. GsAS1 expression was up-regulated by treatment with methyl jasmonate (MeJA) over a period from 6 h to 10 d post treatment. The accumulation oleanolic acid increased after induction by MeJA.

  17. Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases1[W][OA

    PubMed Central

    Hall, Dawn E.; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L.; Yuen, Macaire; Bohlmann, Jörg

    2013-01-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  18. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    PubMed

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  19. Expression of the Rhodobacter sphaeroides hemA and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes.

    PubMed Central

    Neidle, E L; Kaplan, S

    1993-01-01

    The nucleotide sequences of the Rhodobacter sphaeroides hemA and hemT genes, encoding 5-aminolevulinic acid (ALA) synthase isozymes, were determined. ALA synthase catalyzes the condensation of glycine and succinyl coenzyme A, the first and rate-limiting step in tetrapyrrole biosynthesis. The hemA and hemT structural gene sequences were 65% identical to each other, and the deduced HemA and HemT polypeptide sequences were 53% identical, with an additional 16% of aligned amino acids being similar. HemA and HemT were homologous to all characterized ALA synthases, including two human ALA synthase isozymes. In addition, they were evolutionarily related to 7-keto-8-aminopelargonic acid synthetase (BioF) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), enzymes which catalyze similar reactions. Two hemA transcripts were identified, both expressed under photosynthetic conditions at levels approximately three times higher than those found under aerobic conditions. A single transcriptional start point was identified for both transcripts, and a consensus sequence at this location indicated that an Fnr-like protein may be involved in the transcriptional regulation of hemA. Transcription of hemT was not detected in wild-type cells under the physiological growth conditions tested. In a mutant strain in which the hemA gene had been inactivated, however, hemT was expressed. In this mutant, hemT transcripts were characterized by Northern (RNA) hybridization, primer extension, and ribonuclease protection techniques. A small open reading frame of unknown function was identified upstream of, and transcribed in the same direction as, hemA. Images PMID:8468290

  20. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

  1. Inhibitory effects of grape skin extract and resveratrol on fatty acid synthase.

    PubMed

    Liang, Yan; Tian, Weixi; Ma, Xiaofeng

    2013-12-16

    Grape skin, a rich source of phytochemicals, has been reported to possess remarkable anti-obesity activity. Fatty acid synthase (FAS) is a key enzyme catalyzing the synthesis of fatty acid de novo, and has been considered as an anti-obesity target. To elucidate the anti-obesity mechanism of grape skin, we investigated the effects of grape skin extract (GSE) and resveratrol, one of the phytochemicals in GSE, on FAS and FAS over-expressed 3 T3-L1 preadipocyte. Purified FAS was obtained from chicken liver. Dried grape skin was extracted by 50% ethanol and partitioned by ethyl acetate. Inhibitory effects of GSE and resveratrol on FAS including fast-binding inhibition, time-dependent inhibition, and enzyme kinetics were determined. Inhibitory effects of GSE and resveratrol on 3T3-L1 preadipocyte were also measured. GSE inhibited the overall reaction and β-ketoacyl reductase (KR) reaction of FAS with IC50 values of 4.61 μg/ml and 20.3 μg/ml. For inhibition by resveratrol, the relevant IC50 values were 11.1 μg/ml and 21.9 μg/ml, respectively. And both GSE and resveratrol showed time-dependent inhibition for FAS, with the kobs values of 0.028 min-1, and 0.040 min-1 respectively. They inhibited the overall reaction of FAS competitively with acetyl-CoA, noncompetitively with malonyl-CoA and in a mixed manner with NADPH. Moreover, the inhibition on KR domain by resveratrol was time-dependent with kobs value of 0.106 min-1. In 3 T3-L1 preadipocytes, resveratrol reduced lipid accumulation remarkably. GSE and resveratrol are potent FAS inhibitors and they bound reversibly to the KR domain of FAS to inhibit the reduction of the saturated acyl groups in fatty acid synthesis. Based on the valid data and deliberate analysis, we proposed that GSE and resveratrol have great medical potential and officinal value in treating obesity and related diseases.

  2. Inactivation of fatty acid synthase impairs hepatocarcinogenesis driven by AKT in mice and humans

    PubMed Central

    Li, Lei; Pilo, Giulia M.; Li, Xiaolei; Cigliano, Antonio; Latte, Gavinella; Che, Li; Joseph, Christy; Mela, Marta; Wang, Chunmei; Jiang, Lijie; Ribback, Silvia; Simile, Maria M.; Pascale, Rosa M.; Dombrowski, Frank; Evert, Matthias; Semenkovich, Clay F.; Chen, Xin; Calvisi, Diego F.

    2015-01-01

    Background & Aims Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. Results Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway. PMID:26476289

  3. Fatty acid synthase as a potential therapeutic target in feline oral squamous cell carcinoma.

    PubMed

    Walz, J Z; Saha, J; Arora, A; Khammanivong, A; O'Sullivan, M G; Dickerson, E B

    2017-09-04

    Oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy in both feline and human patients. Recent work has demonstrated aberrant expression of fatty acid synthase (FASN) and an increased capacity for lipogenesis in human OSCC and other cancers. In human OSCC, inhibition of FASN decreased cell viability and growth in vitro, and diminished tumour growth and metastasis in murine preclinical models. This study aimed to characterize FASN as a therapeutic target in feline OSCC. Immunohistochemistry revealed high FASN expression in primary feline OSCC tumours, and FASN expression was detected in OSCC cell lines (3 feline and 3 human) by immunoblotting and quantitative real-time-polymerase chain reaction (qRT-PCR). Orlistat, a FASN inhibitor, substantially reduced cell viability in both feline and human OSCC lines, although feline cell lines consistently displayed higher sensitivity to the drug. FASN mRNA expression among cell lines mirrored sensitivity to orlistat, with feline cell lines expressing higher levels of FASN. Consistent with this observation, diminished sensitivity to orlistat treatment and decreased FASN mRNA expression were observed in feline OSCC cells following incubation under hypoxic conditions. Treatment with orlistat did not potentiate sensitivity to carboplatin in the cell lines investigated; instead, combinations of the 2 drugs resulted in additive to antagonistic effects. Our results suggest that FASN inhibition is a viable therapeutic target for feline OSCC. Furthermore, cats may serve as a spontaneous large animal model for human oral cancer, although differences in the regulation of lipogenesis between these 2 species require further investigation. © 2017 John Wiley & Sons Ltd.

  4. Fatty Acid Synthase Polymorphisms, Tumor Expression, Body Mass Index, Prostate Cancer Risk, and Survival

    PubMed Central

    Nguyen, Paul L.; Ma, Jing; Chavarro, Jorge E.; Freedman, Matthew L.; Lis, Rosina; Fedele, Giuseppe; Fiore, Christopher; Qiu, Weiliang; Fiorentino, Michelangelo; Finn, Stephen; Penney, Kathryn L.; Eisenstein, Anna; Schumacher, Fredrick R.; Mucci, Lorelei A.; Stampfer, Meir J.; Giovannucci, Edward; Loda, Massimo

    2010-01-01

    Purpose Fatty acid synthase (FASN) regulates de novo lipogenesis, body weight, and tumor growth. We examined whether common germline single nucleotide polymorphisms (SNPs) in the FASN gene affect prostate cancer (PCa) risk or PCa-specific mortality and whether these effects vary by body mass index (BMI). Methods In a prospective nested case-control study of 1,331 white patients with PCa and 1,267 age-matched controls, we examined associations of five common SNPs within FASN (and 5 kb upstream/downstream, R2 > 0.8) with PCa incidence and, among patients, PCa-specific death and tested for an interaction with BMI. Survival analyses were repeated for tumor FASN expression (n = 909). Results Four of the five SNPs were associated with lethal PCa. SNP rs1127678 was significantly related to higher BMI and interacted with BMI for both PCa risk (Pinteraction = .004) and PCa mortality (Pinteraction = .056). Among overweight men (BMI ≥ 25 kg/m2), but not leaner men, the homozygous variant allele carried a relative risk of advanced PCa of 2.49 (95% CI, 1.00 to 6.23) compared with lean men with the wild type. Overweight patients carrying the variant allele had a 2.04 (95% CI, 1.31 to 3.17) times higher risk of PCa mortality. Similarly, overweight patients with elevated tumor FASN expression had a 2.73 (95% CI, 1.05 to 7.08) times higher risk of lethal PCa (Pinteraction = .02). Conclusion FASN germline polymorphisms were significantly associated with risk of lethal PCa. Significant interactions of BMI with FASN polymorphisms and FASN tumor expression suggest FASN as a potential link between obesity and poor PCa outcome and raise the possibility that FASN inhibition could reduce PCa-specific mortality, particularly in overweight men. PMID:20679621

  5. Cancer cell-associated fatty acid synthase activates endothelial cells and promotes angiogenesis in colorectal cancer

    PubMed Central

    Evers, B.Mark

    2014-01-01

    Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC–EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in ‘normalization’ of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF189, upregulation of antiangiogenic isoform VEGF165b and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. PMID:24510238

  6. Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids.

    PubMed

    Larsen, L N; Dahl, E; Bremer, J

    1996-01-05

    Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein to dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 . 10(3) at 502 nm, the oxidation of less than 1 microM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, gamma-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n-3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.

  7. All trans retinoic acid depresses the content and activity of the mitochondrial ATP synthase in human keratinocytes.

    PubMed

    Papa, F; Lippolis, R; Sardaro, N; Gnoni, A; Scacco, S

    2017-01-08

    Proteomic analysis shows that treatment of keratinocytes cultures with all trans retinoic acid (ATRA), under condition in which it inhibits cell growth, results in marked decrease of the level of the F1-β subunit of the catalytic sector of the mitochondrial FoF1 ATP synthase complex. Enzymatic analysis shows in ATRA-treated keratinocytes a consistent depression of the ATPase activity, with decreased olygomycin sensitivity, indicating an overall alteration of the ATP synthase complex. These findings, together with the previously reported inhibition of respiratory complex I, show that depression of the activity of oxidative phosphorylation enzymes is involved in the cell growth inhibitory action of ATRA. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Fatty acid synthase cooperates with glyoxalase 1 to protect against sugar toxicity.

    PubMed

    Garrido, Damien; Rubin, Thomas; Poidevin, Mickael; Maroni, Brigitte; Le Rouzic, Arnaud; Parvy, Jean-Philippe; Montagne, Jacques

    2015-02-01

    Fatty acid (FA) metabolism is deregulated in several human diseases including metabolic syndrome, type 2 diabetes and cancers. Therefore, FA-metabolic enzymes are potential targets for drug therapy, although the consequence of these treatments must be precisely evaluated at the organismal and cellular levels. In healthy organism, synthesis of triacylglycerols (TAGs)-composed of three FA units esterified to a glycerol backbone-is increased in response to dietary sugar. Saturation in the storage and synthesis capacity of TAGs is associated with type 2 diabetes progression. Sugar toxicity likely depends on advanced-glycation-end-products (AGEs) that form through covalent bounding between amine groups and carbonyl groups of sugar or their derivatives α-oxoaldehydes. Methylglyoxal (MG) is a highly reactive α-oxoaldehyde that is derived from glycolysis through a non-enzymatic reaction. Glyoxalase 1 (Glo1) works to neutralize MG, reducing its deleterious effects. Here, we have used the power of Drosophila genetics to generate Fatty acid synthase (FASN) mutants, allowing us to investigate the consequence of this deficiency upon sugar-supplemented diets. We found that FASN mutants are lethal but can be rescued by an appropriate lipid diet. Rescued animals do not exhibit insulin resistance, are dramatically sensitive to dietary sugar and accumulate AGEs. We show that FASN and Glo1 cooperate at systemic and cell-autonomous levels to protect against sugar toxicity. We observed that the size of FASN mutant cells decreases as dietary sucrose increases. Genetic interactions at the cell-autonomous level, where glycolytic enzymes or Glo1 were manipulated in FASN mutant cells, revealed that this sugar-dependent size reduction is a direct consequence of MG-derived-AGE accumulation. In summary, our findings indicate that FASN is dispensable for cell growth if extracellular lipids are available. In contrast, FA-synthesis appears to be required to limit a cell-autonomous accumulation

  9. Fatty Acid Synthase Cooperates with Glyoxalase 1 to Protect against Sugar Toxicity

    PubMed Central

    Garrido, Damien; Rubin, Thomas; Poidevin, Mickael; Maroni, Brigitte; Le Rouzic, Arnaud; Parvy, Jean-Philippe; Montagne, Jacques

    2015-01-01

    Fatty acid (FA) metabolism is deregulated in several human diseases including metabolic syndrome, type 2 diabetes and cancers. Therefore, FA-metabolic enzymes are potential targets for drug therapy, although the consequence of these treatments must be precisely evaluated at the organismal and cellular levels. In healthy organism, synthesis of triacylglycerols (TAGs)—composed of three FA units esterified to a glycerol backbone—is increased in response to dietary sugar. Saturation in the storage and synthesis capacity of TAGs is associated with type 2 diabetes progression. Sugar toxicity likely depends on advanced-glycation-end-products (AGEs) that form through covalent bounding between amine groups and carbonyl groups of sugar or their derivatives α-oxoaldehydes. Methylglyoxal (MG) is a highly reactive α-oxoaldehyde that is derived from glycolysis through a non-enzymatic reaction. Glyoxalase 1 (Glo1) works to neutralize MG, reducing its deleterious effects. Here, we have used the power of Drosophila genetics to generate Fatty acid synthase (FASN) mutants, allowing us to investigate the consequence of this deficiency upon sugar-supplemented diets. We found that FASN mutants are lethal but can be rescued by an appropriate lipid diet. Rescued animals do not exhibit insulin resistance, are dramatically sensitive to dietary sugar and accumulate AGEs. We show that FASN and Glo1 cooperate at systemic and cell-autonomous levels to protect against sugar toxicity. We observed that the size of FASN mutant cells decreases as dietary sucrose increases. Genetic interactions at the cell-autonomous level, where glycolytic enzymes or Glo1 were manipulated in FASN mutant cells, revealed that this sugar-dependent size reduction is a direct consequence of MG-derived-AGE accumulation. In summary, our findings indicate that FASN is dispensable for cell growth if extracellular lipids are available. In contrast, FA-synthesis appears to be required to limit a cell

  10. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

    PubMed

    Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

    2014-09-01

    Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

  11. The stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the α-protons of amino acids

    PubMed Central

    2004-01-01

    13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen–deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine at pH 7.8. 1H-NMR has also been used to follow the tryptophan-synthase-catalysed hydrogen–deuterium exchange of the α-protons of a range of L- and D-amino acids at pH 7.8. The pKa values of the α-protons of these amino acids have been estimated and we have determined whether or not their exchange rates can be predicted from their pKa values. With the exception of tryptophan and norleucine, the stereospecificities of the first-order α-proton exchange rates are independent of the size and electronegativity of the amino acid R-group. Similar results are obtained with the second-order α-proton exchange rates, except that both L-tryptophan and L-serine have much higher stereospecificities than all the other amino acids studied. PMID:15107013

  12. From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis[W][OA

    PubMed Central

    de Kraker, Jan-Willem; Gershenzon, Jonathan

    2011-01-01

    Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the kcat/Km for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism. PMID:21205930

  13. Role of exogenously supplied ferulic and p-coumaric acids in mimicking the mode of action of acetolactate synthase inhibiting herbicides.

    PubMed

    Orcaray, Luis; Igal, María; Zabalza, Ana; Royuela, Mercedes

    2011-09-28

    Chlorsulfuron and imazethapyr (herbicides that inhibit acetolactate synthase; ALS, EC 4.1.3.18) produced a strong accumulation of hydroxycinnamic acids that was related to the induction of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 2.5.2.54). The exogenous application of two hydroxycinnamic acids, ferulic and p-coumaric acids, to pea plants resulted in their internal accumulation, arrested growth, carbohydrate and quinate accumulation in the leaves, and the induction of ethanolic fermentation. These effects resemble some of the physiological effects detected after acetolactate synthase inhibition and suggest important roles for ferulic and p-coumaric acids in the mode of action of herbicides inhibiting the biosynthesis of branched chain amino acids.

  14. Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.

    PubMed Central

    Chang, A K; Duggleby, R G

    1998-01-01

    Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. PMID:9677339

  15. The beta subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies.

    PubMed

    Peña, P; Garesse, R

    1993-09-15

    The cDNA encoding the Drosophila melanogaster beta subunit of H+ ATP synthase has been cloned and sequenced. The predicted mature protein is highly homologous to the equivalent beta subunits of other organisms and is preceded by a signal peptide of 31 amino acids, that although not conserved at primary sequence level has the characteristics of leader peptides present in other mitochondrial proteins. We have raised polyclonal antibodies that specifically recognize the beta H+ ATP synthase subunit present in Drosophila melanogaster protein extracts. This is the first time that a gene of the ATP synthase complex has been characterized in the invertebrate phyla.

  16. Mutation of L-2,3-diaminopropionic acid synthase genes blocks staphyloferrin B synthesis in Staphylococcus aureus

    PubMed Central

    2011-01-01

    Background Staphylococcus aureus synthesizes two siderophores, staphyloferrin A and staphyloferrin B, that promote iron-restricted growth. Previous work on the biosynthesis of staphyloferrin B has focused on the role of the synthetase enzymes, encoded from within the sbnA-I operon, which build the siderophore from the precursor molecules citrate, alpha-ketoglutarate and L-2,3-diaminopropionic acid. However, no information yet exists on several other enzymes, expressed from the biosynthetic cluster, that are thought to be involved in the synthesis of the precursors (or synthetase substrates) themselves. Results Using mutants carrying insertions in sbnA and sbnB, we show that these two genes are essential for the synthesis of staphyloferrin B, and that supplementation of the growth medium with L-2,3-diaminopropionic acid can bypass the block in staphyloferrin B synthesis displayed by the mutants. Several mechanisms are proposed for how the enzymes SbnA, with similarity to cysteine synthase enzymes, and SbnB, with similarity to amino acid dehydrogenases and ornithine cyclodeaminases, function together in the synthesis of this unusual nonproteinogenic amino acid L-2,3-diaminopropionic acid. Conclusions Mutation of either sbnA or sbnB result in abrogation of synthesis of staphyloferrin B, a siderophore that contributes to iron-restricted growth of S. aureus. The loss of staphyloferrin B synthesis is due to an inability to synthesize the unusual amino acid L-2,3-diaminopropionic acid which is an important, iron-liganding component of the siderophore structure. It is proposed that SbnA and SbnB function together as an L-Dap synthase in the S. aureus cell. PMID:21906287

  17. Fatty Acid Synthase: A Metabolic Enzyme and Candidate Oncogene in Prostate Cancer

    PubMed Central

    Migita, Toshiro; Ruiz, Stacey; Fornari, Alessandro; Fiorentino, Michelangelo; Priolo, Carmen; Zadra, Giorgia; Inazuka, Fumika; Grisanzio, Chiara; Palescandolo, Emanuele; Shin, Eyoung; Fiore, Christopher; Xie, Wanling; Kung, Andrew L.; Febbo, Phillip G.; Subramanian, Aravind; Mucci, Lorelei; Ma, Jing; Signoretti, Sabina; Stampfer, Meir; Hahn, William C.; Finn, Stephen

    2009-01-01

    Background Overexpression of the fatty acid synthase (FASN) gene has been implicated in prostate carcinogenesis. We sought to directly assess the oncogenic potential of FASN. Methods We used immortalized human prostate epithelial cells (iPrECs), androgen receptor–overexpressing iPrECs (AR-iPrEC), and human prostate adenocarcinoma LNCaP cells that stably overexpressed FASN for cell proliferation assays, soft agar assays, and tests of tumor formation in immunodeficient mice. Transgenic mice expressing FASN in the prostate were generated to assess the effects of FASN on prostate histology. Apoptosis was evaluated by Hoechst 33342 staining and by fluorescence-activated cell sorting in iPrEC-FASN cells treated with stimulators of the intrinsic and extrinsic pathways of apoptosis (ie, camptothecin and anti-Fas antibody, respectively) or with a small interfering RNA (siRNA) targeting FASN. FASN expression was compared with the apoptotic index assessed by the terminal deoxynucleotidyltransferase-mediated UTP end-labeling method in 745 human prostate cancer samples by using the least squares means procedure. All statistical tests were two-sided. Results Forced expression of FASN in iPrECs, AR-iPrECs, and LNCaP cells increased cell proliferation and soft agar growth. iPrECs that expressed both FASN and androgen receptor (AR) formed invasive adenocarcinomas in immunodeficient mice (12 of 14 mice injected formed tumors vs 0 of 14 mice injected with AR-iPrEC expressing empty vector (P < .001, Fisher exact test); however, iPrECs that expressed only FASN did not. Transgenic expression of FASN in mice resulted in prostate intraepithelial neoplasia, the incidence of which increased from 10% in 8- to 16-week-old mice to 44% in mice aged 7 months or more (P  = .0028, Fisher exact test), but not in invasive tumors. In LNCaP cells, siRNA-mediated silencing of FASN resulted in apoptosis. FASN overexpression protected iPrECs from apoptosis induced by camptothecin but did not

  18. Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker

    PubMed Central

    Liu, Hailan; Liu, Jing-Yuan; Wu, Xi; Zhang, Jian-Ting

    2010-01-01

    Human fatty acid synthase (FASN) is a 270-kDa cytosolic dimeric enzyme that is responsible for palmitate synthesis. FASN is slowly emerging and rediscovered as a marker for diagnosis and prognosis of human cancers. Recent studies showed that FASN is an oncogene and inhibition of FASN effectively and selectively kill cancer cells. With recent publications of the FASN crystal structure and the new development of FASN inhibitors, targeting FASN opens a new window of opportunity for metabolically combating cancers. In this article, we will review critically the recent progresses in understanding the structure, function, and the role of FASN in cancers and pharmacologically targeting FASN for human cancer treatment. PMID:20706604

  19. A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids.

    PubMed

    Zhu, Mingzhao; Harshbarger, Wayne D; Robles, Omar; Krysiak, Joanna; Hull, Kenneth G; Cho, Sung Wook; Richardson, Robyn D; Yang, Yanyan; Garcia, Andres; Spiegelman, Lindsey; Ramirez, Bianca; Wilson, Christopher T; Yau, Ju Anne; Moore, James T; Walker, Caitlen B; Sacchettini, James C; Liu, Wenshe R; Sieber, Stephan A; Smith, Jeffrey W; Romo, Daniel

    2017-06-01

    The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, β-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding.

    PubMed Central

    Ahmad, P M; Feltman, D S; Ahmad, F

    1982-01-01

    A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4'-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity. PMID:7103949

  1. Novel and potent inhibitors of fatty acid synthase derived from catechins and their inhibition on MCF-7 cells.

    PubMed

    Zhang, Shu-Yan; Ma, Xiao-Feng; Zheng, Chao-Gu; Wang, Yan; Cao, Xue-Li; Tian, Wei-Xi

    2009-06-01

    Fatty acid synthase (FAS) is a potential target for cancer, but potent inhibitors against FAS are scarce. In this study, we found that activities of catechins on inhibiting FAS increased greatly by heating them in acid. The enhancement was positively correlated to H(+) concentration. The inhibitory activities of the final products from different catechins were similar, all of which were less than 1 microg/mL. The product from (-)-epigallocatechin gallate (EGCG) was stable at room temperature, and its inhibitory kinetics and reacting sites on FAS were obviously different from the known FAS inhibitors. It also affected the viability of MCF-7 cells more obviously than EGCG. A putative route of the reaction progress was proposed and the effective inhibitors were deduced to be oligomers of 2-hydroxy-3-(3', 4', 5'-trihydroxyphenyl) propenoic acid by analysis of their spectra. The work affords new and potent FAS inhibitors that would be promising candidates for the treatment of cancer.

  2. Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

    PubMed Central

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

    2012-01-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

  3. The Mechanism of Hsp90 ATPase Stimulation by Aha1

    PubMed Central

    Wolmarans, Annemarie; Lee, Brian; Spyracopoulos, Leo; LaPointe, Paul

    2016-01-01

    Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer. PMID:27615124

  4. A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes.

    PubMed

    Welsch, Ralf; Wüst, Florian; Bär, Cornelia; Al-Babili, Salim; Beyer, Peter

    2008-05-01

    We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

  5. A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes1[W

    PubMed Central

    Welsch, Ralf; Wüst, Florian; Bär, Cornelia; Al-Babili, Salim; Beyer, Peter

    2008-01-01

    We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5′ untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1. PMID:18326788

  6. Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I

    SciTech Connect

    Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik Shivaji; Grininger, Martin

    2015-10-23

    Bacterial and fungal type I fatty-acid synthases (FAS I) are evolutionarily connected, as bacterial FAS I is considered to be the ancestor of fungal FAS I. In this work, the production, crystallization and X-ray diffraction data analysis of a bacterial FAS I are reported. While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.

  7. Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells.

    PubMed

    Jenik, R A; Fisch, J E; Goodridge, A G

    1987-11-01

    The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with 3H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the "lipogenic" enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.

  8. Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

    SciTech Connect

    Mayer, R.; Ross, P.; Weinhouse, H.; Amikam, D.; Volman, G.; Ohana, P.; Benziman, M. ); Calhoon, R.D.; Wong, Hing C.; Emerick, A.W. )

    1991-06-15

    To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

  9. Commercial Herbicides Can Trigger the Oxidative Inactivation of Acetohydroxyacid Synthase.

    PubMed

    Lonhienne, Thierry; Nouwens, Amanda; Williams, Craig M; Fraser, James A; Lee, Yu-Ting; West, Nicholas P; Guddat, Luke W

    2016-03-18

    Acetohydroxyacid synthase (AHAS) inhibitors are highly successful commercial herbicides. New kinetic data show that the binding of these compounds leads to reversible accumulative inhibition of AHAS. Crystallographic data (to a resolution of 2.17 Å) for an AHAS-herbicide complex shows that closure of the active site occurs when the herbicidal inhibitor binds, thus preventing exchange with solvent. This feature combined with new kinetic data shows that molecular oxygen promotes an accumulative inhibition leading to the conclusion that the exceptional potency of these herbicides is augmented by subversion of an inherent oxygenase side reaction. The reactive oxygen species produced by this reaction are trapped in the active site, triggering oxidation reactions that ultimately lead to the alteration of the redox state of the cofactor flavin adenine dinucleotide (FAD), a feature that accounts for the observed reversible accumulative inhibition.

  10. Structure-guided Discovery of Phenyl diketo-acids as Potent Inhibitors of M. tuberculosis Malate Synthase

    PubMed Central

    Krieger, Inna V.; Freundlich, Joel S.; Gawandi, Vijay B.; Roberts, Justin P.; Gawandi, Vidyadhar B.; Sun, Qingan; Owen, Joshua L.; Fraile, Maria T.; Huss, Sofia I.; Lavandera, Jose-Luis; Ioerger, Thomas R.; Sacchettini, James C.

    2012-01-01

    Summary The glyoxylate shunt plays an important role in fatty-acid metabolism, and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of novel phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and over-expression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors were used to guide optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics. PMID:23261599

  11. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    SciTech Connect

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  12. Knockdown of Pyruvate Carboxylase or Fatty Acid Synthase Lowers Numerous Lipids and Glucose-Stimulated Insulin Release in Insulinoma Cells

    PubMed Central

    MacDonald, Michael J.; Hasan, Noaman M.; Dobrzyn, Agnieszka; Stoker, Scott W.; Ntambi, James M.; Liu, Xueqing; Sampath, Harini

    2013-01-01

    We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20–30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated insulin secretion. PMID:23357280

  13. The structure of coral allene oxide synthase reveals a catalase adapted for metabolism of a fatty acid hydroperoxide

    PubMed Central

    Oldham, Michael L.; Brash, Alan R.; Newcomer, Marcia E.

    2005-01-01

    8R-Lipoxygenase and allene oxide synthase (AOS) are parts of a naturally occurring fusion protein from the coral Plexaura homomalla. AOS catalyses the production of an unstable epoxide (an allene oxide) from the fatty acid hydroperoxide generated by the lipoxygenase activity. Here, we report the structure of the AOS domain and its striking structural homology to catalase. Whereas nominal sequence identity between the enzymes had been previously described, the extent of structural homology observed was not anticipated, given that this enzyme activity had been exclusively associated with the P450 superfamily, and conservation of a catalase fold without catalase activity is unprecedented. Whereas the heme environment is largely conserved, the AOS heme is planar and the distal histidine is flanked by two hydrogen-bonding residues. These critical differences likely facilitate the switch from a catalatic activity to that of a fatty acid hydroperoxidase. PMID:15625113

  14. The structure of coral allene oxide synthase reveals a catalase adapted for metabolism of a fatty acid hydroperoxide.

    PubMed

    Oldham, Michael L; Brash, Alan R; Newcomer, Marcia E

    2005-01-11

    8R-Lipoxygenase and allene oxide synthase (AOS) are parts of a naturally occurring fusion protein from the coral Plexaura homomalla. AOS catalyses the production of an unstable epoxide (an allene oxide) from the fatty acid hydroperoxide generated by the lipoxygenase activity. Here, we report the structure of the AOS domain and its striking structural homology to catalase. Whereas nominal sequence identity between the enzymes had been previously described, the extent of structural homology observed was not anticipated, given that this enzyme activity had been exclusively associated with the P450 superfamily, and conservation of a catalase fold without catalase activity is unprecedented. Whereas the heme environment is largely conserved, the AOS heme is planar and the distal histidine is flanked by two hydrogen-bonding residues. These critical differences likely facilitate the switch from a catalatic activity to that of a fatty acid hydroperoxidase.

  15. Use of structure-based drug design approaches to obtain novel anthranilic acid acyl carrier protein synthase inhibitors.

    PubMed

    Joseph-McCarthy, Diane; Parris, Kevin; Huang, Adrian; Failli, Amedeo; Quagliato, Dominick; Dushin, Elizabeth Glasfeld; Novikova, Elena; Severina, Elena; Tuckman, Margareta; Petersen, Peter J; Dean, Charles; Fritz, Christian C; Meshulam, Tova; DeCenzo, Maureen; Dick, Larry; McFadyen, Iain J; Somers, William S; Lovering, Frank; Gilbert, Adam M

    2005-12-15

    Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.

  16. Individualized supplementation of folic acid according to polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) reduced pregnant complications.

    PubMed

    Li, Xiujuan; Jiang, Jing; Xu, Min; Xu, Mei; Yang, Yan; Lu, Wei; Yu, Xuemei; Ma, Jianlin; Pan, Jiakui

    2015-01-01

    This study aimed to detect the genotype distributions and allele frequencies of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine synthase reductase (MTRR) A66G polymorphisms of pregnant women in Jiaodong region in China, and to investigate whether folic acid supplementation affect the pregnancy complications. A total of 7,812 pregnant women from the Jiaodong region in Shandong province in China. By using Taqman-MGB, 2,928 pregnant women (case group) were tested for the genotype distributions and allele frequencies of MTHFR C677T, A1298C and MTRR A66G polymorphisms. Folic acid metabolism ability was ranked at four levels and then pregnant women in different rank group were supplemented with different doses of folic acid. Their pregnancy complications were followed up and compared with 4,884 pregnant women without folic acid supplementation (control group) in the same hospital. The allele frequencies of MTHFR C677T were 49.1 and 50.9%; those of MTHFR A1298C were 80.2 and 19.8%, and those of MTRR A66G were 74.1 and 25.9%. After supplemented with folic acid, the complication rates in different age groups were significantly reduced, especially for gestational diabetes mellitus and hypertension. Periconceptional folic acid supplementation and healthcare following gene polymorphism testing may be a powerful measure to decrease congenital malformations. © 2015 S. Karger AG, Basel.

  17. Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression.

    PubMed

    Goodridge, A G; Jenik, R A; McDevitt, M A; Morris, S M; Winberry, L K

    1984-04-01

    Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.

  18. Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A Randomized Controlled Trial. Addendum

    DTIC Science & Technology

    2011-07-01

    acids ( PUFA ), particularly omega -3 fatty acids , inhibits SREBP-1 activation, resulting in a decreased transcription of FAS. 15. SUBJECT TERMS Prostate...Cancer; Lipid Metabolism; Clinical Trial; Omega -3 Fatty Acids 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...controls, Menendez et al demonstrated that addition of omega -3 fatty acids (-3 FA), docosahexanoic acid (DHA), alpha- linolenic acid

  19. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  20. Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme.

    PubMed

    Li, N; Mattoo, A K

    1994-03-04

    1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

  1. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    SciTech Connect

    Hopperton, Kathryn E.; Duncan, Robin E.; Bazinet, Richard P.; Archer, Michael C.

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  2. An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea

    PubMed Central

    Liu, Yanling; Zhao, Zhongjuan; Xue, Zheyong; Wang, Long; Cai, Yunfei; Wang, Peng; Wei, Tiandi; Gong, Jing; Liu, Zhenhua; Li, Juan; Li, Shuo; Xiang, Fengning

    2016-01-01

    Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1. PMID:27624821

  3. An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea.

    PubMed

    Liu, Yanling; Zhao, Zhongjuan; Xue, Zheyong; Wang, Long; Cai, Yunfei; Wang, Peng; Wei, Tiandi; Gong, Jing; Liu, Zhenhua; Li, Juan; Li, Shuo; Xiang, Fengning

    2016-09-14

    Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1.

  4. The fatty acid synthase inhibitor cerulenin and feeding, like leptin, activate hypothalamic pro-opiomelanocortin (POMC) neurons.

    PubMed

    Shu, I-Wei; Lindenberg, Daniel L; Mizuno, Tooru M; Roberts, James L; Mobbs, Charles V

    2003-09-19

    Hypothalamic POMC neurons mediate catabolic responses such as decreased food intake and increased energy expenditure by, in part, monitoring levels of metabolic factors such as glucose, insulin and leptin. Recently, fatty acid synthase inhibitors were reported to reduce body weight, inhibit food intake, and increase metabolic rate, possibly by acting on hypothalamic neurons through a mechanism involving malonyl-CoA accumulation. Given the observation that leptin mediates similar catabolic effects by, in part, activating hypothalamic POMC neurons, it is possible that other catabolic signals such as feeding and fatty acid synthase inhibition may also activate POMC neurons. To test this hypothesis, hypothalamic sections from mice that were fed or injected with the fatty acid synthase inhibitor cerulenin were examined for Fos (a marker for neuronal activation) and POMC product immunoreactivity and compared with similarly processed sections from leptin-injected mice. Feeding increased Fos immunoreactivity in the lateral peri-arcuate area of the hypothalamus of both wild-type and leptin-deficient ob/ob mice (P<0.05), indicating that nutritional activation of the hypothalamus can be leptin-independent. Furthermore, feeding significantly induced Fos immunoreactivity in neurons expressing POMC (P<0.003), indicating that feeding, like leptin, activates POMC neurons. Injection with cerulenin, like feeding and leptin, also increased Fos immunoreactivity in the lateral peri-arcuate area (P<0.03) and, more specifically, in neurons expressing POMC. In contrast, injection with cerulenin had no grossly observable effects on cortical Fos immunoreactivity and appeared to suppress fasting-induced Fos immunoreactivity by about 35% (although the decrease did not reach statistical significance) in the medial arcuate nucleus, an area associated with anabolic responses such as increased food intake. Injection with cerulenin also decreased Fos immunoreactivity in the granular layer of the

  5. Phytotoxicity of Acetohydroxyacid Synthase Inhibitors Is Not Due to Accumulation of 2-Ketobutyrate and/or 2-Aminobutyrate.

    PubMed Central

    Shaner, D. L.; Singh, B. K.

    1993-01-01

    Acetohydroxyacid synthase (AHAS) is the site of action of herbicides of different chemical classes, such as imidazolinones, sulfonylureas, and triazolopyrimidines. Inhibition of AHAS causes the accumulation of 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) (the transamination product of 2-KB), and it has been proposed that the phytotoxicity of these inhibitors is due to this accumulation. Experiments were done to determine the relationship between accumulation of 2-KB and 2-AB and the phytotoxicity of imazaquin to maize (Zea mays). Imazaquin concentrations that inhibit growth of maize plants also cause the accumulation of 2-KB and 2-AB in the shoots. Supplementation of imazaquin-treated plants with isoleucine reduced the pools of 2-KB and 2-AB in the plant but did not protect plants from the growth inhibitory effects of imazaquin. Conversely, feeding 2-AB to maize plants increased 2-KB and 2-AB pools to much higher levels than those observed in imazaquin-treated plants, yet such high pools of 2-KB and 2-AB in the plant had no significant effect on growth. These results conclusively demonstrate that growth inhibition following imazaquin treatment is not due to accumulation of 2-KB and/or 2-AB in plants. Changes in the amino acid profiles after treatment with imazaquin suggest that starvation for the branched-chain amino acids may be the primary cause of growth retardation of maize. PMID:12232015

  6. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

    PubMed Central

    Paradkar, A S; Jensen, S E

    1995-01-01

    A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon. PMID:7868606

  7. Reduced alpha-lipoic acid synthase gene expression exacerbates atherosclerosis in diabetic apolipoprotein E-deficient mice.

    PubMed

    Yi, Xianwen; Xu, Longquan; Hiller, Sylvia; Kim, Hyung-Suk; Maeda, Nobuyo

    2012-07-01

    To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe(-/-)). Heterozygous lipoic acid synthase gene knockout mice (Lias(+/-)) crossed with Apoe(-/-) mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe(-/-) mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias(+/-)Apoe(-/-) mice. In the aortic lesion area, the Lias(+/-)Apoe(-/-) mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias(+/+)Apoe(-/-) littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta. Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

    PubMed Central

    Mayer, R; Ross, P; Weinhouse, H; Amikam, D; Volman, G; Ohana, P; Calhoon, R D; Wong, H C; Emerick, A W; Benziman, M

    1991-01-01

    To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature. Images PMID:1647035

  9. AHA Moments of Science and Mathematics Pre-Service Teachers

    ERIC Educational Resources Information Center

    Caniglia, Joanne C.; Borgerding, Lisa; Courtney, Scott

    2017-01-01

    This research study explored pre-service teachers' (PST) reflections of their student teaching experiences through AHA moments. Participants included 37 pre-service teachers enrolled in mathematics and science student teaching seminars. Qualitative methods were used to analyze PSTs' written and verbal responses to questions regarding AHA…

  10. AHA Moments of Science and Mathematics Pre-Service Teachers

    ERIC Educational Resources Information Center

    Caniglia, Joanne C.; Borgerding, Lisa; Courtney, Scott

    2017-01-01

    This research study explored pre-service teachers' (PST) reflections of their student teaching experiences through AHA moments. Participants included 37 pre-service teachers enrolled in mathematics and science student teaching seminars. Qualitative methods were used to analyze PSTs' written and verbal responses to questions regarding AHA…

  11. A key role of nuclear factor Y in the refeeding response of fatty acid synthase in adipocytes.

    PubMed

    Nishi-Tatsumi, Makiko; Yahagi, Naoya; Takeuchi, Yoshinori; Toya, Naoki; Takarada, Ayako; Murayama, Yuki; Aita, Yuichi; Sawada, Yoshikazu; Piao, Xiaoying; Oya, Yukari; Shikama, Akito; Masuda, Yukari; Kubota, Midori; Izumida, Yoshihiko; Matsuzaka, Takashi; Nakagawa, Yoshimi; Sekiya, Motohiro; Iizuka, Yoko; Kawakami, Yasushi; Kadowaki, Takashi; Yamada, Nobuhiro; Shimano, Hitoshi

    2017-03-09

    Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around -100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes. This article is protected by copyright. All rights reserved.

  12. Oncogene KRAS activates fatty acid synthase, resulting in specific ERK and lipid signatures associated with lung adenocarcinoma.

    PubMed

    Gouw, Arvin M; Eberlin, Livia S; Margulis, Katherine; Sullivan, Delaney K; Toal, Georgia G; Tong, Ling; Zare, Richard N; Felsher, Dean W

    2017-04-11

    KRAS gene mutation causes lung adenocarcinoma. KRAS activation has been associated with altered glucose and glutamine metabolism. Here, we show that KRAS activates lipogenesis, and this activation results in distinct proteomic and lipid signatures. By gene expression analysis, KRAS is shown to be associated with a lipogenesis gene signature and specific induction of fatty acid synthase (FASN). Through desorption electrospray ionization MS imaging (DESI-MSI), specific changes in lipogenesis and specific lipids are identified. By the nanoimmunoassay (NIA), KRAS is found to activate the protein ERK2, whereas ERK1 activation is found in non-KRAS-associated human lung tumors. The inhibition of FASN by cerulenin, a small molecule antibiotic, blocked cellular proliferation of KRAS-associated lung cancer cells. Hence, KRAS is associated with activation of ERK2, induction of FASN, and promotion of lipogenesis. FASN may be a unique target for KRAS-associated lung adenocarcinoma remediation.

  13. Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I

    PubMed Central

    Enderle, Mathias; McCarthy, Andrew; Paithankar, Karthik Shivaji; Grininger, Martin

    2015-01-01

    While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution. PMID:26527268

  14. Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

    PubMed

    Broadbent, J R; Oberg, T S; Hughes, J E; Ward, R E; Brighton, C; Welker, D L; Steele, J L

    2014-03-01

    Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.

  15. The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas

    PubMed Central

    Seguin, F; Carvalho, M A; Bastos, D C; Agostini, M; Zecchin, K G; Alvarez-Flores, M P; Chudzinski-Tavassi, A M; Coletta, R D; Graner, E

    2012-01-01

    Background: Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells. Methods: The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT–PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells. Results: B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA121, 165, 189, and 165b in SK-MEL-25 and SCC-9 cells. Conclusion: FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells. PMID:22892389

  16. In vitro evidence that D-serine disturbs the citric acid cycle through inhibition of citrate synthase activity in rat cerebral cortex.

    PubMed

    Zanatta, Angela; Schuck, Patrícia Fernanda; Viegas, Carolina Maso; Knebel, Lisiane Aurélio; Busanello, Estela Natacha Brandt; Moura, Alana Pimentel; Wajner, Moacir

    2009-11-17

    The present work investigated the in vitro effects of D-serine (D-Ser) on important parameters of energy metabolism in cerebral cortex of young rats. The parameters analyzed were CO(2) generation from glucose and acetate, glucose uptake and the activities of the respiratory chain complexes I-IV, of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase and of creatine kinase and Na(+),K(+)-ATPase. Our results show that D-Ser significantly reduced CO(2) production from acetate, but not from glucose, reflecting an impairment of the citric acid cycle function. Furthermore, D-Ser did not affect glucose uptake. We also observed that the activity of the mitochondrial enzyme citrate synthase from mitochondrial preparations and purified citrate synthase was significantly inhibited by D-Ser, whereas the other activities of the citric acid cycle as well as the activities of complexes I-III, II-III, II and IV of the respiratory chain, creatine kinase and Na(+),K(+)-ATPase were not affected by this D-amino acid. We also found that L-serine did not affect citrate synthase activity from mitochondrial preparations and purified enzyme. The data indicate that D-Ser impairs the citric acid cycle activity via citrate synthase inhibition, therefore compromising energy metabolism production in cerebral cortex of young rats. Therefore, it is presumed that this mechanism may be involved at least in part in the neurological damage found in patients affected by disorders in which D-Ser metabolism is impaired, with altered cerebral concentrations of this D-amino acid.

  17. Design and structure-activity relationships of potent and selective inhibitors of undecaprenyl pyrophosphate synthase (UPPS): tetramic, tetronic acids and dihydropyridin-2-ones.

    PubMed

    Peukert, Stefan; Sun, Yingchuan; Zhang, Rui; Hurley, Brian; Sabio, Mike; Shen, Xiaoyu; Gray, Christen; Dzink-Fox, JoAnn; Tao, Jianshi; Cebula, Regina; Wattanasin, Sompong

    2008-03-15

    Based on a pharmacophore hypothesis substituted tetramic and tetronic acid 3-carboxamides as well as dihydropyridin-2-one-3-carboxamides were investigated as inhibitors of undecaprenyl pyrophosphate synthase (UPPS) for use as novel antimicrobial agents. Synthesis and structure-activity relationship patterns for this class of compounds are discussed. Selectivity data and antibacterial activities for selected compounds are provided.

  18. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  19. Associations of uric acid with polymorphisms in the delta-aminolevulinic acid dehydratase, vitamin D receptor, and nitric oxide synthase genes in Korean lead workers.

    PubMed

    Weaver, Virginia M; Schwartz, Brian S; Jaar, Bernard G; Ahn, Kyu-Dong; Todd, Andrew C; Lee, Sung-Soo; Kelsey, Karl T; Silbergeld, Ellen K; Lustberg, Mark E; Parsons, Patrick J; Wen, Jiayu; Lee, Byung-Kook

    2005-11-01

    Recent research suggests that uric acid may be nephrotoxic at lower levels than previously recognized and that it may be one mechanism for lead-related nephrotoxicity. Therefore, in understanding mechanisms for lead-related nephrotoxicity, it would be of value to determine whether genetic polymorphisms that are associated with renal outcomes in lead workers and/or modify associations between lead dose and renal function are also associated with uric acid and/or modify associations between lead dose and uric acid. We analyzed data on three such genetic polymorphisms: delta-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR). Mean (+/- SD) tibia, blood, and dimercaptosuccinic acid-chelatable lead levels were 37.2 +/- 40.4 microg/g bone mineral, 32.0+/- 15.0 g/dL, and 0.77+/- 0.86 microg/mg creatinine, respectively, in 798 current and former lead workers. Participants with the eNOSAsp allele had lower mean serum uric acid compared with those with the Glu/Glu genotype. Among older workers (age > or = median of 40.6 years), ALAD genotype modified associations between lead dose and uric acid levels. Higher lead dose was significantly associated with higher uric acid in workers with the ALAD1-1 genotype; associations were in the opposite direction in participants with the variant ALAD1-2 genotype. In contrast, higher tibia lead was associated with higher uric acid in those with the variant VDRB allele; however, modification was dependent on participants with the bb genotype and high tibia lead levels. We conclude that genetic polymorphisms may modify uric acid mediation of lead-related adverse renal effects.

  20. Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components.

    PubMed

    O'Hara, Paul; Slabas, Antoni R; Fawcett, Tony

    2007-05-01

    Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.

  1. Structural Characterization of the Mycobacterium tuberculosis Biotin Biosynthesis Enzymes 7,8-Diaminopelargonic Acid Synthase and Dethiobiotin Synthetase†,‡

    PubMed Central

    Dey, Sanghamitra; Lane, James M.; Lee, Richard E.; Rubin, Eric J.; Sacchettini, James C.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 Å and 1.85 Å. Superimposition of the DAPAS structures bound either to the SAM analog sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 Å, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb. PMID:20565114

  2. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

    PubMed

    Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

    2015-08-07

    Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

  3. Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis.

    PubMed

    Lange, Kerstin; Schmid, Andreas; Julsing, Mattijs K

    2016-09-10

    Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids.

  4. siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown.

    PubMed

    Randeria, Pratik S; Seeger, Mark A; Wang, Xiao-Qi; Wilson, Heather; Shipp, Desmond; Mirkin, Chad A; Paller, Amy S

    2015-05-05

    Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.

  5. Fatty acid synthase as a factor required for exercise-induced cognitive enhancement and dentate gyrus cellular proliferation.

    PubMed

    Chorna, Nataliya E; Santos-Soto, Iván J; Carballeira, Nestor M; Morales, Joan L; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis.

  6. Fatty Acid Synthase as a Factor Required for Exercise-Induced Cognitive Enhancement and Dentate Gyrus Cellular Proliferation

    PubMed Central

    Chorna, Nataliya E.; Santos-Soto, Iván J.; Carballeira, Nestor M.; Morales, Joan L.; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P.; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis. PMID:24223732

  7. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity.

    PubMed

    Hopperton, Kathryn E; Duncan, Robin E; Bazinet, Richard P; Archer, Michael C

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from (14)C-labeled acetate to those supplied exogenously as (14)C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2-3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells.

  8. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    PubMed

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro.

  9. Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

    PubMed Central

    Mafu, Sibongile; Karunanithi, Prema Sambandaswami; Palazzo, Teresa Ann; Harrod, Bronwyn Lee; Rodriguez, Selina Marakana; Mollhoff, Iris Natalie; O’Brien, Terrence Edward; Tong, Shen; Fiehn, Oliver; Tantillo, Dean J.; Bohlmann, Jörg; Zerbe, Philipp

    2017-01-01

    The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined “pseudolaratriene.” Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P. amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene. PMID:28096378

  10. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    PubMed Central

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  11. Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening, under salicylic acid and indole-3-acetic acid treatment, and in diseased fruit.

    PubMed

    Shi, Hai-Yan; Zhang, Yu-Xing

    2014-06-01

    In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.

  12. New Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Inhibitors, Nalidixic Acid Linked to Isatin Schiff Bases via Certain l-Amino Acid Bridges.

    PubMed

    Naglah, Ahmed M; Ahmed, Atallah F; Wen, Zhi-Hong; Al-Omar, Mohamed A; Amr, Abd El-Galil E; Kalmouch, Atef

    2016-04-15

    A series of new Schiff bases were synthesized by condensation of isatins with the nalidixic acid-l-amino acid hydrazides. Prior to hydrazide formation, a peptide linkage has been prepared via coupling of nalidixic acid with appropriate l-amino acid methyl esters to yield 3a-c. The chemical structures of the new Schiff bases (5b and 5d-h) were confirmed by means of IR, NMR, mass spectroscopic, and elemental analyses. The anti-inflammatory activity of these Schiff bases was evaluated via measurement of the expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells model. The Schiff bases exhibited significant dual inhibitory effect against the induction of the pro-inflammatory iNOS and COX-2 proteins with variable potencies. However, they strongly down-regulated the iNOS expression to the level of 16.5% ± 7.4%-42.2% ± 19.6% compared to the effect on COX-2 expression (<56.4% ± 3.1% inhibition) at the same concentration (10 μM). The higher iNOS inhibition activity of the tested Schiff bases, relative to that of COX-2, seems to be a reflection of the combined suppressive effects exerted by their nalidixic acid, isatins (4a-c), and l-amino acid moieties against iNOS expression. These synthesized nalidixic acid-l-amino acid-isatin conjugates can be regarded as a novel class of anti-inflammatory antibacterial agents.

  13. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  14. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  15. Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN) and, cycloxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis

    PubMed Central

    Goswami, Sudeshna; Sharma-Walia, Neelam

    2016-01-01

    The crosstalk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. In our previous study, we discovered Osteoprotegerin (OPG) as one of the cytokines heavily secreted by breast cancer cells. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to normal human mammary epithelial HMEC cells. OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer. Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assays highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of the COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells. Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and fatty acid synthase (FASN). FASN is a multifunctional enzyme involved in lipid biosynthesis. Immunofluorescence staining revealed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be crosstalk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Interestingly, knocking down OPG by CRISPR/Cas9 gene editing in breast cancer cells decreased FASN expression at the protein level. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 and COX-2 inhibitor celecoxib individually decreased the number of lipid bodies/cell, downregulated phosphorylation of ERK

  16. ACTIVATION OF VASCULAR ENDOTHELIAL NITRIC OXIDE SYNTHASE AND HEME OXYGENASE-1 EXPRESSION BY ELECTROPHILIC NITRO-FATTY ACIDS

    PubMed Central

    Khoo, Nicholas K.H.; Rudolph, Volker; Cole, Marsha P.; Golin-Bisello, Franca; Schopfer, Francisco J.; Woodcock, Steven R.; Batthyany, Carlos; Freeman, Bruce A.

    2010-01-01

    Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated byproducts of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yield electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO2) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO2 via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO2-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO2-FAs stimulated the phosphorylation of eNOS at Ser1179. These post-translational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO2-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders. PMID:19857569

  17. A Strategy for Dual Inhibition of the Proteasome and Fatty Acid Synthase with Belactosin C-Orlistat Hybrids

    PubMed Central

    Zhu, Mingzhao; Harshbarger, Wayne D.; Robles, Omar; Krysiak, Joanna; Hull, Kenneth G.; Cho, Sung Wook; Richardson, Robyn D.; Yang, Yanyan; Garcia, Andres; Spiegelman, Lindsey; Ramirez, Bianca; Wilson, Christopher T.; Yau, Ju Anne; Moore, James T.; Walker, Caitlen B.; Sacchettini, James C.; Liu, Wenshe; Sieber, Stephan A.; Smith, Jeffrey W.; Romo, Daniel

    2017-01-01

    The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, β-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and FAS-TE enabling a new approach for the development of drug-candidates with potential to overcome resistance. PMID:28236510

  18. Body mass index and risk of colorectal cancer according to fatty acid synthase expression in the nurses' health study.

    PubMed

    Kuchiba, Aya; Morikawa, Teppei; Yamauchi, Mai; Imamura, Yu; Liao, Xiaoyun; Chan, Andrew T; Meyerhardt, Jeffrey A; Giovannucci, Edward; Fuchs, Charles S; Ogino, Shuji

    2012-03-07

    Fatty acid synthase (FASN) plays an important role in energy metabolism of fatty acids and is overexpressed in some colon cancers. We investigated whether associations between body mass index (BMI) and risk of colorectal cancer varied according to FASN expression. During follow-up of 109,051 women in the ongoing prospective Nurses' Health Study, a total of 1351 incident colon and rectal cancers were diagnosed between 1986 and 2004. We constructed tissue microarrays of the available resected tumor samples (n = 536), and FASN expression was analyzed by immunohistochemistry. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazards regression models. All statistical tests were two-sided. High BMI was associated with an increased risk of FASN-negative (no or weak expression) colorectal cancer compared with normal BMI (high BMI [≥ 30 kg/m(2)], ie, obese vs normal BMI [18.5-22.9 kg/m(2)], HR = 2.25, 95% CI = 1.49 to 3.40, P(trend) < .001) but not with FASN-positive (moderate to strong expression) colorectal cancer. A statistically significant heterogeneity in colorectal cancer risks was observed between FASN-negative and FASN-positive tumors (P(heterogeneity) = .033). The age-adjusted incidence rates for FASN-positive and FASN-negative colorectal cancers were 10.9 and 7.1, respectively, per 100,000 person-years. This molecular pathological epidemiology study supports a role of energy metabolism in colorectal cancer pathogenesis.

  19. Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

    PubMed Central

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-01-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops. PMID:21948846

  20. Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase.

    PubMed

    Tian, Yong-Sheng; Xu, Jing; Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Peng, Ri-He; Yao, Quan-Hong

    2011-12-01

    A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

  1. Inhibitory effects of onion (Allium cepa L.) extract on proliferation of cancer cells and adipocytes via inhibiting fatty acid synthase.

    PubMed

    Wang, Yi; Tian, Wei-Xi; Ma, Xiao-Feng

    2012-01-01

    Onions (Allium cepa L.) are widely used in the food industry for its nutritional and aromatic properties. Our studies showed that ethyl acetate extract of onion (EEO) had potent inhibitory effects on animal fatty acid synthase (FAS), and could induce apoptosis in FAS over-expressing human breast cancer MDA-MB-231 cells. Furthermore, this apoptosis was accompanied by reduction of intracellular FAS activity and could be rescued by 25 mM or 50 mM exogenous palmitic acids, the final product of FAS catalyzed synthesis. These results suggest that the apoptosis induced by EEO occurs via inhibition of FAS. We also found that EEO could suppress lipid accumulation during the differentiation of 3T3-L1 adipocytes, which was also related to its inhibition of intracellular FAS activity. Since obesity is closely related to breast cancer and obese patients are at elevated risk of developing various cancers, these findings suggested that onion might be useful for preventing obesity-related malignancy.

  2. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    PubMed

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction.

  3. Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

    PubMed

    Dye, J F; Vause, S; Johnston, T; Clark, P; Firth, J A; D'Souza, S W; Sibley, C P; Glazier, J D

    2004-01-01

    We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low.

  4. The ketogenic diet component decanoic acid increases mitochondrial citrate synthase and complex I activity in neuronal cells.

    PubMed

    Hughes, Sean David; Kanabus, Marta; Anderson, Glenn; Hargreaves, Iain P; Rutherford, Tricia; O'Donnell, Maura; Cross, J Helen; Rahman, Shamima; Eaton, Simon; Heales, Simon J R

    2014-05-01

    The Ketogenic diet (KD) is an effective treatment with regards to treating pharmaco-resistant epilepsy. However, there are difficulties around compliance and tolerability. Consequently, there is a need for refined/simpler formulations that could replicate the efficacy of the KD. One of the proposed hypotheses is that the KD increases cellular mitochondrial content which results in elevation of the seizure threshold. Here, we have focussed on the medium-chain triglyceride form of the diet and the observation that plasma octanoic acid (C8) and decanoic acid (C10) levels are elevated in patients on the medium-chain triglyceride KD. Using a neuronal cell line (SH-SY5Y), we demonstrated that 250-μM C10, but not C8, caused, over a 6-day period, a marked increase in the mitochondrial enzyme, citrate synthase along with complex I activity and catalase activity. Increased mitochondrial number was also indicated by electron microscopy. C10 is a reported peroxisome proliferator activator receptor γ agonist, and the use of a peroxisome proliferator activator receptor γ antagonist was shown to prevent the C10-mediated increase in mitochondrial content and catalase. C10 may mimic the mitochondrial proliferation associated with the KD and raises the possibility that formulations based on this fatty acid could replace a more complex diet. We propose that decanoic acid (C10) results in increased mitochondrial number. Our data suggest that this may occur via the activation of the PPARγ receptor and its target genes involved in mitochondrial biogenesis. This finding could be of significant benefit to epilepsy patients who are currently on a strict ketogenic diet. Evidence that C10 on its own can modulate mitochondrial number raises the possibility that a simplified and less stringent C10-based diet could be developed.

  5. Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.).

    PubMed

    Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A; Schwab, Wilfried

    2011-08-01

    One of the most common types of modification of secondary metabolites is the acylation of oxygen- and nitrogen-containing substrates to produce esters and amides, respectively. Among the known acyltransferases, the members of the plant BAHD family are capable of acylating a wide variety of substrates. Two full-length acyltransferase cDNAs (LaAT1 and 2) were isolated from lavender flowers (Lavandula angustifolia L.) by reverse transcriptase-PCR using degenerate primers based on BAHD sequences. Recombinant LaAT1 exhibited a broad substrate tolerance accepting (hydroxy)cinnamoyl-CoAs as acyl donors and not only tyramine, tryptamine, phenylethylamine and anthranilic acid but also shikimic acid and 4-hydroxyphenyllactic acid as acceptors. Thus, LaLT1 forms esters and amides like its phylogenetic neighbors. In planta LaAT1 might be involved in the biosynthesis of rosmarinic acid, the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, a major constituent of lavender flowers. LaAT2 is one of three members of clade VI with unknown function.

  6. Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle.

    PubMed

    Traaseth, Nathaniel; Elfering, Sarah; Solien, Joseph; Haynes, Virginia; Giulivi, Cecilia

    2004-07-23

    An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either L-Arg (to sustain a mtNOS activity) or N(G)-monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K(0.5) of 0.16 microM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K(0.5) of 1.2 microM intramitochondrial free calcium was obtained, similar to the K(0.5) (0.9 microM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.

  7. Discovery and characterization of [(cyclopentyl)ethyl]benzoic acid inhibitors of microsomal prostaglandin E synthase-1.

    PubMed

    Partridge, Katherine M; Antonysamy, Stephen; Bhattachar, Shobha N; Chandrasekhar, Srinivasan; Fisher, Matthew J; Fretland, Adrian; Gooding, Karen; Harvey, Anita; Hughes, Norman E; Kuklish, Steven L; Luz, John G; Manninen, Peter R; McGee, James E; Mudra, Daniel R; Navarro, Antonio; Norman, Bryan H; Quimby, Steven J; Schiffler, Matthew A; Sloan, Ashley V; Warshawsky, Alan M; Weller, Jennifer M; York, Jeremy S; Yu, Xiao-Peng

    2017-03-15

    We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC80 of 24nM for inhibition of PGE2 formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC80 in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.

  8. Interactions between large and small subunits of different acetohydroxyacid synthase isozymes of Escherichia coli.

    PubMed

    Vyazmensky, Maria; Zherdev, Yuri; Slutzker, Alex; Belenky, Inna; Kryukov, Olga; Barak, Ze'ev; Chipman, David M

    2009-09-15

    The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

  9. Neuronal nitric oxide synthase modulation of intracellular Ca(2+) handling overrides fatty acid potentiation of cardiac inotropy in hypertensive rats.

    PubMed

    Jin, Chun Li; Yin, Ming Zhe; Paeng, Jin Chul; Ha, Seunggyun; Lee, Jeong Hoon; Jin, Peng; Jin, Chun Zi; Zhao, Zai Hao; Wang, Yue; Kang, Keon Wook; Leem, Chae Hun; Park, Jong-Wan; Kim, Sung Joon; Zhang, Yin Hua

    2017-05-22

    Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca(2+) homeostasis and contractility of healthy and diseased hearts. Here, we examined the effects of nNOS on fatty acid (FA) regulation of left ventricular (LV) myocyte contraction in sham and angiotensin II (Ang II)-induced hypertensive (HTN) rats. Our results showed that palmitic acid (PA, 100 μM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyltransferase-1 inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA potentiation of sarcomere shortening only in sham. PA increased nNOS-derived NO only in HTN. Inhibition of nNOS with S-methyl-L-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA potentiation of myocyte contraction in HTN. Mechanistically, PA increased intracellular Ca(2+) transient ([Ca(2+)]i) without changing Ca(2+) influx via L-type Ca(2+) channel (I-LTCC) and reduced myofilament Ca(2+) sensitivity in sham. nNOS inhibition increased [Ca(2+)]i, I-LTCC and reduced myofilament Ca(2+) sensitivity prior to PA supplementation; as such, normalized PA increment of [Ca(2+)]i. In HTN, PA reduced I-LTCC without affecting [Ca(2+)]i or myofilament Ca(2+) sensitivity. However, PA increased I-LTCC, [Ca(2+)]i and reduced myofilament Ca(2+) sensitivity following nNOS inhibition. Myocardial FA oxidation ((18)F-fluoro-6-thia-heptadecanoic acid, (18)F-FTHA) was comparable between groups, but nNOS inhibition increased it only in HTN. Collectively, PA increases myocyte contraction through stimulating [Ca(2+)]i and mitochondrial activity in healthy hearts. PA-dependent cardiac inotropy was limited by nNOS in HTN, predominantly due to its modulatory effect on [Ca(2+)]i handling.

  10. Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

    PubMed

    Rossato, Franco A; Zecchin, Karina G; La Guardia, Paolo G; Ortega, Rose M; Alberici, Luciane C; Costa, Rute A P; Catharino, Rodrigo R; Graner, Edgard; Castilho, Roger F; Vercesi, Aníbal E

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce

  11. Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses.

    PubMed

    Ohol, Yamini M; Wang, Zhaoti; Kemble, George; Duke, Gregory

    2015-01-01

    Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.

  12. Fatty Acid Synthase Inhibitors Induce Apoptosis in Non-Tumorigenic Melan-A Cells Associated with Inhibition of Mitochondrial Respiration

    PubMed Central

    Rossato, Franco A.; Zecchin, Karina G.; La Guardia, Paolo G.; Ortega, Rose M.; Alberici, Luciane C.; Costa, Rute A. P.; Catharino, Rodrigo R.; Graner, Edgard; Castilho, Roger F.; Vercesi, Aníbal E.

    2014-01-01

    The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat

  13. Effect of nitric oxide synthase inhibition on the exchange of glucose and fatty acids in human skeletal muscle

    PubMed Central

    2013-01-01

    Background The role of nitric oxide in controlling substrate metabolism in humans is incompletely understood. Methods The present study examined the effect of nitric oxide blockade on glucose uptake, and free fatty acid and lactate exchange in skeletal muscle of eight healthy young males. Exchange was determined by measurements of muscle perfusion by positron emission tomography and analysis of arterial and femoral venous plasma concentrations of glucose, fatty acids and lactate. The measurements were performed at rest and during exercise without (control) and with blockade of nitric oxide synthase (NOS) with NG-monomethyl-l-arginine (L-NMMA). Results Glucose uptake at rest was 0.40 ± 0.21 μmol/100 g/min and increased to 3.71 ± 2.53 μmol/100 g/min by acute one leg low intensity exercise (p < 0.01). Prior inhibition of NOS by L-NMMA did not affect glucose uptake, at rest or during exercise (0.40 ± 0.26 and 4.74 ± 2.69 μmol/100 g/min, respectively). In the control trial, there was a small release of free fatty acids from the limb at rest (−0.05 ± 0.09 μmol/100 g/min), whereas during inhibition of NOS, there was a small uptake of fatty acids (0.04 ± 0.05 μmol/100 g/min, p < 0.05). During exercise fatty acid uptake was increased to (0.89 ± 1.07 μmol/100 g/min), and there was a non-significant trend (p = 0.10) for an increased FFA uptake with NOS inhibition 1.23 ± 1.48 μmol/100 g/min) compared to the control condition. Arterial concentrations of all substrates and exchange of lactate over the limb at rest and during exercise remained unaltered during the two conditions. Conclusion In conclusion, inhibition of nitric oxide synthesis does not alter muscle glucose uptake during low intensity exercise, but affects free fatty acid exchange especially at rest, and may thus be involved in the modulation of energy metabolism in the human skeletal muscle. PMID:23773265

  14. Inhibition of Fatty Acid Synthase in Prostate Cancer by Orlistat, a Novel Therapeutic

    DTIC Science & Technology

    2007-11-01

    sphingosine) Ceramides ( ceramide ) Sterol lipids Sterols Cholesterol Steroids C18 steroids (estrogens) C19 steroids (androgens) C21 steroids (gluco...School of Medicine, December 2006, Targeting fatty acid synthesis in tumor cells. 3. W. Todd Lowther, Molecular and Cellular Biology of Lipids , Gordon...July 22-27, 2007 4. 10th International Conference on Bioactive Lipids in Cancer, Inflammation and Related Diseases, Montreal, Canada, September 16

  15. Fatty Acid Synthase Inhibitors Engage the Cell Death Program Through the Endoplasmic Reticulum

    DTIC Science & Technology

    2007-12-01

    Vitamin D3 Skin Fig. 4 Generation of isoprenoid, cholesterol, steroids and bile acids. Farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and...vitamin D3 in the skin . These metabolites exert important signaling functions by binding to families of nuclear receptors which regulate gene expression...cholesterol in the skin 607 exposed to sunlight (Fig. 4); however, individuals with 608 limited sun exposure may require dietary supplementation. 609

  16. Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

    DTIC Science & Technology

    2013-07-01

    be used for membrane biogenesis , protein acylation, synthesis of lipid signaling mediators, maintenance of the cellular redox state, and storage of...free their bound energy for use in other cellular tasks. This process, known as β- oxidation, occurs in the mitochondria . Acylglycerols are hydrolyzed...to free fatty acids which are translocated into the mitochondria by the tightly regulated enzyme carnitine o- palmitoyltransferase (CPT)-1. In a

  17. Thermal conversions of fatty acid peroxides to cyclopentenones: a biomimetic model for allene oxide synthase pathway.

    PubMed

    Mukhtarova, Lucia S; Mukhitova, Fakhima K; Grechkin, Alexander N

    2013-01-01

    The trimethylsilyl (TMS) peroxides of linoleic acid 9(S)-hydroperoxide (TMS or Me esters) were subjected to gas chromatography-mass spectrometry (GC-MS) analyses. The cyclopentenones, trans- and cis-10-oxo-11-phytoenoic acid (10-oxo-PEA, Me or TMS esters) were first time detected as the products of TMS-peroxide thermal conversions. The major products were ketodienes, epoxyalcohols, hemiacetals and decadienals. For further study of thermal cyclopentenone formation, 9(S)- or 13(S)-hydroperoxides of linoleic acid (Me esters) were sealed in ampoules and heated at 230 °C for 15 or 30 min. The products were separated by HPLC. The cyclopentenone fractions were collected and analyzed by GC-MS. Trans-10-oxo-PEA (Me) and 10-oxo-9(13)-PEA (Me) were formed during the thermal conversion of 9-hydroperoxide (Me ester). Similarly, the cyclopentenones trans-12-oxo-PEA (Me) and 12-oxo-9(13)-PEA (Me) were detected after the heating of 13-hydroperoxide (Me ester). Thermal formation of cyclopentenones can be considered as a biomimetic model of AOS pathway, providing new insights into the mechanisms of allene oxide formation and cyclization.

  18. Divergence of cuticular hydrocarbons in two sympatric grasshopper species and the evolution of fatty acid synthases and elongases across insects

    PubMed Central

    Finck, Jonas; Berdan, Emma L.; Mayer, Frieder; Ronacher, Bernhard; Geiselhardt, Sven

    2016-01-01

    Cuticular hydrocarbons (CHCs) play a major role in the evolution of reproductive isolation between insect species. The CHC profiles of two closely related sympatric grasshopper species, Chorthippus biguttulus and C. mollis, differ mainly in the position of the first methyl group in major methyl-branched CHCs. The position of methyl branches is determined either by a fatty acid synthase (FAS) or by elongases. Both protein families showed an expansion in insects. Interestingly, the FAS family showed several lineage-specific expansions, especially in insect orders with highly diverse methyl-branched CHC profiles. We found five putative FASs and 12 putative elongases in the reference transcriptomes for both species. A dN/dS test showed no evidence for positive selection acting on FASs and elongases in these grasshoppers. However, one candidate FAS showed species-specific transcriptional differences and may contribute to the shift of the methyl-branch position between the species. In addition, transcript levels of four elongases were expressed differentially between the sexes. Our study indicates that complex methyl-branched CHC profiles are linked to an expansion of FASs genes, but that species differences can also mediated at the transcriptional level. PMID:27677406

  19. Pharmacophore Modeling and Virtual Screening for Novel Acidic Inhibitors of Microsomal Prostaglandin E2 Synthase-1 (mPGES-1)

    PubMed Central

    2011-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes prostaglandin E2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 μM, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis. PMID:21466167

  20. Fatty acid synthase 2 contributes to diapause preparation in a beetle by regulating lipid accumulation and stress tolerance genes expression

    PubMed Central

    Tan, Qian-Qian; Liu, Wen; Zhu, Fen; Lei, Chao-Liang; Wang, Xiao-Ping

    2017-01-01

    Diapause, also known as dormancy, is a state of arrested development that allows insects to survive unfavorable environmental conditions. Diapause-destined insects store large amounts of fat when preparing for diapause. However, the extent to which these accumulated fat reserves influence diapause remains unclear. To address this question, we investigated the function of fatty acid synthase (FAS), which plays a central role in lipid synthesis, in stress tolerance, the duration of diapause preparation, and whether insects enter diapause or not. In diapause-destined adult female cabbage beetles, Colaphellus bowringi, FAS2 was more highly expressed than FAS1 at the peak stage of diapause preparation. FAS2 knockdown suppressed lipid accumulation and subsequently affected stress tolerance genes expression and water content. However, silencing FAS2 had no significant effects on the duration of diapause preparation or the incidence of diapause. FAS2 transcription was suppressed by juvenile hormone (JH) and the JH receptor methoprene-tolerant (Met). These results suggest that the absence of JH-Met induces FAS2 expression, thereby promoting lipid storage in diapause-destined female beetles. These results demonstrate that fat reserves regulate stress tolerance genes expression and water content, but have no significant effect on the duration of diapause preparation or the incidence of diapause. PMID:28071706

  1. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    SciTech Connect

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  2. Liver dominant expression of fatty acid synthase (FAS) gene in two chicken breeds during intramuscular-fat development.

    PubMed

    Cui, H X; Zheng, M Q; Liu, R R; Zhao, G P; Chen, J L; Wen, J

    2012-04-01

    Fatty acid synthase (FAS) is a key enzyme of lipogenesis. In this study, the FAS mRNA expression patterns were examined in three fat related tissues (liver, breast and thigh) at different developmental stages in two chicken breeds (Beijing-You, BJY and Arbor Acres broiler, AA). Results of the Real time-qPCR showed that the expression of FAS mRNA level in liver was significantly higher (P < 0.01) than that in breast and thigh in both two chicken breeds. Significant differences of FAS mRNA expression in liver were found between BJY and AA chickens during different developmental stages. After the contents of intramuscular-fat (IMF) and the liver fat were measured, the correlation analysis was performed. In liver, the FAS mRNA level was highly correlated with hepatic fat content (r = 0.891, P < 0.01 for BJY; r = 0.901, P < 0.01 for AA). On the contrary, the FAS expression level in both breast and thigh tissues were relatively low, stable and there was no correlation between the FAS mRNA level and IMF content in breast and thigh tissues of each breed. The results here can contribute to the knowledge on the developmental expression pattern of FAS mRNA and facilitate the further research on the molecular mechanism underlying IMF deposition in chicken.

  3. Divergent cyclooxygenase responses to fatty acid structure and peroxide level in fish and mammalian prostaglandin H synthases.

    PubMed

    Liu, Wen; Cao, Dazhe; Oh, Sungwhan F; Serhan, Charles N; Kulmacz, Richard J

    2006-06-01

    Prostanoid synthesis in mammalian tissues is regulated at the level of prostaglandin H synthase (PGHS) cyclooxygenase catalysis by the availability and structure of substrate fatty acid and the availability of peroxide activator. Two major PGHS isoforms, with distinct pathophysiological functions and catalytic regulation, have been characterized in mammals; a functionally homologous PGHS isoform pair has been cloned from an evolutionarily distant vertebrate, brook trout. The cyclooxygenase activities of recombinant brook trout PGHS-1 and -2 were characterized to test the generality of mammalian regulatory paradigms for substrate specificity, peroxide activation, and product shifting by aspirin. Both trout cyclooxygenases had much more restrictive substrate specificities than their mammalian counterparts, with pronounced discrimination toward arachidonate (20:4n-6) and against eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), the latter two prominent in trout tissue lipids. Aspirin treatment did not increase lipoxygenase-type catalysis by either trout enzyme. Both trout enzymes had higher requirements for peroxide activator than their mammalian counterparts, though the preferential peroxide activation of PGHS-2 over PGHS-1 seen in mammals was conserved in the fish enzymes. The divergence in cyclooxygenase characteristics between the trout and mammalian PGHS proteins may reflect accomodations to differences among vertebrates in tissue lipid composition and general redox state.

  4. Report of the American Heart Association (AHA) Scientific Sessions 2016, New Orleans.

    PubMed

    Amaki, Makoto; Konagai, Nao; Fujino, Masashi; Kawakami, Shouji; Nakao, Kazuhiro; Hasegawa, Takuya; Sugano, Yasuo; Tahara, Yoshio; Yasuda, Satoshi

    2016-12-22

    The American Heart Association (AHA) Scientific Sessions 2016 were held on November 12-16 at the Ernest N. Morial Convention Center, New Orleans, LA. This 5-day event featured cardiovascular clinical practice covering all aspects of basic, clinical, population, and translational content. One of the hot topics at AHA 2016 was precision medicine. The key presentations and highlights from the AHA Scientific Sessions 2016, including "precision medicine" as one of the hot topics, are herein reported.

  5. Coding and 3' non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme

    PubMed Central

    Reimold, Ursula; Kröger, Manfred; Kreuzaler, Fritz; Hahlbrock, Klaus

    1983-01-01

    The nucleotide sequence of an almost complete cDNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) has been determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A-rich stretch of the 5' non-coding region and the complete 3' non-coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N-terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent mol. wt. of the subunit of the purified enzyme. PMID:16453477

  6. The Effects of C75, an Inhibitor of Fatty Acid Synthase, on Sleep and Metabolism in Mice

    PubMed Central

    Pellinen, Jacob; Szentirmai, Éva

    2012-01-01

    Sleep is greatly affected by changes in metabolic state. A possible mechanism where energy-sensing and sleep-regulatory functions overlap is related to lipid metabolism. Fatty acid synthase (FAS) plays a central role in lipid metabolism as a key enzyme in the formation of long-chain fatty acids. We studied the effects of systemic administration of C75, an inhibitor of FAS, on sleep, behavioral activity and metabolic parameters in mice. Since the effects of C75 on feeding and metabolism are the opposite of ghrelin's and C75 suppresses ghrelin production, we also tested the role of ghrelin signaling in the actions of C75 by using ghrelin receptor knockout (KO) mice. After a transient increase in wakefulness, C75 elicited dose-dependent and long lasting inhibition of REMS, motor activity and feeding. Simultaneously, C75 significantly attenuated slow-wave activity of the electroencephalogram. Energy expenditure, body temperature and respiratory exchange ratio were suppressed. The diurnal rhythm of feeding was completely abolished by C75. There was significant correlation between the anorectic effects, the decrease in motor activity and the diminished energy expenditure after C75 injection. We found no significant difference between wild-type and ghrelin receptor KO mice in their sleep and metabolic responses to C75. The effects of C75 resemble to what was previously reported in association with visceral illness. Our findings suggest that sleep and metabolic effects of C75 in mice are independent of the ghrelin system and may be due to its aversive actions in mice. PMID:22348016

  7. Mice with heterozygous deficiency of lipoic acid synthase have an increased sensitivity to lipopolysaccharide-induced tissue injury

    PubMed Central

    Yi, Xianwen; Kim, Kuikwon; Yuan, Weiping; Xu, Longquan; Kim, Hyung-Suk; Homeister, Jonathon W.; Key, Nigel S.; Maeda, Nobuyo

    2009-01-01

    α-Lipoic acid (1, 2-dithiolane-3-pentanoic acid; LA), synthesized in mitochondria by LA synthase (Lias), is a potent antioxidant and a cofactor for metabolic enzyme complexes. In this study, we examined the effect of genetic reduction of LA synthesis on its antioxidant and anti-inflammatory properties using a model of LPS-induced inflammation in Lias+/– mice. The increase of plasma proinflammatory cytokine, TNF-α, and NF-κB at an early phase following LPS injection was greater in Lias+/– mice compared with Lias+/+ mice. The circulating blood white blood cell (WBC) and platelet counts dropped continuously during the initial 4 h. The counts subsequently recovered partially in Lias+/+ mice, but the recovery was impaired totally in Lias+/– mice. Administration of exogenous LA normalized the recovery of WBC counts in Lias+/– mice but not platelets. Enhanced neutrophil sequestration in the livers of Lias+/– mice was associated with increased hepatocyte injury and increased gene expression of growth-related oncogene, E-selectin, and VCAM-1 in the liver and/or lung. Lias gene expression in tissues was 50% of normal expression in Lias+/– mice and reduced further by LPS treatment. Decreased Lias expression was associated with diminished hepatic LA and tissue oxidative stress. Finally, Lias+/– mice displayed enhanced mortality when exposed to LPS-induced sepsis. These data demonstrate the importance of endogenously produced LA for preventing leukocyte accumulation and tissue injury that result from LPS-induced inflammation. PMID:18845616

  8. A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples.

    PubMed

    Cascini, Fidelia; Passerotti, Stella; Martello, Simona

    2012-04-10

    In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested.

  9. Regulation of fatty acid synthase (FAS) and apoptosis in estrogen-receptor positive and negative breast cancer cells by conjugated linoleic acids.

    PubMed

    Song, H-J; Sneddon, A A; Heys, S D; Wahle, K W J

    2012-12-01

    Conjugated linoleic acids (CLAs) are natural dairy food components that exhibit a unique body of potential health benefits in animals and man, including anti-cardiovascular disease and anti-cancer effects. Several studies have demonstrated that fatty acid synthase (FAS) levels (protein and mRNA) are over expressed in many carcinomas. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate genes involved in lipid metabolism, including FAS. Breast cancer cell lines, MCF-7 and MDA-MB-231 were treated with CLAs to investigate the regulation of SREBP-1c and FAS expression. In MDA-MB-231 cells, SREBP-1c and FAS were co-ordinately decreased by treatment with 25 μM CLA 9-11 and 10-12. In MCF-7 cells, the decrease in SREBP-1c and FAS expression was dependant on the concentration of CLA used. The data suggest a differential effect of CLAs on SREBP-1c and FAS in estrogen receptor-positive (MCF-7) compared to estrogen receptor-negative (MDA-MB-231) breast cancer cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Molecular characterization and expression analysis of GlHMGS, a gene encoding hydroxymethylglutaryl-CoA synthase from Ganoderma lucidum (Ling-zhi) in ganoderic acid biosynthesis pathway.

    PubMed

    Ren, Ang; Ouyang, Xiang; Shi, Liang; Jiang, Ai-Liang; Mu, Da-Shuai; Li, Meng-Jiao; Han, Qin; Zhao, Ming-Wen

    2013-03-01

    A hydroxymethylglutaryl-CoA synthase gene, designated as GlHMGS (GenBank accession No. JN391469) involved in ganoderic acid (GA) biosynthesis pathway was cloned from Ganoderma lucidum. The full-length cDNA of GlHMGS (GenBank accession No. JN391468) was found to contain an open reading frame of 1,413 bp encoding a polypeptide of 471 amino acid residues. The deduced amino acid sequence of GlHMGS shared high homology with other known hydroxymethylglutaryl-CoA synthase (HMGS) enzymes. In addition, functional complementation of GlHMGS in a mutant yeast strain YSC1021 lacking HMGS activity demonstrated that the cloned cDNA encodes a functional HMGS. A 1,561 bp promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. GlHMGS expression profile analysis revealed that salicylic acid, abscisic acid and methyl jasmonate up-regulated GlHMGS transcript levels over the control. Further expression analysis revealed that the developmental stage and carbon source had significant effects on GlHMGS transcript levels. GlHMGS expression peaked on day 16 before decreasing with prolonged culture time. The highest mRNA level was observed when the carbon source was maltose. Overexpression of GlHMGS enhanced GA content in G. lucidum. This study provides useful information for further studying this gene and on its function in the ganoderic acid biosynthetic pathway in G. lucidum.

  11. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  12. Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

    PubMed

    Selle, Kurt; Goh, Yong J; Johnson, Brant R; O'Flaherty, Sarah; Andersen, Joakim M; Barrangou, Rodolphe; Klaenhammer, Todd R

    2017-01-01

    Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

  13. Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

    PubMed Central

    Selle, Kurt; Goh, Yong J.; Johnson, Brant R.; O’Flaherty, Sarah; Andersen, Joakim M.; Barrangou, Rodolphe; Klaenhammer, Todd R.

    2017-01-01

    Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall. PMID:28443071

  14. Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

    PubMed

    Dezfulian, Mohammad H; Foreman, Curtis; Jalili, Espanta; Pal, Mrinal; Dhaliwal, Rajdeep K; Roberto, Don Karl A; Imre, Kathleen M; Kohalmi, Susanne E; Crosby, William L

    2017-04-07

    Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. Taken together the

  15. Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

    PubMed

    Gannoun-Zaki, Laila; Alibaud, Laeticia; Kremer, Laurent

    2013-01-01

    The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

  16. Tetra- and Pentacyclic Triterpene Acids from the Ancient Anti-inflammatory Remedy Frankincense as Inhibitors of Microsomal Prostaglandin E2 Synthase-1

    PubMed Central

    2014-01-01

    The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3–30 μM), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3α-acetoxy-8,24-dienetirucallic acid (6) and 3α-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 μM, each. Structure–activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 μM). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

  17. Structure of 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid synthase, a catalyst in the archaeal pathway for the biosynthesis of aromatic amino acids.

    PubMed

    Morar, Mariya; White, Robert H; Ealick, Steven E

    2007-09-18

    Genes responsible for the generation of 3-dehydroquinate (DHQ), an early metabolite in the established shikimic pathway of aromatic amino acid biosynthesis, are absent in most euryarchaeotes. Alternative gene products, Mj0400 and Mj1249, have been identified in Methanocaldococcus jannaschii as the enzymes involved in the synthesis of DHQ. 2-Amino-3,7-dideoxy-d-threo-hept-6-ulosonic acid (ADH) synthase, the product of the Mj0400 gene, catalyzes a transaldol reaction between 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde to yield ADH. Dehydroquinate synthase II, the product of the Mj1249 gene, then catalyzes deamination and cyclization of ADH, resulting in DHQ, which is fed into the canonical pathway. Three crystal structures of ADH synthase were determined in this work: a complex with a substrate analogue, fructose 1,6-bisphosphate, a complex with dihydroxyacetone phosphate (DHAP), thought to be a product of fructose 1-phosphate cleavage, and a native structure containing copurified ligands, modeled as DHAP and glycerol. On the basis of the structural analysis and comparison of the enzyme with related aldolases, ADH synthase is classified as a new member of the class I aldolase superfamily. The description of the active site allows for the identification and characterization of possible catalytic residues, Lys184, which is responsible for formation of the Schiff base intermediate, and Asp33 and Tyr153, which are candidates for the general acid/base catalysis.

  18. Induction of aminolevulinic acid synthase gene expression and enhancement of metabolite, protoporphyrin IX, excretion by organic germanium.

    PubMed

    Nakamura, Takashi; Saito, Miki; Shimada, Yasuhiro; Fukaya, Haruhiko; Shida, Yasuo; Tokuji, Yoshihiko

    2011-02-25

    Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132 is a water-soluble organic germanium compound. Oral intake of dietary Ge-132 changes fecal color and we attempted to identify the fecal red pigment, which increased by the intake of dietary Ge-132. Sprague Dawley rats were given diets containing Ge-132 from 0 to 0.5% concentration. Fecal red pigment was extracted and purified for optical and structural studies. We examined the fecal red pigment content by high performance liquid chromatography (HPLC), and hepatic gene expressions relating to heme synthesis by reverse transcription polymerase chain reaction (RT-PCR). The purified red pigment had particular optical characteristics on the ultraviolet (UV)-visible spectrum (Soret band absorbance at 400 nm) and fluorescence emission at 600 nm by 400 nm excitation, and was identified as protoporphyrin IX by LC-MS analysis. Protoporphyrin IX significantly (P<0.05) increased 2.4-fold in the feces by the intake of a 0.5% Ge-132 diet. Gene expression analysis of the liver explained the increase of protoporphyrin IX by dietary Ge-132 as it enhanced (P<0.05) aminolevulinic acid synthase 1 (Alas1), a rate-limiting enzyme of heme synthesis, expression 1.8-fold, but decreased ferrochelatase (Fech) expression 0.6-fold (P<0.05). The results show that the intake of dietary Ge-132 is related to heme metabolism. Because protoporphyrin IX is used to treat chronic hepatitis, Ge-132 may be a beneficial substance to increase protoporphyrin IX in the liver.

  19. 18β-Glycyrrhetinic Acid Suppresses Cell Proliferation through Inhibiting Thromboxane Synthase in Non-Small Cell Lung Cancer

    PubMed Central

    Huang, Qing-Chun; Chen, Xiu-Min; Jiang, Ze-Bo; Zhang, Xian; Zeng, Xing

    2014-01-01

    18β-glycyrrhetinic acid (18β-GA) is a bioactive component of licorice. The anti-cancer activity of 18β-GA has been studied in many cancer types, whereas its effects in lung cancer remain largely unknown. We first showed that 18β-GA effectively suppressed cell proliferation and inhibited expression as well as activity of thromboxane synthase (TxAS) in non-small cell lung cancer (NSCLC) cells A549 and NCI-H460. In addition, the administration of 18β-GA did not have any additional inhibitory effect on the decrease of cell proliferation induced by transfection with TxAS small interference RNA (siRNA). Moreover, 18β-GA failed to inhibit cell proliferation in the immortalized human bronchial epithelial cells 16HBE-T and another NSCLC cell line NCI-H23, both of which expressed minimal level of TxAS as compared to A549 and NCI-H460. However, 18β-GA abolished the enhancement of cell proliferation induced by transfection of NCI-H23 with pCMV6-TxAS plasmid. Further study found that the activation of both extracellular signal-regulated kinase (ERK)1/2 and cyclic adenosine monophosphate response element binding protein (CREB) induced by TxAS cDNA transfection could be totally blocked by 18β-GA. Altogether, we have delineated that, through inhibiting TxAS and its initiated ERK/CREB signaling, 18β-GA suppresses NSCLC cell proliferation. Our study has highlighted the significance of 18β-GA with respect to prevention and treatment of NSCLC. PMID:24695790

  20. Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between japonica and indica rice varieties.

    PubMed

    Nakamura, Yasunori; Francisco, Perigio B; Hosaka, Yuko; Sato, Aya; Sawada, Takayuki; Kubo, Akiko; Fujita, Naoko

    2005-05-01

    Four amino acids were variable between the 'active' indica-type and 'inactive' japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the 'active' indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B(1) chains by elongating short A and B(1) chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.

  1. Colonic Mucosal Fatty Acid Synthase as an Early Biomarker for Colorectal Neoplasia: Modulation by Obesity and Gender

    PubMed Central

    Cruz, Mart Dela; Wali, Ramesh K.; Bianchi, Laura K.; Radosevich, Andrew J.; Crawford, Susan E.; Jepeal, Lisa; Goldberg, Michael J.; Weinstein, Jaclyn; Momi, Navneet; Roy, Priya; Calderwood, Audrey H.; Backman, Vadim; Roy, Hemant K.

    2015-01-01

    Background We have previously reported that colonic peri-cryptal microvascular blood flow is augmented in the premalignant colonic epithelium, highlighting the increased metabolic demand of the proliferative epithelium as a marker of field carcinogenesis. However, its molecular basis is unexplored. In this study, we assessed the expression of a regulator of the “lipogenic switch”, fatty acid synthase (FASN), in early colon carcinogenesis for its potential biomarker utility for concurrent neoplasia. Methods FASN expression (IHC) in the colonic epithelium from azoxymethane and Pirc rat models of CRC was studied. FASN mRNA expression from endoscopically normal rectal mucosa was evaluated and correlated with colonoscopic findings (pathological confirmation of neoplasia). Results FASN expression progressively increased from premalignant to malignant stage in the azoxymethane-model (1.9 to 2.5 fold; p<0.0001) and was also higher in the adenomas compared to adjacent uninvolved mucosa (1.8 to 3.4 fold; p<0.001) in the pirc-model. Furthermore, FASN was significantly overexpressed in rectal biopsies from patients harboring adenomas compared to those with no adenomas. These effects were accentuated in male (~2 fold) and obese patients (1.4 fold compared to those with BMI <30). Overall, the performance of rectal FASN was excellent (AUROC of 0.81). Conclusions FASN is altered in the premalignant colonic mucosa and may serve as a marker for colonic neoplasia present elsewhere. The enhanced effects in men and obesity may have implications for identifying patient subgroups at risk for early onset neoplasia. Impact These findings support the role of rectal FASN expression as a reliable biomarker of colonic neoplasia. PMID:25155760

  2. The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase.

    PubMed

    Baldini, Steffi F; Wavelet, Cindy; Hainault, Isabelle; Guinez, Céline; Lefebvre, Tony

    2016-08-14

    Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases.

  3. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

  4. [Inhibition of triclosan to fatty acid synthase from goose uropygial glands and human breast cancer cells in vitro].

    PubMed

    Wang, Ying-Qiang; Lai, Bing-Sen; Anderson, Vernon E

    2003-03-01

    It has been indicated that fatty acid synthase (FAS) is abnormally overexpressed in human breast cancer compared with normal human tissue. Inhibition of FAS induces apoptosis of human breast cancer cells. The aim of this study was to observe the inhibition of triclosan on FAS from goose uropygial glands for establishing the method and to study the inhibition of triclosan on FAS from human breast cancer SKBr3 cells in vitro. The goose uropygial glands FAS was purified by ultra-centrifugation and Superdex PG 200 chromatography; the human breast cancer SKBr3 cell FAS was partially purified by ultra-centrifugation. The FAS was interacted with different concentrations of Triclosan with different times before catalyzing. Then the substrates of FAS were added to the reaction system. The inhibitory activities of triclosan against the FAS were investigated using spectrophotometric assays. In the goose uropygial gland group, FAS was purified as a single band at 250kDa with SDS-PAGE. The inhibitory activities of triclosan(12.5 micromol/L) at 0, 5, and 10 minute on FAS were 26.40%, 28.30%, and 43.93%, respectively. The inhibitory activities of triclosan (25.00 micromol/L) at 0, 5, and 10 minute on FAS were 46.22%, 50.28%, and 97.05%, respectively. The inhibitory activities of triclosan (100.00 micromol/L) at 0, 5, and 10 minute on FAS were 98.11%, 97.75%, and 97.37%, respectively. In human SKBr3 breast cancer cell group, the inhibitory activities of triclosan (25, 50, 100, and 200 micromol/L) at 5 minute on FAS were 20.00%, 26.67%, 60.00%, and 100%, respectively. Triclosan inhibits the FAS from goose uropygial glands and human breast cancer SKBr3 cells. The inhibitory activities depended on the concentrations of triclosan and the interaction times between triclosan and FAS before catalyzing.

  5. Differential regulation of hyaluronic acid synthase isoforms in human saphenous vein smooth muscle cells: possible implications for vein graft stenosis.

    PubMed

    van den Boom, M; Sarbia, M; von Wnuck Lipinski, K; Mann, P; Meyer-Kirchrath, J; Rauch, B H; Grabitz, K; Levkau, B; Schrör, K; Fischer, J W

    2006-01-06

    Autologous saphenous vein bypass grafts (SVG) are frequently compromised by neointimal thickening and subsequent atherosclerosis eventually leading to graft failure. Hyaluronic acid (HA) generated by smooth muscle cells (SMC) is thought to augment the progression of atherosclerosis. The aim of the present study was (1) to investigate HA accumulation in native and explanted arterialized SVG, (2) to identify factors that regulate HA synthase (HAS) expression and HA synthesis, and (3) to study the function of the HAS2 isoform. In native SVG, expression of all 3 HAS isoforms was detected by RT-PCR. Histochemistry revealed that native and arterialized human saphenous vein segments were characterized by marked deposition of HA in association with SMC. Interestingly, in contrast to native SVG, cyclooxygenase (COX)-2 expression by SMC and macrophages was detected only in arterialized SVG. In vitro in human venous SMC HAS isoforms were found to be differentially regulated. HAS2, HAS1, and HA synthesis were strongly induced by vasodilatory prostaglandins via Gs-coupled prostaglandin receptors. In addition, thrombin induced HAS2 via activation of PAR1 and interleukin 1beta was the only factor that induced HAS3. By small interfering RNA against HAS2, it was shown that HAS2 mediated HA synthesis is critically involved in cell cycle progression through G1/S phase and SMC proliferation. In conclusion, the present study shows that HA-rich extracellular matrix is maintained after arterialization of vein grafts and might contribute to graft failure because of its proproliferative function in venous SMC. Furthermore, COX-2-dependent prostaglandins may play a key role in the regulation of HA synthesis in arterialized vein grafts.

  6. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys

    PubMed Central

    Nawrocki, Andrea R.; Zhou, Dan; Wu, Margaret; Previs, Stephen; Miller, Corey; Liu, Haiying; Hines, Catherine D. G.; Madeira, Maria; Cao, Jin; Herath, Kithsiri; Wang, Liangsu; Kelley, David E.; Li, Cai

    2016-01-01

    Objectives Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. Methods We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. Results The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. Conclusions These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic

  7. Early Growth Response1and Fatty Acid Synthase Expression is Altered in Tumor Adjacent Prostate Tissue and Indicates Field Cancerization

    PubMed Central

    Jones, Anna C.; Trujillo, Kristina A.; Phillips, Genevieve K.; Fleet, Trisha M.; Murton, Jaclyn K.; Severns, Virginia; Shah, Satyan K.; Davis, Michael S.; Smith, Anthony Y.; Griffith, Jeffrey K.; Fischer, Edgar G.; Bisoffi, Marco

    2011-01-01

    BACKGROUND Field cancerization denotes the occurrence of molecular alterations in histologically normal tissues adjacent to tumors. In prostate cancer, identification of field cancerization has several potential clinical applications. However, prostate field cancerization remains ill defined. Our previous work has shown up-regulated mRNA of the transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS) in tissues adjacent to prostate cancer. METHODS Immunofluorescence data were analyzed quantitatively by spectral imaging and linear unmixing to determine the protein expression levels of EGR-1 and FAS in human cancerous, histologically normal adjacent, and disease-free prostate tissues. RESULTS EGR-1 expression was elevated in both structurally intact tumor adjacent (1.6× on average) and in tumor (3.0× on average) tissues compared to disease-free tissues. In addition, the ratio of cytoplasmic versus nuclear EGR-1 expression was elevated in both tumor adjacent and tumor tissues. Similarly, FAS expression was elevated in both tumor adjacent (2.7× on average) and in tumor (2.5× on average) compared to disease-free tissues. CONCLUSIONS EGR-1 and FAS expression is similarly deregulated in tumor and structurally intact adjacent prostate tissues and defines field cancerization. In cases with high suspicion of prostate cancer but negative biopsy, identification of field cancerization could help clinicians target areas for repeat biopsy. Field cancerization at surgical margins on prostatectomy specimen should also be looked at as a predictor of cancer recurrence. EGR-1 and FAS could also serve as molecular targets for chemoprevention. PMID:22127986

  8. Gibberellic acid, synthetic auxins, and ethylene differentially modulate alpha-L-Arabinofuranosidase activities in antisense 1-aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs.

    PubMed

    Sozzi, Gabriel O; Greve, L Carl; Prody, Gerry A; Labavitch, John M

    2002-07-01

    Alpha-L-Arabinofuranosidases (alpha-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different alpha-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. alpha-Af I and II are active throughout fruit ontogeny. alpha-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. alpha-Af II activity accounts for over 80% of the total alpha-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, alpha-Af III is ethylene dependent and specifically active during ripening. alpha-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas alpha-Af II and III acted on Na(2)CO(3)-soluble pectins. Different alpha-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. alpha-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only alpha-Af III activity. Results suggest that tomato alpha-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.

  9. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    PubMed Central

    Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

    2002-01-01

    α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

  10. Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin.

    PubMed

    White, James; Guerin, Theresa; Swanson, Hollie; Post, Steven; Zhu, Haining; Gong, Ming; Liu, Jun; Everson, William V; Li, Xiang-An; Graf, Gregory A; Ballard, Hubert O; Ross, Stuart A; Smart, Eric J

    2008-01-01

    In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

  11. Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues.

    PubMed

    Saadat, D; Harrison, D H

    1998-07-14

    Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.

  12. Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin.

    PubMed

    Sidgwick, Gary P; Iqbal, Syed A; Bayat, Ardeshir

    2013-05-01

    Keloid disease (KD) is a fibroproliferative disorder characterised partly by an altered extracellular matrix (ECM) profile. In fetal scarring, hyaluronic acid (HA) expression is increased, but is reduced in KD tissue compared with normal skin (NS). The expression of Hyaluronan Synthase (HAS) and hyaluronidase (HYAL) in KD and NS tissue were investigated for the first time using a range of techniques. Hyaluronan synthase and HYAL mRNA expression were significantly increased in NS tissue compared with KD tissue (P < 0.05). Immunohistological analysis of tissue indicated an accumulation of HAS and HYAL protein expression in KD compared with NS due to the thicker epidermis. No differences were observed in mRNA or protein expression in KD and NS fibroblasts. Reduced expression of HAS and HYAL may alter HA synthesis, degradation and accumulation in KD. Better understanding of the role of HA in KD may lead to novel therapeutic approaches to address the resulting ECM imbalance.

  13. Acetohydroxyacid synthase FgIlv2 and FgIlv6 are involved in BCAA biosynthesis, mycelial and conidial morphogenesis, and full virulence in Fusarium graminearum.

    PubMed

    Liu, Xin; Han, Qi; Xu, Jianhong; Wang, Jian; Shi, Jianrong

    2015-11-10

    In this study, we characterized FgIlv2 and FgIlv6, the catalytic and regulatory subunits of acetohydroxyacid synthase (AHAS) from the important wheat head scab fungus Fusarium graminearum. AHAS catalyzes the first common step in the parallel pathways toward branched-chain amino acids (BCAAs: isoleucine, leucine, valine) and is the inhibitory target of several commercialized herbicides. Both FgILV2 and FgILV6 deletion mutants were BCAA-auxotrophic and showed reduced aerial hyphal growth and red pigmentation when cultured on PDA plates. Conidial formation was completely blocked in the FgILV2 deletion mutant ΔFgIlv2-4 and significantly reduced in the FgILV6 deletion mutant ΔFgIlv6-12. The auxotrophs of ΔFgIlv2-4 and ΔFgIlv6-12 could be restored by exogenous addition of BCAAs but relied on the designated nitrogen source the medium contained. Deletion of FgILV2 or FgILV6 also leads to hypersensitivity to various cellular stresses and reduced deoxynivalenol production. ΔFgIlv2-4 lost virulence completely on flowering wheat heads, whereas ΔFgIlv6-12 could cause scab symptoms in the inoculated spikelet but lost its aggressiveness. Taken together, our study implies the potential value of antifungals targeting both FgIlv2 and FgIlv6 in F. graminearum.

  14. Crystal Structure of Arachidonic Acid Bound to a Mutant of Prostaglandin Endoperoxide Synthase-1 that Forms Predominantly 11-HPETE

    SciTech Connect

    Harman, C.; Rieke, C.J.; Garavito, R.M.; Smith, W.L.

    2010-03-05

    Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G{sub 2} (PGG{sub 2}), 11Rhydroperoxyeicosatetraenoic acid (HPETE), and 15R,SHPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11RHPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co{sup 3+}-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 {angstrom}. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val{sup 349} and Trp{sup 387} help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG{sub 2}. In the V349A/W387F oPGHS-1 {center_dot} AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG{sub 2}. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG{sub 2}.

  15. Effects of fatty acid synthase inhibitors on lymphatic vessels: an in vitro and in vivo study in a melanoma model.

    PubMed

    Bastos, Débora C; Paupert, Jenny; Maillard, Catherine; Seguin, Fabiana; Carvalho, Marco A; Agostini, Michelle; Coletta, Ricardo D; Noël, Agnès; Graner, Edgard

    2017-02-01

    Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on

  16. Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid.

    PubMed Central

    Man, W J; Li, Y; O'Connor, C D; Wilton, D C

    1991-01-01

    The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563]. PMID:1684105

  17. The 10t,12c isomer of conjugated linoleic acid inhibits fatty acid synthase expression and enzyme activity in human breast, colon, and prostate cancer cells.

    PubMed

    Lau, Dominic S Y; Archer, Michael C

    2010-01-01

    The objective of this study was to determine whether downregulation of fatty acid synthase (FAS) expression and/or inhibition of its activity by the two major CLA isomers, 10t,12c and 9c,11t CLA, could contribute to their inhibitory effect on the growth of human breast (MCF-7), colon (HT-29) and prostate (LNCaP) cancer cell lines. We first confirmed and extended the results of others showing that the inhibitory action of CLA on proliferation is dependent on the cell type as well as the structure of the isomer, the 10,12 isomer being a more potent inhibitor than the 9,11 isomer in the concentration range 25-100 microM. By Western analysis, we showed that 10,12 CLA downregulated FAS expression in all of the cell lines in a concentration-dependent manner, but the 9,11 isomer had no effect. Both isomers inhibited FAS enzyme activity, but 10,12 CLA was again more potent than the 9,11 isomer. Our results suggest that downregulation of FAS by 10,12 CLA, but not by the 9,11 isomer, as well as inhibition of FAS enzyme activity by both isomers, may contribute to growth inhibition of cancer cells but only at relatively high concentrations.

  18. Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck.

    PubMed

    Poulose, A J; Rogers, L; Cheesbrough, T M; Kolattukudy, P E

    1985-12-15

    In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.

  19. Operational Definition of Active and Healthy Aging (AHA): The European Innovation Partnership (EIP) on AHA Reference Site Questionnaire: Montpellier October 20-21, 2014, Lisbon July 2, 2015.

    PubMed

    Bousquet, Jean; Malva, Joao; Nogues, Michel; Mañas, Leocadio Rodriguez; Vellas, Bruno; Farrell, John

    2015-12-01

    A core operational definition of active and healthy aging (AHA) is needed to conduct comparisons. A conceptual AHA framework proposed by the European Innovation Partnership on Active and Healthy Ageing Reference Site Network includes several items such as functioning (individual capability and underlying body systems), well-being, activities and participation, and diseases (including noncommunicable diseases, frailty, mental and oral health disorders). The instruments proposed to assess the conceptual framework of AHA have common applicability and availability attributes. The approach includes core and optional domains/instruments depending on the needs and the questions. A major common domain is function, as measured by the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0). WHODAS 2.0 can be used across all diseases and healthy individuals. It covers many of the AHA dimensions proposed by the Reference Site network. However, WHODAS 2.0 does not include all dimensions proposed for AHA assessment. The second common domain is health-related quality of life (HRQoL). A report of the AHA questionnaire in the form of a spider net has been proposed to facilitate usual comparisons across individuals and groups of interest. Copyright © 2015. Published by Elsevier Inc.

  20. Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

    PubMed Central

    Stoops, J K; Kolodziej, S J; Schroeter, J P; Bretaudiere, J P; Wakil, S J

    1992-01-01

    The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis. Images PMID:1631160

  1. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    PubMed

    Jiang, Wen; Jiang, Yanfang; Bentley, Gayle J; Liu, Di; Xiao, Yi; Zhang, Fuzhong

    2015-08-01

    Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

  2. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  3. Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I.

    PubMed

    Li, Heng; Liu, Nan; Wang, Wen-Ting; Wang, Ji-Yu; Gao, Wen-Yun

    2016-01-01

    There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 μmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Developmental changes in the expression of S-acyl fatty acid synthase thioesterase gene and lipid composition in the uropygial gland of mallard ducks (Anas platyrhynchos).

    PubMed

    Kolattukudy, P E; Bohnet, S; Sasaki, G; Rogers, L

    1991-01-01

    Developmental changes in the composition of the uropygial gland secretory lipids of the postembryonic mallard ducks (Anas platyrhynchos) were determined. During the first 3 weeks after hatching, the composition of the secretory lipids remained constant; the lipids consisted of long-chain wax esters composed of a complex mixture of n-, monomethyl, and dimethyl fatty acids esterified to n-C16 and n-C18 fatty alcohols. Afterward, as the ducks began to acquire adult feathers, short-chain wax esters composed of 2- and 4-monomethyl fatty acids began to appear with 2-methylhexanoyl and 4-methylhexanoyl as the major acyl components; esters of short-chain monomethyl fatty acids (less than or equal to C12) constituted 90% of the lipids when the ducks were 2 months old and had acquired adult plumage. The appearance of the short-chain acids in the acyl portion of the wax esters was accompanied by the appearance of S-acyl fatty acid synthase thioesterase, which can hydrolytically release short-chain acids from fatty acid synthase in the gland. Northern blot analysis showed that the gland-specific thioesterase gene transcripts began to appear in the gland only 3 weeks after hatching. The appearance of the transcripts and immunologically detectable thioesterase protein reached maximum levels 2 months after hatching, with the acquisition of the adult plumage. Thus, the developmental changes in lipid composition correlated with the changes in the level of expression of the thioesterase gene. Expression of other gland-specific genes has been previously found to begin just prior to hatching. The gland-specific thioesterase is the first case of delayed expression of a gland-specific gene.

  5. Cystathionine-β-Synthase Inhibition for Colon Cancer: Enhancement of the Efficacy of Aminooxyacetic Acid via the Prodrug Approach

    PubMed Central

    Chao, Celia; Zatarain, John R; Ding, Ye; Coletta, Ciro; Mrazek, Amy A; Druzhyna, Nadiya; Johnson, Paul; Chen, Haiying; Hellmich, Judy L; Asimakopoulou, Antonia; Yanagi, Kazunori; Olah, Gabor; Szoleczky, Petra; Törö, Gabor; Bohanon, Fredrick J; Cheema, Minal; Lewis, Rachel; Eckelbarger, David; Ahmad, Akbar; Módis, Katalin; Untereiner, Ashley; Szczesny, Bartosz; Papapetropoulos, Andreas; Zhou, Jia; Hellmich, Mark R; Szabo, Csaba

    2016-01-01

    Colon cancer cells contain high levels of cystathionine-β-synthase (CBS). Its product, hydrogen sulfide (H2S), promotes the growth and proliferation of colorectal tumor cells. To improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle or AOAA (0.1, 0.5 or 1 mg/kg/d) for 3 wks. Tumor growth was significantly reduced by 9 mg/kg/d AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/d. Thus, the in vivo efficacy of YD0171 is nine times higher than that of AOAA. YD0171 (1 mg/kg/d) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/d) also reduced tumor growth in patient-derived tumor xenograft bearing athymic mice. YD0171 (3 mg/kg/d) induced the regression of established HCT116 tumors in vivo. A 5-d safety study in mice demonstrated that YD0171 at 20 mg/kg/d (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory

  6. Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down-regulation of ghKAS2 encoding β-ketoacyl-ACP synthase II (KASII).

    PubMed

    Liu, Qing; Wu, Man; Zhang, Baolong; Shrestha, Pushkar; Petrie, James; Green, Allan G; Singh, Surinder P

    2017-01-01

    Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed-specific RNAi-mediated down-regulation of β-ketoacyl-ACP synthase II (KASII) catalysing the elongation of palmitoyl-ACP to stearoyl-ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high-palmitic (HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols (TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn-2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high-oleic (HO) and high-stearic (HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid.

  7. Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in ent-kaurene synthase of Physcomitrella patens.

    PubMed

    Kawaide, Hiroshi; Hayashi, Ken-ichiro; Kawanabe, Ryo; Sakigi, Yuka; Matsuo, Akihiko; Natsume, Masahiro; Nozaki, Hiroshi

    2011-01-01

    ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Recently, the moss Physcomitrella patens ent-kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent-kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent-kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent-kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent-kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only ent-kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to ent-kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.

  8. Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-β-d-Glucan Synthase and Callose Synthesis 1

    PubMed Central

    Kauss, Heinrich; Jeblick, Wolfgang

    1986-01-01

    The activity of 1,3-β-d-glucan synthase assayed in the presence of digitonin in a microsomal preparation from suspension-cultured cells of Glycine max can be fully inhibited by unsaturated fatty acids, trienoic acids being most effective. Lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B can also fully inhibit the enzyme. Inhibition is observed both when the enzyme is activated by Ca2+ or by trypsinization. At low amounts some of the substances can also cause stimulation. These effects all may result from a displacement of certain endogenous phospholipids necessary for optimal activity of the 1,3-β-d-glucan synthase. In the absence of digitonin the enzyme activity is greatly stimulated by lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B within a certain concentration range, presumably by rendering the microsomal vesicles permeable to the substrate and Ca2+. Dibucaine does not cause such an effect. Acylcarnitine and Echinocandin B at low concentrations can induce callose synthesis in vivo; this effect is enhanced by chitosan. At higher concentrations the two substances and polyunsaturated fatty acids cause severe electrolyte leakage. The effects are discussed in regard to the induction of callose synthesis by enforced Ca2+ influx, and its modulation by membrane lipids. PMID:16664610

  9. 4217C>A polymorphism in carbamoyl-phosphate synthase 1 gene may not associate with hyperammonemia development during valproic acid-based therapy.

    PubMed

    Inoue, Kazuyuki; Suzuki, Eri; Takahashi, Toshiki; Yamamoto, Yoshiaki; Yazawa, Rei; Takahashi, Yukitoshi; Imai, Katsumi; Miyakawa, Kou; Inoue, Yushi; Tsuji, Daiki; Hayashi, Hideki; Itoh, Kunihiko

    2014-08-01

    Valproic acid, which is widely used to treat various types of epilepsy, may cause severe hyperammonemia. However, the mechanism responsible for this side effect is not readily apparent. Polymorphisms in the genes encoding carbamoyl-phosphate synthase 1 (CPS1) and N-acetylglutamate synthase (NAGS) were recently reported to be risk factors for the development of hyperammonemia during valproic acid-based therapy. This study aimed to examine the influence of patient characteristics, including polymorphisms in CPS1 4217C>A and NAGS -3064C>A, on the development of hyperammonemia in Japanese pediatric epilepsy patients. The study included 177 pediatric epilepsy patients. The presence of a 4217C>A polymorphism in CPS1 was determined using an allele-specific polymerase chain reaction (PCR)-based method, and the presence of a -3064C>A polymorphism in NAGS was determined using a PCR-based restriction fragment length polymorphism method. Hyperammonemia was defined as a plasma ammonia level exceeding 200 μg/dL. We observed a significant difference between the combination of valproic acid with phenytoin and the development of hyperammonemia in both univariate and multivariate analyses. With regard to the CPS1 4217C>A polymorphism, we did not observe a significant association with the development of hyperammonemia. In conclusion, CPS1 4217C>A polymorphism may not be associated with the development of hyperammonemia in Japanese population. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. 1-(Fluoroalkylidene)-1,1-bisphosphonic Acids are Potent and Selective Inhibitors of the Enzymatic Activity of Toxoplasma gondii Farnesyl Pyrophosphate Synthase

    PubMed Central

    Szajnman, Sergio H.; Rosso, Valeria S.; Malayil, Leena; Smith, Alyssa; Moreno, Silvia N. J.; Docampo, Roberto

    2012-01-01

    α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease and against Toxoplasma gondii, the responsible agent of toxoplasmosis and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) has proven to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range exhibiting an IC50 of 30 nM. This compound was two-fold more potent than risedronate (IC50 = 74 nM) taken as a positive control. This enzymatic activity was associated to a strong cell growth inhibition against tachyzoites of T. gondii having an IC50 value of 2.7 μM. PMID:22215028

  11. A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

    PubMed Central

    Lassner, M W; Lardizabal, K; Metz, J G

    1996-01-01

    beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils. PMID:8742713

  12. The AHA Moment: Assessment of the Redox Stability of Ionic Liquids Based on Aromatic Heterocyclic Anions (AHAs) for Nuclear Separations and Electric Energy Storage.

    PubMed

    Shkrob, Ilya A; Marin, Timothy W

    2015-11-19

    Because of their extended conjugated bond network, aromatic compounds generally have higher redox stability than less saturated compounds. We conjectured that ionic liquids (ILs) consisting of aromatic heterocyclic anions (AHAs) may exhibit improved radiation and electrochemical stability. Such properties are important in applications of these ILs as diluents in radionuclide separations and electrolytes in the electric energy storage devices. In this study, we systematically examine the redox chemistry of the AHAs. Three classes of these anions have been studied: (i) simple 5-atom ring AHAs, such as the pyrazolide and triazolides, (ii) AHAs containing an adjacent benzene ring, and (iii) AHAs containing electron-withdrawing groups that were introduced to reduce their basicity and interaction with metal ions. It is shown that fragmentation in the reduced and oxidized states of these AHAs does not generally occur, and the two main products, respectively, are the H atom adduct and the imidyl radical. The latter species occurs either as an N σ-radical or as an N π-radical, depending on the length of the N-N bond, and the state that is stabilized in the solid matrix is frequently different from that having the lowest energy in the gas phase. In some instances, the formation of the sandwich π-stack dimer radical anions has been observed. For trifluoromethylated anions, H adduct formation did not occur; instead, there was facile loss of fluoride from their fluorinated groups. The latter can be problematic in nuclear separations, but beneficial in batteries. Overall, our study suggests that AHA-based ILs are viable candidates for use as radiation-exposed diluents and electrolytes.

  13. Generation and the subjective feeling of "aha!" are independently related to learning from insight.

    PubMed

    Kizilirmak, Jasmin M; Galvao Gomes da Silva, Joana; Imamoglu, Fatma; Richardson-Klavehn, Alan

    2016-11-01

    It has been proposed that sudden insight into the solutions of problems can enhance long-term memory for those solutions. However, the nature of insight has been operationalized differently across studies. Here, we examined two main aspects of insight problem-solving-the generation of a solution and the subjective "aha!" experience-and experimentally evaluated their respective relationships to long-term memory formation (encoding). Our results suggest that generation (generated solution vs. presented solution) and the "aha!" experience ("aha!" vs. no "aha!") are independently related to learning from insight, as well as to the emotional response towards understanding the solution during encoding. Moreover, we analyzed the relationship between generation and the "aha!" experience and two different kinds of later memory tests, direct (intentional) and indirect (incidental). Here, we found that the generation effect was larger for indirect testing, reflecting more automatic retrieval processes, while the relationship with the occurrence of an "aha!" experience was somewhat larger for direct testing. Our results suggest that both the generation of a solution and the subjective experience of "aha!" indicate processes that benefit long-term memory formation, though differently. This beneficial effect is possibly due to the intrinsic reward associated with sudden comprehension and the detection of schema-consistency, i.e., that novel information can be easily integrated into existing knowledge.

  14. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner*

    PubMed Central

    Tirodkar, Tejas S.; Lu, Ping; Bai, Aiping; Scheffel, Matthew J.; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-01-01

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14–26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3–5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  15. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

  16. Recalibration of the ACC/AHA Risk Score in Two Population-Based German Cohorts

    PubMed Central

    de las Heras Gala, Tonia; Geisel, Marie Henrike; Peters, Annette; Thorand, Barbara; Baumert, Jens; Lehmann, Nils; Jöckel, Karl-Heinz; Moebus, Susanne; Erbel, Raimund; Meisinger, Christine

    2016-01-01

    Background The 2013 ACC/AHA guidelines introduced an algorithm for risk assessment of atherosclerotic cardiovascular disease (ASCVD) within 10 years. In Germany, risk assessment with the ESC SCORE is limited to cardiovascular mortality. Applicability of the novel ACC/AHA risk score to the German population has not yet been assessed. We therefore sought to recalibrate and evaluate the ACC/AHA risk score in two German cohorts and to compare it to the ESC SCORE. Methods We studied 5,238 participants from the KORA surveys S3 (1994–1995) and S4 (1999–2001) and 4,208 subjects from the Heinz Nixdorf Recall (HNR) Study (2000–2003). There were 383 (7.3%) and 271 (6.4%) first non-fatal or fatal ASCVD events within 10 years in KORA and in HNR, respectively. Risk scores were evaluated in terms of calibration and discrimination performance. Results The original ACC/AHA risk score overestimated 10-year ASCVD rates by 37% in KORA and 66% in HNR. After recalibration, miscalibration diminished to 8% underestimation in KORA and 12% overestimation in HNR. Discrimination performance of the ACC/AHA risk score was not affected by the recalibration (KORA: C = 0.78, HNR: C = 0.74). The ESC SCORE overestimated by 5% in KORA and by 85% in HNR. The corresponding C-statistic was 0.82 in KORA and 0.76 in HNR. Conclusions The recalibrated ACC/AHA risk score showed strongly improved calibration compared to the original ACC/AHA risk score. Predicting only cardiovascular mortality, discrimination performance of the commonly used ESC SCORE remained somewhat superior to the ACC/AHA risk score. Nevertheless, the recalibrated ACC/AHA risk score may provide a meaningful tool for estimating 10-year risk of fatal and non-fatal cardiovascular disease in Germany. PMID:27732641

  17. What about False Insights? Deconstructing the Aha! Experience along Its Multiple Dimensions for Correct and Incorrect Solutions Separately.

    PubMed

    Danek, Amory H; Wiley, Jennifer

    2016-01-01

    The subjective Aha! experience that problem solvers often report when they find a solution has been taken as a marker for insight. If Aha! is closely linked to insightful solution processes, then theoretically, an Aha! should only be experienced when the correct solution is found. However, little work has explored whether the Aha! experience can also accompany incorrect solutions ("false insights"). Similarly, although the Aha! experience is not a unitary construct, little work has explored the different dimensions that have been proposed as its constituents. To address these gaps in the literature, 70 participants were presented with a set of difficult problems (37 magic tricks), and rated each of their solutions for Aha! as well as with regard to Suddenness in the emergence of the solution, Certainty of being correct, Surprise, Pleasure, Relief, and Drive. Solution times were also used as predictors for the Aha!

  18. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  19. What about False Insights? Deconstructing the Aha! Experience along Its Multiple Dimensions for Correct and Incorrect Solutions Separately

    PubMed Central

    Danek, Amory H.; Wiley, Jennifer

    2017-01-01

    The subjective Aha! experience that problem solvers often report when they find a solution has been taken as a marker for insight. If Aha! is closely linked to insightful solution processes, then theoretically, an Aha! should only be experienced when the correct solution is found. However, little work has explored whether the Aha! experience can also accompany incorrect solutions (“false insights”). Similarly, although the Aha! experience is not a unitary construct, little work has explored the different dimensions that have been proposed as its constituents. To address these gaps in the literature, 70 participants were presented with a set of difficult problems (37 magic tricks), and rated each of their solutions for Aha! as well as with regard to Suddenness in the emergence of the solution, Certainty of being correct, Surprise, Pleasure, Relief, and Drive. Solution times were also used as predictors for the Aha! experience. This study reports three main findings: First, false insights exist. Second, the Aha! experience is multidimensional and consists of the key components Pleasure, Suddenness and Certainty. Third, although Aha! experiences for correct and incorrect solutions share these three common dimensions, they are also experienced differently with regard to magnitude and quality, with correct solutions emerging faster, leading to stronger Aha! experiences, and higher ratings of Pleasure, Suddenness, and Certainty. Solution correctness proffered a slightly different emotional coloring to the Aha! experience, with the additional perception of Relief for correct solutions, and Surprise for incorrect ones. These results cast some doubt on the assumption that the occurrence of an Aha! experience can serve as a definitive signal that a true insight has taken place. On the other hand, the quantitative and qualitative differences in the experience of correct and incorrect solutions demonstrate that the Aha! experience is not a mere epiphenomenon. Strong Aha

  20. The Plasma Membrane H+-ATPase AHA1 Plays a Major Role in Stomatal Opening in Response to Blue Light.

    PubMed

    Yamauchi, Shota; Takemiya, Atsushi; Sakamoto, Tomoaki; Kurata, Tetsuya; Tsutsumi, Toshifumi; Kinoshita, Toshinori; Shimazaki, Ken-Ichiro

    2016-08-01

    Stomata open in response to a beam of weak blue light under strong red light illumination. A blue light signal is perceived by phototropins and transmitted to the plasma membrane H(+)-ATPase that drives stomatal opening. To identify the components in this pathway, we screened for mutants impaired in blue light-dependent stomatal opening. We analyzed one such mutant, provisionally named blus2 (blue light signaling2), and found that stomatal opening in leaves was impaired by 65%, although the magnitude of red light-induced opening was not affected. Blue light-dependent stomatal opening in the epidermis and H(+) pumping in guard cell protoplasts were inhibited by 70% in blus2 Whole-genome resequencing identified a mutation in the AHA1 gene of the mutant at Gly-602. T-DNA insertion mutants of AHA1 exhibited a similar phenotype to blus2; this phenotype was complemented by the AHA1 gene. We renamed blus2 as aha1-10 T-DNA insertion mutants of AHA2 and AHA5 did not show any impairment in stomatal response, although the transcript levels of AHA2 and AHA5 were higher than those of AHA1 in wild-type guard cells. Stomata in ost2, a constitutively active AHA1 mutant, did not respond to blue light. A decreased amount of H(+)-ATPase in aha1-10 accounted for the reduced stomatal blue light responses and the decrease was likely caused by proteolysis of misfolded AHA1. From these results, we conclude that AHA1 plays a major role in blue light-dependent stomatal opening in Arabidopsis and that the mutation made the AHA1 protein unstable in guard cells. © 2016 American Society of Plant Biologists. All Rights Reserved.

  1. Evidence for an ionic intermediate in the transformation of fatty acid hydroperoxide by a catalase-related allene oxide synthase from the Cyanobacterium Acaryochloris marina.

    PubMed

    Gao, Benlian; Boeglin, William E; Zheng, Yuxiang; Schneider, Claus; Brash, Alan R

    2009-08-14

    Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

  2. Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol.

    PubMed

    Engel, Stanislav; Vyazmensky, Maria; Geresh, Shimona; Barak, Ze'ev; Chipman, David M

    2003-09-30

    We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.

  3. Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype.

    PubMed

    Onofri, Chiara; de Meijer, Etienne P M; Mandolino, Giuseppe

    2015-08-01

    Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.

  4. Severing of a hydrogen bond disrupts amino acid networks in the catalytically active state of the alpha subunit of tryptophan synthase

    PubMed Central

    Axe, Jennifer M; O'Rourke, Kathleen F; Kerstetter, Nicole E; Yezdimer, Eric M; Chan, Yan M; Chasin, Alexander; Boehr, David D

    2015-01-01

    Conformational changes in the β2α2 and β6α6 loops in the alpha subunit of tryptophan synthase (αTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (βTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the β6α6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli αTS in the absence of the βTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the β2α2 and β6α6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the αTS-βTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations. PMID:25377949

  5. Application of chromatography technology in the separation of active alkaloids from Hypecoum leptocarpum and their inhibitory effect on fatty acid synthase.

    PubMed

    Zhang, Qiulong; Luan, Guangxiang; Ma, Tao; Hu, Na; Suo, Yourui; Wang, Xiaoyan; Ma, Xiaofeng; Ding, Chenxu

    2015-12-01

    A method that involved the combination of pH-zone-refining counter-current chromatography and semipreparative reversed-phase liquid chromatography has been established for the preparative separation of alkaloids from Hypecoum leptocarpum. From 1.2 g of crude sample, 31 mg N-feruloyltyramine, 27 mg oxohydrastinine, 47 mg hydroprotopine, 25 mg leptopidine, and 18 mg hypecocarpine have been obtained. The structure of the new compound, hypecocarpine, is confirmed based on the analysis of spectroscopic data, including NMR, UV, and IR spectroscopy and positive electrospray ionization mass spectrometry. The known chemical structures were characterized on the basis of (1) H and (13) C NMR spectroscopy. The purities of the five alkaloids are all over 92.7% as determined by high-performance liquid chromatography. The alkaloids' cytotoxicity in breast cancer cells is assessed by using a Cell Counting Kit assay and their inhibitory effect on fatty acid synthase expression is assessed by a Western blot assay. These results suggest that leptopidine could suppress growth and induce cytotoxicity in breast cancer cells and that the cytotoxicity of leptopidine may be related to its inhibitory effect on fatty acid synthase expression.

  6. Plasma lipids and fatty acid synthase activity are regulated by short-chain fructo-oligosaccharides in sucrose-fed insulin-resistant rats.

    PubMed

    Agheli, N; Kabir, M; Berni-Canani, S; Petitjean, E; Boussairi, A; Luo, J; Bornet, F; Slama, G; Rizkalla, S W

    1998-08-01

    The aim of this study was to evaluate the chronic effects of a short-chain fructo-oligosaccharide (FOS)-containing diet on plasma lipids and the activity of fatty acid synthase (FAS) in insulin-resistant rats. Normal male Sprague-Dawley rats, 5 wk old, were randomly assigned to two groups and fed either a sucrose-rich diet (S, 575 g sucrose /kg diet and 140 g lipids/kg diet) or a sucrose-rich diet supplemented with 10 g/100 g short-chain fructo-oligosaccharides (S/FOS). A third reference group (R) was fed a standard nonpurified diet (g/kg, 575 g starch, 50 g fat). After 3 wk the sucrose-fed rats (compared with the R group) were characterized by the following: 1) higher insulin responses after a glucose challenge (P < 0.05); 2) heavier liver (P < 0.001) and retroperitoneal adipose tissue (P < 0.01); 3) hypertriglyceridemia (P < 0.0001) and higher plasma free fatty acids (P < 0.0001); and 4) higher fatty acid synthase activity in the liver but a low activity in the adipose tissue (P < 0.001). The addition of FOS to the diet resulted in 11% lower liver weight than in the S group (P < 0.05) and tended to result in lower adipose tissue weight (P < 0.11). Plasma triglycerides and plasma free fatty acids were lower in S/FOS- than in S-fed rats (P < 0.05). Chylomicrons + VLDL, and intermediate density lipoprotein (IDL) concentrations did not differ between groups, nor was plasma cholesterol influenced by diet. Hepatic FAS activity was lower in S/FOS-fed rats than in the S-fed rats (P < 0.05). In adipose tissue, however, this activity tended to be greater in rats fed S/FOS than in rats fed the S diet (P < 0.07). In conclusion, in a rat model of diet-induced (57.5% sucrose and 14% lipids) insulin resistance, the addition of short-chain FOS prevented some lipid disorders, lowered fatty acid synthase activity in the liver and tended to raise this activity in the adipose tissue. Short-chain FOS, in addition to being a nondigestible sweetener with good bulking capacity, might be

  7. Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy.

    PubMed

    Marks, G T; Harris, T K; Massiah, M A; Mildvan, A S; Harrison, D H

    2001-06-12

    Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share neither sequence nor structural similarities, yet the reactions catalyzed by both enzymes are similar, in that both initially convert dihydroxyacetone phosphate to a cis-enediolic intermediate. This enediolic intermediate is formed from the abstraction of the pro-S C3 proton of DHAP by Asp-71 of MGS or the pro-R C3 proton of DHAP by Glu-165 of TIM. MGS then catalyzes the elimination of phosphate from this enediolic intermediate to form the enol of methylglyoxal, while TIM catalyzes proton donation to C2 to form D-glyceraldehyde phosphate. A competitive inhibitor of TIM, phosphoglycolohydroxamic acid (PGH) is found to be a tight binding competitive inhibitor of MGS with a K(i) of 39 nM. PGH's high affinity for MGS may be due in part to a short, strong hydrogen bond (SSHB) from the NOH of PGH to the carboxylate of Asp-71. Evidence for this SSHB is found in X-ray, 1H NMR, and fractionation factor data. The X-ray structure of the MGS homohexamer complexed with PGH at 2.0 A resolution shows this distance to be 2.30-2.37 +/- 0.24 A. 1H NMR shows a PGH-dependent 18.1 ppm signal that is consistent with a hydrogen bond length of 2.49 +/- 0.02 A. The D/H fractionation factor (phi = 0.43 +/- 0.02) is consistent with a hydrogen bond length of 2.53 +/- 0.01 A. Further, 15N NMR suggests a significant partial positive charge on the nitrogen atom of bound PGH, which could strengthen hydrogen bond donation to Asp-71. Both His-98 and His-19 are uncharged in the MGS-PGH complex on the basis of the chemical shifts of their Cdelta and C(epsilon) protons. The crystal structure reveals that Asp-71, on the re face of PGH, and His-19, on the si face of PGH, both approach the NO group of the analogue, while His-98, in the plane of PGH, approaches the carbonyl oxygen of the analogue. The phosphate group of PGH accepts nine hydrogen bonds from seven residues and is tilted out of the imidate plane of PGH toward the re face

  8. Isolation of streptococcal hyaluronate synthase.

    PubMed

    Prehm, P; Mausolf, A

    1986-05-01

    Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.

  9. Isolation of streptococcal hyaluronate synthase.

    PubMed Central

    Prehm, P; Mausolf, A

    1986-01-01

    Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography. Images Fig. 1. Fig. 2. PMID:3092808

  10. Elevated urinary excretion of beta-aminoisobutyric acid and delta-aminolevulinic acid (ALA) and the inhibition of ALA-synthase and ALA-dehydratase activities in both liver and kidney in mice exposed to lead.

    PubMed

    Tomokuni, K; Ichiba, M; Hirai, Y

    1991-12-01

    Urinary excretion of beta-aminoisobutyric acid (ABA) and delta-aminolevulinic acid (ALA) was investigated in mice exposed to lead (500 p.p.m.) in drinking water for 14 days. Concentrations of both urinary ABA and urinary ALA increased significantly in the lead-exposed mice. However, the degree of increasing excretion was higher in urinary ALA (10-fold of the control) than in urinary ABA (2-fold of the control). On the other hand, it was demonstrated that ALA dehydratase in liver and kidney is inhibited by exposure to lead, while ALA synthase in these tissues has no inhibitory effect.

  11. Investigation of a 6-MSA Synthase Gene Cluster in Aspergillus aculeatus Reveals 6-MSA-derived Aculinic Acid, Aculins A-B and Epi-Aculin A.

    PubMed

    Petersen, Lene M; Holm, Dorte K; Gotfredsen, Charlotte H; Mortensen, Uffe H; Larsen, Thomas O

    2015-10-12

    Aspergillus aculeatus, a filamentous fungus belonging to the Aspergillus clade Nigri, is an industrial workhorse in enzyme production. Recently we reported a number of secondary metabolites from this fungus; however, its genetic potential for the production of secondary metabolites is vast. In this study we identified a 6-methylsalicylic acid (6-MSA) synthase from A. aculeatus, and verified its functionality by episomal expression in A. aculeatus and heterologous expression in A. nidulans. Feeding studies with fully (13) C-labeled 6-MSA revealed that 6-MSA is incorporated into aculinic acid, which further incorporates into three compounds that we name aculins A and B, and epi-aculin A, described here for the first time. Based on NMR data and bioinformatic studies we propose the structures of the compounds as well as a biosynthetic pathway leading to formation of aculins from 6-MSA.

  12. Fibroblast growth factor-1 induces phosphofructokinase, fatty acid synthase and Ca(2+)-ATPase mRNA expression in NIH 3T3 cells.

    PubMed

    Hsu, D K; Donohue, P J; Alberts, G F; Winkles, J A

    1993-12-30

    Polypeptide growth factors act in part by inducing the expression of specific proteins that perform functions critical to cell cycle progression. We have used a differential display technique to identify genes that are expressed at higher levels following fibroblast growth factor (FGF)-1 (acidic FGF) stimulation of quiescent murine NIH 3T3 fibroblasts. Three such genes--liver (B-type) phosphofructokinase (PFK), fatty acid synthase (FAS) and sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2 (SERCA2)--are described in this report. The level of FAS and SERCA2 mRNA expression is increased rapidly after FGF-1 addition; in contrast, PFK mRNA is induced with kinetics more typical of delayed-early genes. These results indicate that enhanced expression of the PFK, FAS and SERCA2 proteins may be important for FGF-1-stimulated cell proliferation.

  13. Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes.

    PubMed

    Shin, So-Yeon; Han, Nam Soo; Park, Yong-Cheol; Kim, Myoung-Dong; Seo, Jin-Ho

    2011-01-05

    Resveratrol is a well-known polyphenol present in red wine and exerts antioxidative and anti-carcinogenic effects on the human body. To produce resveratrol in a food-grade yeast, the 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned and transformed into Saccharomyces cerevisiae W303-1A. The resveratrol produced was unglycosylated and secreted into the culture medium. A batch culture with 15.3mg/l p-coumaric acid used as precursor resulted in the production of 3.1mg/l resveratrol with 14.4 mol% yield. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production.

  14. In Planta Recapitulation of Isoprene Synthases Evolution from Ocimene Synthases.

    PubMed

    Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

    2017-06-16

    Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated.We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated in vivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause in vivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms.Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Hybrid polyketide synthases

    DOEpatents

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  16. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cadmium uptake, translocation, and tolerance in AHA1OX Arabidopsis thaliana.

    PubMed

    Hou, Lingyan; Shi, Weiming; Wei, Wenhui; Shen, Hong

    2011-02-01

    Information on cadmium (Cd) uptake and transport is essential to understand better the physiology of Cd tolerance in plants. In this study, Cd uptake, translocation, and tolerance were investigated in AHA1 (Arabidopsis plasma membrane H(+)-ATPase gene) overexpressed plants. Exposed to 10 µM CdCl(2), AHA1OX showed a higher root elongation, accumulated more Cd, and maintained better integrity of nucleus membrane of root tips in comparison to the control plant (WT), suggesting that AHA1OX was more Cd tolerant than WT. To investigate Cd tolerance mechanism of AHA1OX plants, we measured the activity of plasma membrane H(+)-ATPase and the secretion of citrate. Results indicated that treatment with 10 µM of Cd stimulated the activity of plasma membrane H(+)-ATPase and the secretion of citrate, while 30 µM of Cd inhibited them. AHA1OX had higher activity of H(+)-ATPase and secretion of citrate than WT. Addition of citrate enhanced root-to-shoot translocation of Cd significantly. A higher root-to-shoot Cd translocation was observed in AHA1OX than in WT plants. Treatment with low temperature or metabolic inhibitor (carbonyl cyanide m-chlorophenylhydrazone) inhibited Cd uptake and translocation. The study of Cd forms using sequential extraction indicated that Cd was mainly present as a protein-bound form, and AHA1OX had more water-soluble Cd than WT. Taken together, our results suggested that the Cd tolerance of AHA1OX was associated with its root-to-shoot Cd translocation and secretion of citrate, which converts Cd(2+) into less toxic and more easily transportable forms in plant cells.

  18. A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions*

    PubMed Central

    McMillan, Duncan G. G.; Ferguson, Scott A.; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T.; Ronimus, Ron S.; Meier, Thomas; Janssen, Peter H.; Cook, Gregory M.

    2011-01-01

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A1Ao-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A1Ao-ATP synthase (MbbrA1Ao) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA1Ao was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na+-binding signature made up of identical amino acid residues. The purified MbbrA1Ao was stimulated by sodium ions, and Na+ provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A1Ao-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions. PMID:21953465

  19. Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus

    PubMed Central

    Sheldrick, E. Linda R.; Derecka, Kamila; Marshall, Elaine; Chin, Evonne C.; Hodges, Louise; Wathes, D. Claire; Abayasekara, D. Robert E.; Flint, Anthony P. F.

    2007-01-01

    Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARα and PPARδ (also known as PPARβ) (but not PPARγ). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARα (but not PPARδ or PPARγ) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4β-PMA and PGF2α, and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4β-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4β-PMA in the absence of a PPAR ligand was decreased by the NF-κB (nuclear factor κB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-κB in addition to PPAR phosphorylation. Use of NF-κB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARα to increase PTGS2 levels in bovine endometrial stromal cells. PMID:17516915

  20. Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus.

    PubMed

    Sheldrick, E Linda R; Derecka, Kamila; Marshall, Elaine; Chin, Evonne C; Hodges, Louise; Wathes, D Claire; Abayasekara, D Robert E; Flint, Anthony P F

    2007-08-15

    Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.

  1. Activation of AMP-activated protein kinase and phosphorylation of glycogen synthase kinase3 β mediate ursolic acid induced apoptosis in HepG2 liver cancer cells.

    PubMed

    Son, Hyun-Soo; Kwon, Hee Young; Sohn, Eun Jung; Lee, Jang-Hoon; Woo, Hong-Jung; Yun, Miyong; Kim, Sung-Hoon; Kim, Young-Chul

    2013-11-01

    Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

    PubMed Central

    Mosher, Roy H.; Paradkar, Ashish S.; Anders, Cecilia; Barton, Barry; Jensen, Susan E.

    1999-01-01

    Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis. PMID:10223939

  3. β-Ketoacyl-acyl Carrier Protein Synthase I (KASI) Plays Crucial Roles in the Plant Growth and Fatty Acids Synthesis in Tobacco

    PubMed Central

    Yang, Tianquan; Xu, Ronghua; Chen, Jianghua; Liu, Aizhong

    2016-01-01

    Fatty acids serve many functions in plants, but the effects of some key genes involved in fatty acids biosynthesis on plants growth and development are not well understood yet. To understand the functions of 3-ketoacyl-acyl-carrier protein synthase I (KASI) in tobacco, we isolated two KASI homologs, which we have designated NtKASI-1 and NtKASI-2. Expression analysis showed that these two KASI genes were transcribed constitutively in all tissues examined. Over-expression of NtKASI-1 in tobacco changed the fatty acid content in leaves, whereas over-expressed lines of NtKASI-2 exhibited distinct phenotypic features such as slightly variegated leaves and reduction of the fatty acid content in leaves, similar to the silencing plants of NtKASI-1 gene. Interestingly, the silencing of NtKASI-2 gene had no discernibly altered phenotypes compared to wild type. The double silencing plants of these two genes enhanced the phenotypic changes during vegetative and reproductive growth compared to wild type. These results uncovered that these two KASI genes had the partially functional redundancy, and that the KASI genes played a key role in regulating fatty acids synthesis and in mediating plant growth and development in tobacco. PMID:27509494

  4. Inhibition of hippocampal long-term potentiation by high-fat diets: is it related to an effect of palmitic acid involving glycogen synthase kinase-3?

    PubMed

    Contreras, Ana; Del Rio, Danila; Martínez, Ana; Gil, Carmen; Morales, Lidia; Ruiz-Gayo, Mariano; Del Olmo, Nuria

    2017-04-12

    High-fat diets (HFD) impair hippocampal-dependent learning and memory and produce important changes in synaptic transmission by enhancing glutamate uptake, decreasing synaptic efficacy, and inhibiting plasticity mechanisms such as N-methyl-D-aspartate-mediated long-term depression (LTD) within the hippocampus. Adolescent animals seem to be particularly susceptible to the detrimental effect of HFD as dietary treatments carried out between weaning and early adulthood are much more efficient in terms of hippocampal damage that those carried out during the adult period. As palmitic acid is the most abundant saturated fatty acid in HFD, its effect on hippocampal function needs to be studied. However, glycogen synthase kinase-3 (GSK-3), a pleiotropic enzyme highly expressed in the central nervous system, modulates both hippocampal long-term potentiation (LTP) and LTD, and has been implicated in neurological disorders including Alzheimer's disease. In this study, we have characterized in mice hippocampus the effect of (i) a 48 h HFD intervention and (ii) in-vitro palmitic acid, as well as the possible involvement of GSK-3 in the above-mentioned plasticity mechanisms. Our results show that both 48 h HFD and palmitic acid inhibit LTP in hippocampal slices, whereas no effect on LTD was observed. Moreover, tideglusib, an ATP-noncompetitive inhibitor of GSK-3, induced hippocampal LTP and partially reversed the impairment of LTP induced by palmitic acid.

  5. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  6. Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases: a single amino acid substitution reverses their cyclization specificity.

    PubMed

    Satou, Ryutaro; Miyanaga, Akimasa; Ozawa, Hiroki; Funa, Nobutaka; Katsuyama, Yohei; Miyazono, Ken-ichi; Tanokura, Masaru; Ohnishi, Yasuo; Horinouchi, Sueharu

    2013-11-22

    Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.

  7. p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase.

    PubMed

    Chang, Z; Sun, Y; He, J; Vining, L C

    2001-08-01

    Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.

  8. A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

    PubMed

    Brosnan, James T; Vargas, Jose J; Breeden, Gregory K; Grier, Logan; Aponte, Raphael A; Tresch, Stefan; Laforest, Martin

    2016-01-01

    This is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides. Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.

  9. Cloning, expression, and characterization of para-aminobenzoic acid (PABA) synthase from Agaricus bisporus 02, a thermotolerant mushroom strain.

    PubMed

    Deng, Li-Xin; Shen, Yue-Mao; Song, Si-Yang

    2015-01-01

    The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.

  10. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  11. Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

    DOE PAGES

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; ...

    2016-05-26

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

  12. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase

    PubMed Central

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; Sisson, Lyle A.; Schneider, Philip E.; Posewitz, Matthew C.

    2016-01-01

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. PMID:27303412

  13. Increased thymidylate synthase in L1210 cells possessing acquired resistance to N10-propargyl-5,8-dideazafolic acid (CB3717): development, characterization, and cross-resistance studies

    SciTech Connect

    Jackman, A.L.; Alison, D.L.; Calvert, A.H.; Harrap, K.R.

    1986-06-01

    The properties are described of a mutant L1210 cell line (L1210:C15) with acquired resistance (greater than 200-fold) to the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid. TS was overproduced 45-fold and was accompanied by a small increase in the activity of dihydrofolate reductase (2.6-fold). Both the level of resistance and enzyme activities were maintained in drug-free medium (greater than 300 generations). Failure of N10-propargyl-5,8-dideazafolic acid to suppress the (/sup 3/H)-2'-deoxyuridine incorporation into the acid-precipitable material of the resistant line supported the evidence that TS overproduction was the mechanism of resistance; consequently the L1210:C15 cells were largely cross-resistant to another (but weaker) TS inhibitor, 5,8-dideazafolic acid. Minimal cross-resistance was observed to the dihydrofolate reductase inhibitors methotrexate and 5-methyl-5,8-dideazaaminopterin (5- and 2-fold, respectively). L1210 and L1210:C15 cells were, however, equally sensitive to 5-fluorodeoxyuridine (FdUrd), an unexpected finding since a metabolite, 5-fluorodeoxyuridine monophosphate, is a potent TS inhibitor; however, this cytotoxicity against the L1210:C15 cells was antagonized by coincubation with 5 microM folinic acid although folinic acid potentiated the cytotoxicity of FdUrd to the N10-propargyl-5,8-dideazafolic acid-sensitive L1210 line. Thymidine was much less effective as a FdUrd protecting agent in the L1210:C15 when compared with the L1210 cells; however, a combination of thymidine plus hypoxanthine was without any additional effect (compared with thymidine alone) against the sensitive line but effectively protected L1210:C15 cells.

  14. Improvement of manganese peroxidase production by the hyper lignin-degrading fungus Phanerochaete sordida YK-624 by recombinant expression of the 5-aminolevulinic acid synthase gene.

    PubMed

    Hirai, Hirofumi; Misumi, Kenta; Suzuki, Tomohiro; Kawagishi, Hirokazu

    2013-12-01

    The manganese peroxidase (MnP) gene (mnp4) promoter of Phanerochaete sordida YK-624 was used to drive expression of 5-aminolevulinic acid synthase (als), which is a key heme biosynthesis enzyme. The expression plasmid pMnP4pro-als was transformed into P. sordida YK-624 uracil auxotrophic mutant UV-64, and 14 recombinant als expressing-transformants were generated. Average cumulative MnP activities in the transformants were 1.18-fold higher than that of control transformants. In particular, transformants A-14 and A-61 showed significantly higher MnP activity (approximately 2.8-fold) than wild type. RT-PCR analysis indicated that the increased MnP activity was caused by elevated recombinant als expression. These results suggest that the production of MnP is improved by high expression of als.

  15. Associations of patella lead with polymorphisms in the vitamin D receptor, delta-aminolevulinic acid dehydratase and endothelial nitric oxide synthase genes.

    PubMed

    Theppeang, Keson; Schwartz, Brian S; Lee, Byung-Kook; Lustberg, Mark E; Silbergeld, Ellen K; Kelsey, Karl T; Parsons, Patrick J; Todd, Andrew C

    2004-06-01

    A cross-sectional analysis was performed to evaluate associations of polymorphisms in the vitamin D receptor (VDR), delta-aminolevulinic acid dehydratase (ALAD), and endothelial nitric oxide synthase (eNOS) genes with patella lead concentrations in 652 lead workers in the Republic of Korea. There was a wide range of patella lead (from below detection limit to 946 microg Pb/g bone mineral), with a mean (standard deviation) of 75.2 (101.0). There were no associations of ALAD or eNOS genotypes with patella lead, but workers with the VDR B allele had significantly (P value < 0.05) higher patella lead (on average, 25% or approximately 6.6 microg Pb/g bone mineral) than lead workers with the VDR bb genotype. There was evidence that the relation between age and patella lead was modified by both the VDR and eNOS genotypes.

  16. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    PubMed Central

    Zhou, Tao; Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Sato, Seizo; Igarashi, Yasuhiro

    2015-01-01

    The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS) domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides. PMID:25603349

  17. Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation.

    PubMed

    Chiang, Chun-Te; Way, Tzong-Der; Tsai, Shang-Jie; Lin, Jen-Kun

    2007-12-22

    Fatty acid synthase (FAS) expression is markedly elevated in HER2-overexpressing breast cancer cells. In this study, diosgenin, a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin inhibited the phosphorylation of Akt and mTOR, and enhanced phosphorylation of JNK. The use of pharmacological inhibitors revealed that the modulation of Akt, mTOR and JNK phosphorylation was required for diosgenin-induced FAS suppression. Finally, we showed that diosgenin could enhance paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results suggested that diosgenin has the potential to advance as chemopreventive or chemotherapeutic agent for cancers that overexpress HER2.

  18. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

    Dung Pham, Van; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

  19. A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

    PubMed Central

    2011-01-01

    Introduction Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development. PMID:22177475

  20. Pu-erh tea, green tea, and black tea suppresses hyperlipidemia, hyperleptinemia and fatty acid synthase through activating AMPK in rats fed a high-fructose diet.

    PubMed

    Huang, Hsiu-Chen; Lin, Jen-Kun

    2012-02-01

    Although green tea extract has been reported to suppress hyperlipidemia, it is unclear how tea extracts prepared from green, oolong, black and pu-erh teas modulate fatty acid synthase expression in rats fed on a high-fructose diet. In this animal study, we evaluated the hypolipidemic and hypoleptinemia effect of these four different tea leaves fed to male Wistar rats for 12 weeks. The results showed that a fructose-rich diet significantly elevated serum triacylglycerols, cholesterol, insulin, and leptin concentrations, as compared with those in the control group. Interestingly, consuming tea leaves for 12 weeks almost normalized the serum triacylglycerols concentrations. Again, rats fed with fructose/green tea and fructose/pu-erh tea showed the greatest reduction in serum TG, cholesterol, insulin and leptin levels. In contrast, serum cholesterol and insulin concentrations of the fructose/oolong tea-fed rats did not normalize. The relative epididymal adipose tissue weight was lower in all rats supplemented with tea leaves than those fed with fructose alone. There was molecular evidence of improved lipid homeostasis according to fatty acid synthase (FAS) protein expression. Furthermore, supplementation of green, black, and pu-erh tea leaves significantly decreased hepatic FAS mRNA and protein levels, and increased AMPK phosphorylation, compared with those of rats fed with fructose only. These findings suggest that the intake of green, black, and pu-erh tea leaves ameliorated the fructose-induced hyperlipidemia and hyperleptinemia state in part through the suppression of FAS protein levels and increased AMPK phosphorylation.

  1. AHaH Computing–From Metastable Switches to Attractors to Machine Learning

    PubMed Central

    Nugent, Michael Alexander; Molter, Timothy Wesley

    2014-01-01

    Modern computing architecture based on the separation of memory and processing leads to a well known problem called the von Neumann bottleneck, a restrictive limit on the data bandwidth between CPU and RAM. This paper introduces a new approach to computing we call AHaH computing where memory and processing are combined. The idea is based on the attractor dynamics of volatile dissipative electronics inspired by biological systems, presenting an attractive alternative architecture that is able to adapt, self-repair, and learn from interactions with the environment. We envision that both von Neumann and AHaH computing architectures will operate together on the same machine, but that the AHaH computing processor may reduce the power consumption and processing time for certain adaptive learning tasks by orders of magnitude. The paper begins by drawing a connection between the properties of volatility, thermodynamics, and Anti-Hebbian and Hebbian (AHaH) plasticity. We show how AHaH synaptic plasticity leads to attractor states that extract the independent components of applied data streams and how they form a computationally complete set of logic functions. After introducing a general memristive device model based on collections of metastable switches, we show how adaptive synaptic weights can be formed from differential pairs of incremental memristors. We also disclose how arrays of synaptic weights can be used to build a neural node circuit operating AHaH plasticity. By configuring the attractor states of the AHaH node in different ways, high level machine learning functions are demonstrated. This includes unsupervised clustering, supervised and unsupervised classification, complex signal prediction, unsupervised robotic actuation and combinatorial optimization of procedures–all key capabilities of biological nervous systems and modern machine learning algorithms with real world application. PMID:24520315

  2. AHaH computing-from metastable switches to attractors to machine learning.

    PubMed

    Nugent, Michael Alexander; Molter, Timothy Wesley

    2014-01-01

    Modern computing architecture based on the separation of memory and processing leads to a well known problem called the von Neumann bottleneck, a restrictive limit on the data bandwidth between CPU and RAM. This paper introduces a new approach to computing we call AHaH computing where memory and processing are combined. The idea is based on the attractor dynamics of volatile dissipative electronics inspired by biological systems, presenting an attractive alternative architecture that is able to adapt, self-repair, and learn from interactions with the environment. We envision that both von Neumann and AHaH computing architectures will operate together on the same machine, but that the AHaH computing processor may reduce the power consumption and processing time for certain adaptive learning tasks by orders of magnitude. The paper begins by drawing a connection between the properties of volatility, thermodynamics, and Anti-Hebbian and Hebbian (AHaH) plasticity. We show how AHaH synaptic plasticity leads to attractor states that extract the independent components of applied data streams and how they form a computationally complete set of logic functions. After introducing a general memristive device model based on collections of metastable switches, we show how adaptive synaptic weights can be formed from differential pairs of incremental memristors. We also disclose how arrays of synaptic weights can be used to build a neural node circuit operating AHaH plasticity. By configuring the attractor states of the AHaH node in different ways, high level machine learning functions are demonstrated. This includes unsupervised clustering, supervised and unsupervised classification, complex signal prediction, unsupervised robotic actuation and combinatorial optimization of procedures-all key capabilities of biological nervous systems and modern machine learning algorithms with real world application.

  3. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice.

    PubMed

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-02-05

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence

    PubMed Central

    Jiao, Yuntong; Xu, Weirong; Duan, Dong; Wang, Yuejin; Nick, Peter

    2016-01-01

    Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. ‘Carigane’ (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis. PMID:27702992

  5. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice*♦

    PubMed Central

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-01-01

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. PMID:26670611

  6. Tamoxifen-induced anorexia is associated with fatty acid synthase inhibition in the ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA.

    PubMed

    López, Miguel; Lelliott, Christopher J; Tovar, Sulay; Kimber, Wendy; Gallego, Rosalía; Virtue, Sam; Blount, Margaret; Vázquez, Maria J; Finer, Nick; Powles, Trevor J; O'Rahilly, Stephen; Saha, Asish K; Diéguez, Carlos; Vidal-Puig, Antonio J

    2006-05-01

    Fatty acid metabolism in the hypothalamus has recently been shown to regulate feeding. The selective estrogen receptor modulator tamoxifen (TMX) exerts a potent anorectic effect. Here, we show that the anorectic effect of TMX is associated with the accumulation of malonyl-CoA in the hypothalamus and inhibition of fatty acid synthase (FAS) expression specifically in the ventromedial nucleus of the hypothalamus (VMN). Furthermore, we demonstrate that FAS mRNA expression is physiologically regulated by fasting and refeeding in the VMN but not in other hypothalamic nuclei. Thus, the VMN appears to be the hypothalamic site where regulation of FAS and feeding converge. Supporting the potential clinical relevance of these observations, reanalysis of a primary breast cancer prevention study showed that obese women treated with TMX gained significantly less body weight over a 6-year period than obese women given placebo. The finding that TMX can modulate appetite through alterations in FAS expression and malonyl-CoA levels suggests a link between hypothalamic sex steroid receptors, fatty acid metabolism, and feeding behavior.

  7. In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

    PubMed Central

    Val, Dale L.; Cronan, John E.

    1998-01-01

    Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine. PMID:9573148

  8. Building Bridges for Innovation in Ageing: Synergies between Action Groups of the EIP on AHA.

    PubMed

    Bousquet, J; Bewick, M; Cano, A; Eklund, P; Fico, G; Goswami, N; Guldemond, N A; Henderson, D; Hinkema, M J; Liotta, G; Mair, A; Molloy, W; Monaco, A; Monsonis-Paya, I; Nizinska, A; Papadopoulos, H; Pavlickova, A; Pecorelli, S; Prados-Torres, A; Roller-Wirnsberger, R E; Somekh, D; Vera-Muñoz, C; Visser, F; Farrell, J; Malva, J; Andersen Ranberg, K; Camuzat, T; Carriazo, A M; Crooks, G; Gutter, Z; Iaccarino, G; Manuel de Keenoy, E; Moda, G; Rodriguez-Mañas, L; Vontetsianos, T; Abreu, C; Alonso, J; Alonso-Bouzon, C; Ankri, J; Arredondo, M T; Avolio, F; Bedbrook, A; Białoszewski, A Z; Blain, H; Bourret, R; Cabrera-Umpierrez, M F; Catala, A; O'Caoimh, R; Cesari, M; Chavannes, N H; Correia-da-Sousa, J; Dedeu, T; Ferrando, M; Ferri, M; Fokkens, W J; Garcia-Lizana, F; Guérin, O; Hellings, P W; Haahtela, T; Illario, M; Inzerilli, M C; Lodrup Carlsen, K C; Kardas, P; Keil, T; Maggio, M; Mendez-Zorrilla, A; Menditto, E; Mercier, J; Michel, J P; Murray, R; Nogues, M; O'Byrne-Maguire, I; Pappa, D; Parent, A S; Pastorino, M; Robalo-Cordeiro, C; Samolinski, B; Siciliano, P; Teixeira, A M; Tsartara, S I; Valiulis, A; Vandenplas, O; Vasankari, T; Vellas, B; Vollenbroek-Hutten, M; Wickman, M; Yorgancioglu, A; Zuberbier, T; Barbagallo, M; Canonica, G W; Klimek, L; Maggi, S; Aberer, W; Akdis, C; Adcock, I M; Agache, I; Albera, C; Alonso-Trujillo, F; Angel Guarcia, M; Annesi-Maesano, I; Apostolo, J; Arshad, S H; Attalin, V; Avignon, A; Bachert, C; Baroni, I; Bel, E; Benson, M; Bescos, C; Blasi, F; Barbara, C; Bergmann, K C; Bernard, P L; Bonini, S; Bousquet, P J; Branchini, B; Brightling, C E; Bruguière, V; Bunu, C; Bush, A; Caimmi, D P; Calderon, M A; Canovas, G; Cardona, V; Carlsen, K H; Cesario, A; Chkhartishvili, E; Chiron, R; Chivato, T; Chung, K F; d'Angelantonio, M; De Carlo, G; Cholley, D; Chorin, F; Combe, B; Compas, B; Costa, D J; Costa, E; Coste, O; Coupet, A-L; Crepaldi, G; Custovic, A; Dahl, R; Dahlen, S E; Demoly, P; Devillier, P; Didier, A; Dinh-Xuan, A T; Djukanovic, R; Dokic, D; Du Toit, G; Dubakiene, R; Dupeyron, A; Emuzyte, R; Fiocchi, A; Wagner, A; Fletcher, M; Fonseca, J; Fougère, B; Gamkrelidze, A; Garces, G; Garcia-Aymeric, J; Garcia-Zapirain, B; Gemicioğlu, B; Gouder, C; Hellquist-Dahl, B; Hermosilla-Gimeno, I; Héve, D; Holland, C; Humbert, M; Hyland, M; Johnston, S L; Just, J; Jutel, M; Kaidashev, I P; Khaitov, M; Kalayci, O; Kalyoncu, A F; Keijser, W; Kerstjens, H; Knezović, J; Kowalski, M; Koppelman, G H; Kotska, T; Kovac, M; Kull, I; Kuna, P; Kvedariene, V; Lepore, V; MacNee, W; Maggio, M; Magnan, A; Majer, I; Manning, P; Marcucci, M; Marti, T; Masoli, M; Melen, E; Miculinic, N; Mihaltan, F; Milenkovic, B; Millot-Keurinck, J; Mlinarić, H; Momas, I; Montefort, S; Morais-Almeida, M; Moreno-Casbas, T; Mösges, R; Mullol, J; Nadif, R; Nalin, M; Navarro-Pardo, E; Nekam, K; Ninot, G; Paccard, D; Pais, S; Palummeri, E; Panzner, P; Papadopoulos, N K; Papanikolaou, C; Passalacqua, G; Pastor, E; Perrot, M; Plavec, D; Popov, T A; Postma, D S; Price, D; Raffort, N; Reuzeau, J C; Robine, J M; Rodenas, F; Robusto, F; Roche, N; Romano, A; Romano, V; Rosado-Pinto, J; Roubille, F; Ruiz, F; Ryan, D; Salcedo, T; Schmid-Grendelmeier, P; Schulz, H; Schunemann, H J; Serrano, E; Sheikh, A; Shields, M; Siafakas, N; Scichilone, N; Siciliano, P; Skrindo, I; Smit, H A; Sourdet, S; Sousa-Costa, E; Spranger, O; Sooronbaev, T; Sruk, V; Sterk, P J; Todo-Bom, A; Touchon, J; Tramontano, D; Triggiani, M; Tsartara, S I; Valero, A L; Valovirta, E; van Ganse, E; van Hage, M; van den Berge, M; Vandenplas, O; Ventura, M T; Vergara, I; Vezzani, G; Vidal, D; Viegi, G; Wagemann, M; Whalley, B; Wickman, M; Wilson, N; Yiallouros, P K; Žagar, M; Zaidi, A; Zidarn, M; Hoogerwerf, E J; Usero, J; Zuffada, R; Senn, A; de Oliveira-Alves, B

    2017-01-01

    The Strategic Implementation Plan of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA) proposed six Action Groups. After almost three years of activity, many achievements have been obtained through commitments or collaborative work of the Action Groups. However, they have often worked in silos and, consequently, synergies between Action Groups have been proposed to strengthen the triple win of the EIP on AHA. The paper presents the methodology and current status of the Task Force on EIP on AHA synergies. Synergies are in line with the Action Groups' new Renovated Action Plan (2016-2018) to ensure that their future objectives are coherent and fully connected. The outcomes and impact of synergies are using the Monitoring and Assessment Framework for the EIP on AHA (MAFEIP). Eight proposals for synergies have been approved by the Task Force: Five cross-cutting synergies which can be used for all current and future synergies as they consider overarching domains (appropriate polypharmacy, citizen empowerment, teaching and coaching on AHA, deployment of synergies to EU regions, Responsible Research and Innovation), and three cross-cutting synergies focussing on current Action Group activities (falls, frailty, integrated care and chronic respiratory diseases).

  9. Diagnosis of Insulin Resistance in Hypertensive Patients by the Metabolic Syndrome: AHA vs. IDF Definitions

    PubMed Central

    Hwu, Chii-Min; Hsiung, Chao A.; Wu, Kwan-Dun; Lee, Wen-Jane; Shih, Kuang-Chung; Grove, John; Chen, Yii-Der I.; Rodriguez, Beatriz L.; Curb, J David

    2008-01-01

    SUMMARY Background Subjects with the metabolic syndrome are accompanied by insulin resistance (IR). However, it is not clear how well the newly-defined metabolic syndrome identifying IR specifically in hypertensive subjects. Aims The purpose of the study is to evaluate the performance of the metabolic syndrome, defined by the American Heart Association (AHA) and the International Diabetes Federation (IDF) definitions, in identifying IR in hypertension. Methods The analysis is a cross-sectional study. Totally, 228 hypertensive patients and 92 non-diabetic normotensive controls who received insulin suppressive tests to directly evaluate their insulin sensitivity were included from the Stanford Asia and Pacific Program for Hypertension and IR. McNemar’s tests were used to compare sensitivity and specificity of the AHA-defined with the IDF-defined metabolic syndrome in diagnosis of IR. Results The sensitivity of the metabolic syndrome for IR in hypertension was 89.7 % and the specificity 45.9 % by the AHA definition. Using the IDF definition, the sensitivity was 77.6 %, and the specificity increased to 63.5 %. The diagnostic power of individual components of the syndrome was also modest. The predictive discrimination of wider waist circumference was similar to that of the AHA-defined metabolic syndrome. Conclusions Use of the metabolic syndrome by the AHA definition provided good sensitivity but low specificity to diagnose IR in hypertension. The IDF definition improved in false positive rate, but it was still not specific enough to identify IR in hypertension. PMID:18564200

  10. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  11. Acetohydroxyacid synthases: evolution, structure, and function.

    PubMed

    Liu, Yadi; Li, Yanyan; Wang, Xiaoyuan

    2016-10-01

    Acetohydroxyacid synthase, a thiamine diphosphate-dependent enzyme, can condense either two pyruvate molecules to form acetolactate for synthesizing L-valine and L-leucine or pyruvate with 2-ketobutyrate to form acetohydroxybutyrate for synthesizing L-isoleucine. Because the key reaction catalyzed by acetohydroxyacid synthase in the biosynthetic pathways of branched-chain amino acids exists in plants, fungi, archaea, and bacteria, but not in animals, acetohydroxyacid synthase becomes a potential target for developing novel herbicides and antimicrobial compounds. In this article, the evolution, structure, and catalytic mechanism of acetohydroxyacid synthase are summarized.

  12. Partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indoleacetic acid-inositol synthase)

    NASA Technical Reports Server (NTRS)

    Kesy, J. M.; Bandurski, R. S.

    1990-01-01

    A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-beta-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-beta-d-glucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.

  13. Partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (indoleacetic acid-inositol synthase)

    NASA Technical Reports Server (NTRS)

    Kesy, J. M.; Bandurski, R. S.

    1990-01-01

    A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-beta-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-beta-d-glucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.

  14. Diets high in monounsaturated and polyunsaturated fatty acids decrease fatty acid synthase protein levels in adipose tissue but do not alter other markers of adipose function and inflammation in diet-induced obese rats.

    PubMed

    Enns, Jennifer E; Hanke, Danielle; Park, Angela; Zahradka, Peter; Taylor, Carla G

    2014-01-01

    This study investigates the effects of monounsaturated and polyunsaturated fatty acids from different fat sources (High Oleic Canola, Canola, Canola-Flaxseed (3:1 blend), Safflower, or Soybean Oil, or a Lard-based diet) on adipose tissue function and markers of inflammation in Obese Prone rats fed high-fat (55% energy) diets for 12 weeks. Adipose tissue fatty acid composition reflected the dietary fatty acid profiles. Protein levels of fatty acid synthase, but not mRNA levels, were lower in adipose tissue of all groups compared to the Lard group. Adiponectin and fatty acid receptors GPR41 and GPR43 protein levels were also altered, but other metabolic and inflammatory mediators in adipose tissue and serum were unchanged among groups. Overall, rats fed vegetable oil- or lard-based high-fat diets appear to be largely resistant to major phenotypic changes when the dietary fat composition is altered, providing little support for the importance of specific fatty acid profiles in the context of a high-fat diet. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides.

    PubMed

    Blum, Mathias; Gamper, Hannes A; Waldner, Maya; Sierotzki, Helge; Gisi, Ulrich

    2012-04-01

    Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood. In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  16. The influence of bovine growth hormone and growth hormone releasing factor on acetyl-CoA carboxylase and fatty acid synthase in primiparous Holstein cows.

    PubMed

    Beswick, N S; Kennelly, J J

    1998-08-01

    Primiparous Holstein cows received recombinant bovine growth hormone (bGH), bovine growth hormone-releasing factor (bGRF), or no treatment from 118 to 181 +/- 1 d. Milk yield was significantly increased with no change in milk fat percentage or composition. The mRNA and protein abundance of the key lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were measured in the mammary gland and adipose tissue. We hypothesized that bGH and bGRF treatment would increase the mRNA and protein abundance of ACC and FAS in the mammary gland, with an associated decrease in adipose tissue. Analysis of ACC mRNA and protein abundance in the mammary gland revealed that there was no significant influence of either bGH or bGRF treatment. Analysis of FAS mRNA in mammary gland revealed that both bGH and bGRF significantly increased the abundance. However, quantitation of FAS protein in the mammary gland revealed that neither treatment resulted in increased abundance. In adipose tissue, the mRNA and protein abundance of both ACC and FAS were significantly reduced. The increased substrate required for increased milk fatty acid yield may be provided through redirection of nutrients to the mammary gland away from adipose tissue and through overall increased metabolism of the mammary gland.

  17. Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

    PubMed

    Villajuana-Bonequi, Mitzi; Elrouby, Nabil; Nordström, Karl; Griebel, Thomas; Bachmair, Andreas; Coupland, George

    2014-07-01

    Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

  18. Metformin-induced killing of triple-negative breast cancer cells is mediated by reduction in fatty acid synthase via miRNA-193b.

    PubMed

    Wahdan-Alaswad, Reema S; Cochrane, Dawn R; Spoelstra, Nicole S; Howe, Erin N; Edgerton, Susan M; Anderson, Steven M; Thor, Ann D; Richer, Jennifer K

    2014-12-01

    The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells, and triple-negative breast cancer (TNBC) cell lines are more sensitive to the effects of metformin as compared to luminal breast cancer. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin has not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24 h of treatment, and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis and is important for the survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b directly targets the FASN 3'UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into metformin-induced killing of TNBC.

  19. Defect of synthesis of very long-chain fatty acids confers resistance to growth inhibition by inositol phosphorylceramide synthase repression in yeast Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2010-11-01

    Aureobasidin A (AbA) inhibits Aur1p, an enzyme catalysing the formation of inositol phosphorylceramide in the yeast Saccharomyces cerevisiae. AbA treatment results not only in reductions in complex sphingolipid levels but also in accumulation of ceramides, both of which are believed to lead to the growth defect caused by this inhibitor. We screened for mutants showing resistance to this drug, and found that a lack of ELO3, the gene involved in synthesis of very long-chain fatty acids, confers resistance to the inhibitor. The resistance as to growth inhibition by reduction in Aur1p activity was also confirmed by repression of AUR1 expression under the control of a tetracycline-regulatable promoter. Under the AUR1-repressive conditions, the ELO3 mutant showed reduction in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. However, with repression of LCB1 encoding serine palmitoyltransferase or LIP1 encoding the ceramide synthase subunit, the ELO3 mutation did not confer resistance to growth inhibition induced by the impaired sphingolipid biosynthesis. Therefore, it is suggested that the ELO3 mutant shows resistance as to accumulation of ceramides, implying that the chain lengths of fatty acids in ceramide are a critical factor for the ceramide-induced growth defect under AUR1-repressive conditions.

  20. Replacement of two amino acids of 9R-dioxygenase-allene oxide synthase of Aspergillus niger inverts the chirality of the hydroperoxide and the allene oxide.

    PubMed

    Sooman, Linda; Wennman, Anneli; Hamberg, Mats; Hoffmann, Inga; Oliw, Ernst H

    2016-02-01

    The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ~50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.

  1. Buffered l-ascorbic acid, alone or bound to KMUP-1 or sildenafil, reduces vascular endothelium growth factor and restores endothelium nitric oxide synthase in hypoxic pulmonary artery.

    PubMed

    Wu, Jiunn-Ren; Kao, Li-Pin; Wu, Bin-Nan; Dai, Zen-Kong; Wang, Yi-Ya; Chai, Chee-Yin; Chen, Ing-Jun

    2015-05-01

    Ascorbic acid bound to KMUP-1 and sildenafil were examined for their antioxidant effects on vascular endothelium growth factor (VEGF) and endothelium nitric oxide synthase (eNOS) in hypoxic pulmonary artery (PA). Inhaled KMUP-1 and oral sildenafil released NO from eNOS. The effect of buffered l-ascorbic acid, alone and bound to KMUP-1 or sildenafil, for treating pulmonary arterial hypertension (PAH) is unclear. In this study, the antioxidant capacity of ascorbic acid increased the beneficial effects of KMUP-1 on PAH. KMUP-1A and sildenafil-A (5 mg/kg/d) were administered to hypoxic PAH rats. Pulmonary artery blood pressure, and VEGF, Rho kinase II (ROCK II), eNOS, soluble guanylate cyclase (sGC-α), and protein kinase G expression in lung tissues were measured to link PAH and right ventricular hypertrophy. Hypoxic rats had higher pulmonary artery blood pressure, greater PA medial wall thickness and cardiac weight, and a higher right ventricle/left ventricle + septum [RV/(LV+S)] ratio than normoxic rats. Oral KMUP-1A or sildenafil-A for 21 days in hypoxia prevented the rarefaction of eNOS in immunohistochemistry (IHC), reduced the IHC of VEGF in PAs, restored eNOS/protein kinase G/phosphodiesterase 5A; unaffected sGC-α and inactivated ROCK II expression were also found in lung tissues. In normoxic PA, KMUP-1A/Y27632 (10μM) increased eNOS and reduced ROCK II. ROCK II/reactive oxidative species was increased and eNOS was reduced after long-term hypoxia for 21 days. KMUP-1A or Y27632 blunted ROCK II in short-term hypoxic PA at 24 hours. l-Ascorbic acid + l-sodium ascorbate (40, 80μM) buffer alone directly inhibited the IHC of VEGF in hypoxic PA. Finally, KMUP-1A or sildenafil-A reduced PAH and associated right ventricular hypertrophy.

  2. Transparency with a catch. AHA urges hospital price disclosure, but wants protection from lawsuits and broad definition of community benefits.

    PubMed

    Zigmond, Jessica; Evans, Melanie

    2006-05-08

    The AHA last week added its two cents to the transparency debate, approving a policy that supports a patient's right to know pricing. James Springfield, left, CEO of Valley Baptist Health System, who backs the AHA's policy, says the changes come at a time when "the public is clamoring ... to learn more about the mystical world of healthcare.

  3. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGES

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; ...

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  4. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  5. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxygenase with a bacterial type-I fatty acid synthase in E. coli.

    PubMed

    Coursolle, Dan; Lian, Jiazhang; Shanklin, John; Zhao, Huimin

    2015-09-01

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L(-1) long chain alcohol/alkane products including a 57 mg L(-1) titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  6. On the inhibition of AHAS by chlorimuron ethyl: A theoretical study

    NASA Astrophysics Data System (ADS)

    Jaña, Gonzalo A.; Alderete, Joel B.; Delgado, Eduardo J.

    2011-11-01

    The reaction between chlorimuron ethyl (CE) herbicide and the HEThDP- intermediate is addressed with the tools of computational chemistry, molecular dynamics and quantum chemistry, in order to check the postulated hypothesis that the inhibition of AHAS could proceed via a nucleophilic aromatic substitution reaction between HEThDP- and chlorimuron ethyl. The obtained results agree well with the empirical evidence for the inhibition process of AHAS by sulfonylureas, supporting the idea that the inhibition process could proceed by this via. It is also found that the nucleophilic attack of the Cα of the intermediate on the ipso carbon of the herbicide pyrimidinic ring is the rate-limiting step.

  7. Polyester synthases: natural catalysts for plastics.

    PubMed Central

    Rehm, Bernd H A

    2003-01-01

    Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

  8. Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress.

    PubMed

    Yuan, Wei; Zhang, Dongping; Song, Tao; Xu, Feiyun; Lin, Sheng; Xu, Weifeng; Li, Qianfeng; Zhu, Yiyong; Liang, Jiansheng; Zhang, Jianhua

    2017-03-01

    Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression.

    PubMed

    Ventura, Richard; Mordec, Kasia; Waszczuk, Joanna; Wang, Zhaoti; Lai, Julie; Fridlib, Marina; Buckley, Douglas; Kemble, George; Heuer, Timothy S

    2015-08-01

    Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20-200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K-AKT-mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive

  10. Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

    PubMed Central

    Tran, Lee; Hanavan, Paul D.; Campbell, Latoya E.; De Filippis, Elena; Lake, Douglas F.; Coletta, Dawn K.; Roust, Lori R.; Mandarino, Lawrence J.; Carroll, Chad C.; Katsanos, Christos S.

    2016-01-01

    Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish

  11. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

    PubMed Central

    Ventura, Richard; Mordec, Kasia; Waszczuk, Joanna; Wang, Zhaoti; Lai, Julie; Fridlib, Marina; Buckley, Douglas; Kemble, George; Heuer, Timothy S.

    2015-01-01

    Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Research in context Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for

  12. Adherence index based on the AHA 2006 diet and lifestyle recommendations is associated with select cardiovascular disease risk factors in older Puerto Ricans

    USDA-ARS?s Scientific Manuscript database

    Background: The effect of adherence to the American Heart Association (AHA) 2006 Diet and Lifestyle recommendations is unknown. Objective: To develop a unique diet and lifestyle score based on the AHA 2006 Diet and Lifestyle (AHA DL) recommendations. We evaluated this score in relation to available ...

  13. MpFAE3, a beta-ketoacyl-CoA synthase gene in the liverwort Marchantia polymorpha L., is preferentially involved in elongation of palmitic acid to stearic acid.

    PubMed

    Kajikawa, Masataka; Yamato, Katsuyuki T; Kanamaru, Hiroyuki; Sakuradani, Eiji; Shimizu, Sakayu; Fukuzawa, Hideya; Sakai, Yasuyoshi; Ohyama, Kanji

    2003-08-01

    Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.

  14. Multiple allelic forms of acetohydroxyacid synthase are responsible for herbicide resistance in Setaria viridis.

    PubMed

    Laplante, Julie; Rajcan, Istvan; Tardif, François J

    2009-08-01

    In weed species, resistance to herbicides inhibiting acetohydroxyacid synthase (AHAS) is often conferred by genetic mutations at one of six codons in the AHAS gene. These mutations provide plants with various levels of resistance to different chemical classes of AHAS inhibitors. Five green foxtail [Setaria viridis (L.) Beauv.] populations were reported in Ontario with potential resistance to the AHAS-inhibiting herbicide imazethapyr. The objectives of this study were to confirm resistance, establish the resistance spectrum for each of the five populations, and determine its genetic basis. Dose response curves were generated for whole plant growth and enzyme activity, and the AHAS gene was sequenced. Resistance was confirmed by determining the resistance factor to imazethapyr in the five resistant green foxtail populations for whole plant dose response experiments (21- to 182-fold) and enzyme assays (15- to 260-fold). All five imazethapyr-resistant populations showed cross-resistance to nicosulfuron and flucarbazone while only three populations had cross-resistance to pyrithiobac. Sequence analyses revealed single base-pair mutations in the resistant populations of green foxtail. These mutations were coded for Thr, Asn, or Ile substitution at Ser(653). In addition, a new mutation was found in one population that coded for an Asp substitution at Gly(654). There is an agreement between the spectra of resistance observed and the type of resistance known to be conferred by these substitutions. Moreover, it indicates that, under similar selection pressure (imazethapyr), a variety of mutations can be selected for different populations, making the resistance pattern difficult to predict from herbicide exposure history.

  15. Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

    PubMed

    Thuresson, E D; Lakkides, K M; Rieke, C J; Sun, Y; Wingerd, B A; Micielli, R; Mulichak, A M; Malkowski, M G; Garavito, R M; Smith, W L

    2001-03-30

    Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually

  16. Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine▿

    PubMed Central

    Subashchandrabose, Sargurunathan; LeVeque, Rhiannon M.; Wagner, Trevor K.; Kirkwood, Roy N.; Kiupel, Matti; Mulks, Martha H.

    2009-01-01

    In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens. PMID:19703979

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

    PubMed

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

    2011-01-01

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

  18. Clinical significance of elevated serum alpha-fetoprotein (AFP) level in acute viral hepatitis A (AHA).

    PubMed

    Seo, Seung In; Kim, Su Sun; Choi, Bo Youn; Lee, Sang Ho; Kim, Sung Jun; Park, Hye Won; Kim, Hyoung Su; Shin, Woon Geon; Kim, Kyung Ho; Lee, Jin Heon; Kim, Hak Yang; Jang, Myoung Kuk

    2013-10-01

    The clinical course of acute viral hepatitis A (AHA) is highly variable. Serum alphafetoprotein (AFP) level is often elevated in various types of acute liver injuries, indicating active liver regeneration. This study was aimed to investigate the clinical significance of serum AFP level in the aspect of the early recovery in AHA. A total of 238 patients with AHA, confirmed by IgM anti-hepatitis A virus, were included. The patients were classified according to serum AFP level. Multivariate analysis by Cox proportional hazards model using dichotomized clinical variables was performed to identify the independent predictors for early recovery (ALT normalization within 2 weeks). The median age (range) was 30 (17-50) years and male dominant (62%, 147/238). Compared to low AFP group, high AFP group (>10 ng/mL) had significantly lower platelet counts (p <0.0001), lower albumin (p =0.003), lower AST (p <0.001), lower ALT (p = 0.001), higher total bilirubin level (p <0.0001) on univariate analysis. On Cox regression analysis, high AFP level (>10 ng/mL) was the only independent predictor for early recovery (Hazard ratio (HR); 2.392, 95% CI; 1.564-3.659, p = 0.0001). High serum AFP level (>10 ng/mL) may indicate the already-started recovery through active liver regeneration or the early recovery within 2 weeks in AHA.

  19. Infective endocarditis prophylaxis and the current AHA, BSAC, NICE and Australian guidelines.

    PubMed

    Rahman, Naomi; Rogers, Seamus; Ryan, David; Healy, Claire; Flint, Stephen

    The latest guidelines from the American Heart Association (AHA) 2007, the Journal of the American Dental Association (JADA) 2008, the Australian Prevention of Endocarditis Guidelines 2008, the British Society for Antimicrobial Chemotherapy (BSAC) 2006, and the National Institute for Clinical Excellence (NICE) 2008 were reviewed for this article. As a result of recent literature reviews by the AHA and NICE committees, both groups made recommendations regarding antibiotic prophylaxis for dental treatment. While both agree that the benefit of prophylaxis for dental treatment is unproven, the NICE committee has recommended no antibiotic cover for any patients previously classified as 'at risk' of infective endocarditis (IE), while the AHA has recommended cover only for patients deemed to be at high risk of developing IE and with the poorest outcome in the event of IE development. The BSAC guidelines and the recently published Australian Therapeutic Guidelines on Prevention of Endocarditis 2008 fall broadly into line with the AHA guidelines. This paper will review all the separate guidelines and advocate a regimen for treating at-risk patients.

  20. Radioisotope assay for 1-aminocyclopropane-1-carboxylic acid synthase: s-adenosylhomocysteine analogs as inhibitors of the enzyme involved in plant senescence

    SciTech Connect

    Miura, G.A.; Chiang, P.K.

    1985-01-01

    A simple and rapid radioisotopic assay for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase was developed, an enzyme involved in the biosynthesis of the plant hormone ethylene. The assay utilizes an AG50-X4(NH4 (+)) column which separates S-adenosyl-L-(carboxyl-/sup 14/C)methionine (AdoMet) from the product (/sup 14/C)acc, since the latter is not bound to the resin while (/sup 14/C)adoMet is. As opposed to other assays, this procedure measures ACC directly and does not require further conversion to ethylene. When an enzyme preparation from ripe-tomato fruits (Lycopersicon esculentum Mill) was assayed, an I/sub 50/ of 2.5 + or - 0.8 micrometers for sinefungin and a K/sub m/ of 27 + or - 2 micrometers for AdoMet were obtained; these values were in good agreement with previous previous determinations made with a gas-chromatographic assay. When other nucleosides were tested as inhibitors the following order of decreasing activity was found: sinefungin, S-adenosylhomocysteine (AdoHcy), AdoHcy sulfoxide, S-n-butyladenosine, 3-deaza-adenosylhomocysteine, S-isobutyladenosine, S-isobutyladenosine, S-isobutyl-l-deazaadenosine. In contrast, S-isobutyl-3-deazaadenosine, S-isobutyl-7-deazaadenosine, 3-deazaadenosine, and adenodine were not inhibitory.

  1. Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

    PubMed Central

    Li, Yin; Li, Ze-Xu; Jin, Tan; Wang, Zhan-You; Zhao, Pu

    2017-01-01

    Hyaluronic acid (HA) is the backbone of the extracellular matrix (ECM) and provides biochemical and physical support to aggrecan-based perineuronal nets (PNNs), which are associated with the selective vulnerability of neurons in Alzheimer’s disease (AD). Here, we showed that HA synthases (HASs), including Has1, Has2, and Has3, were widely expressed in murine central nervous system. All types of HASs were localized to cell bodies of neurons; only Has1 existed in the membranes of neural axons. By using TauP301S transgenic (Tg) mouse model, we found that the axonal-localization of Has1 was abolished in TauP301S overexpressed mouse brain, and the redistribution of Has1 was also observed in human AD brains, suggesting that the localization of Has1 is dependent on intact microtubules which are regulated partially by the phosphorylation and dephosphorylation cycles of tau proteins. Furthermore, Has1 was reduced and Has3 was increased in TauP301S Tg mouse brain, resulting in the upregulation of shorter-chain HA in the ECM. These findings suggest that by abolishing the axonal-localization of Has1 and promoting the expression of Has3 and the synthesis of shorter-chain HA, the tau pathology breaks the balance of ECM components, promotes the reorganization of the ECM, and inhibits the formation of PNNs in the hippocampus, and then regulates neuronal plasticity during the progression of AD. PMID:28234253

  2. Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission

    PubMed Central

    Menzel, Tila R.; Weldegergis, Berhane T.; David, Anja; Boland, Wilhelm; Gols, Rieta; van Loon, Joop J. A.; Dicke, Marcel

    2014-01-01

    Jasmonic acid (JA) plays a central role in induced plant defence e.g. by regulating the biosynthesis of herbivore-induced plant volatiles that mediate the attraction of natural enemies of herbivores. Moreover, exogenous application of JA can be used to elicit plant defence responses similar to those induced by biting-chewing herbivores and mites that pierce cells and consume their contents. In the present study, we used Lima bean (Phaseolus lunatus) plants to explore how application of a low dose of JA followed by minor herbivory by spider mites (Tetranychus urticae) affects transcript levels of P. lunatus (E)-β-ocimene synthase (PlOS), emission of (E)-β-ocimene and nine other plant volatiles commonly associated with herbivory. Furthermore, we investigated the plant’s phytohormonal response. Application of a low dose of JA increased PlOS transcript levels in a synergistic manner when followed by minor herbivory for both simultaneous and sequential infestation. Emission of (E)-β-ocimene was also increased, and only JA, but not SA, levels were affected by treatments. Projection to latent structures-discriminant analysis (PLS-DA) of other volatiles showed overlap between treatments. Thus, a low-dose JA application results in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defence after herbivore infestation. PMID:25318119

  3. The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Espinoza, Ingrid; Lupu, Ruth

    2016-01-01

    The angiogenic inducer CCN1 (Cysteine-rich 61, CYR61) is differentially activated in metastatic breast carcinomas. However, little is known about the precise mechanisms that underlie the pro-metastatic actions of CCN1. Here, we investigated the impact of CCN1 expression on fatty acid synthase (FASN), a metabolic oncogene thought to provide cancer cells with proliferative and survival advantages. Forced expression of CCN1 in MCF-7 cells robustly up-regulated FASN protein expression and also significantly increased FASN gene promoter activity 2- to 3-fold, whereas deletion of the sterol response element-binding protein (SREBP) binding site in the FASN promoter completely abrogated CCN1-driven transcriptional activation. Pharmacological blockade of MAPK or PI-3'K activation similarly prevented the ability of CCN1 to induce FASN gene activation. Pharmacological inhibition of FASN activity with the mycotoxin cerulenin or the small compound C75 reversed CCN1-induced acquisition of estrogen independence and resistance to hormone therapies such as tamoxifen and fulvestrant in anchorage-independent growth assays. This study uncovers FASNdependent endogenous lipogenesis as a new mechanism controlling the metastatic phenotype promoted by CCN1. Because estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer, this previously unrecognized CCN1-driven lipogenic phenotype represents a novel metabolic target to clinically manage metastatic disease progression.

  4. The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells

    PubMed Central

    Menendez, Javier A.; Vellon, Luciano; Espinoza, Ingrid; Lupu, Ruth

    2016-01-01

    The angiogenic inducer CCN1 (Cysteine-rich 61, CYR61) is differentially activated in metastatic breast carcinomas. However, little is known about the precise mechanisms that underlie the pro-metastatic actions of CCN1. Here, we investigated the impact of CCN1 expression on fatty acid synthase (FASN), a metabolic oncogene thought to provide cancer cells with proliferative and survival advantages. Forced expression of CCN1 in MCF-7 cells robustly up-regulated FASN protein expression and also significantly increased FASN gene promoter activity 2- to 3-fold, whereas deletion of the sterol response element-binding protein (SREBP) binding site in the FASN promoter completely abrogated CCN1-driven transcriptional activation. Pharmacological blockade of MAPK or PI-3'K activation similarly prevented the ability of CCN1 to induce FASN gene activation. Pharmacological inhibition of FASN activity with the mycotoxin cerulenin or the small compound C75 reversed CCN1-induced acquisition of estrogen independence and resistance to hormone therapies such as tamoxifen and fulvestrant in anchorage-independent growth assays. This study uncovers FASNdependent endogenous lipogenesis as a new mechanism controlling the metastatic phenotype promoted by CCN1. Because estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer, this previously unrecognized CCN1-driven lipogenic phenotype represents a novel metabolic target to clinically manage metastatic disease progression. PMID:27713913

  5. Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy

    PubMed Central

    Silva Miranda, Mayra; Rodríguez, Kendy Wek; Martínez Cordero, Erasmo; Rojas-Espinosa, Oscar

    2006-01-01

    Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression. PMID:17222216

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

    PubMed

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

    2008-05-01

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

  7. The Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene Expression during the Transition from System-1 to System-2 Ethylene Synthesis in Tomato1

    PubMed Central

    Barry, Cornelius S.; Llop-Tous, M. Immaculada; Grierson, Donald

    2000-01-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the key regulatory enzymes involved in the synthesis of the hormone ethylene and is encoded by a multigene family containing at least eight members in tomato (Lycopersicon esculentum). Increased ethylene production accompanies ripening in tomato, and this coincides with a change in the regulation of ethylene synthesis from auto-inhibitory to autostimulatory. The signaling pathways that operate to bring about this transition from so-called system-1 to system-2 ethylene production are unknown, and we have begun to address these by investigating the regulation of ACS expression during ripening. Transcripts corresponding to four ACS genes, LEACS1A, LEACS2, LEACS4, and LEACS6, were detected in tomato fruit, and expression analysis using the ripening inhibitor (rin) mutant in combination with ethylene treatments and the Never-ripe (Nr) mutant has demonstrated that each is regulated in a unique way. A proposed model suggests that system-1 ethylene is regulated by the expression of LEACS1A and LEACS6. In fruit a transition period occurs in which the RIN gene plays a pivotal role leading to increased expression of LEACS1A and induction of LEACS4. System-2 ethylene synthesis is subsequently initiated and maintained by ethylene-dependent induction of LEACS2. PMID:10889246

  8. Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy.

    PubMed

    Silva Miranda, Mayra; Rodríguez, Kendy Wek; Martínez Cordero, Erasmo; Rojas-Espinosa, Oscar

    2006-12-01

    Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.

  9. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    SciTech Connect

    Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  10. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  11. The central administration of C75, a fatty acid synthase inhibitor, activates sympathetic outflow and thermogenesis in interscapular brown adipose tissue.

    PubMed

    Cassolla, Priscila; Uchoa, Ernane Torres; Mansur Machado, Frederico Sander; Guimarães, Juliana Bohnen; Rissato Garófalo, Maria Antonieta; de Almeida Brito, Nilton; Kagohara Elias, Lucila Leico; Coimbra, Cândido Celso; do Carmo Kettelhut, Isis; Carvalho Navegantes, Luiz Carlos

    2013-12-01

    The present work investigated the participation of interscapular brown adipose tissue (IBAT), which is an important site for thermogenesis, in the anti-obesity effects of C75, a synthetic inhibitor of fatty acid synthase (FAS). We report that a single intracerebroventricular (i.c.v.) injection of C75 induced hypophagia and weight loss in fasted male Wistar rats. Furthermore, C75 induced a rapid increase in core body temperature and an increase in heat dissipation. In parallel, C75 stimulated IBAT thermogenesis, which was evidenced by a marked increase in the IBAT temperature that preceded the rise in the core body temperature and an increase in the mRNA levels of uncoupling protein-1. As with C75, an i.c.v. injection of cerulenin, a natural FAS inhibitor, increased the core body and IBAT temperatures. The sympathetic IBAT denervation attenuated all of the thermoregulatory effects of FAS inhibitors as well as the C75 effect on weight loss and hypophagia. C75 induced the expression of Fos in the paraventricular nucleus, preoptic area, dorsomedial nucleus, ventromedial nucleus, and raphé pallidus, all of which support a central role of FAS in regulating IBAT thermogenesis. These data indicate a role for IBAT in the increase in body temperature and hypophagia that is induced by FAS inhibitors and suggest new mechanisms explaining the weight loss induced by these compounds.

  12. Inhibition of thalidomide teratogenicity by acetylsalicylic acid: evidence for prostaglandin H synthase-catalyzed bioactivation of thalidomide to a teratogenic reactive intermediate.

    PubMed

    Arlen, R R; Wells, P G

    1996-06-01

    Thalidomide is a teratogenic sedative-hypnotic drug that is structurally similar to phenytoin, which is thought to be bioactivated by prostaglandin H synthase (PHS) and other peroxidases to a teratogenic reactive intermediate. The relevance of this mechanism to thalidomide teratogenicity was evaluated in pregnant New Zealand White rabbits treated with thalidomide at 11:00 A.M. on gestational days 8 to 11, with day 0 indicating the time when sperm were observed in the vaginal fluid. Thalidomide (7.5 mg/kg i.v.) produced mainly fetal limb anomalies analogous to those observed in humans. Thalidomide (25-200 mg/kg i.p.), produced a dose-related increase in a spectrum of fetal anomalies, and in postpartum lethality, but did not produce a reliable incidence of limb anomalies. In subsequent studies, pregnant does received the irreversible PHS inhibitor acetylsalicylic acid (ASA), 75 mg/kg i.p., or its vehicle, followed 2 hr later by thalidomide, 7.5 mg/kg i.v., or its vehicle. ASA pretreatment was remarkably embryoprotective, resulting in respective 61.2 and 61.4% decreases in thalidomide-initiated fetal limb anomalies (P = .002) and postpartum fetal lethality (P < .02), and a small but significant reduction in thalidomide-initiated fetal weight loss. ASA alone did not produce significant embryopathy. These results show that ASA can protect the embryo from thalidomide teratogenicity, suggesting that thalidomide may be bioactivated by PHS to a teratogenic reactive intermediate.

  13. Valproic Acid Modifies Synaptic Structure and Accelerates Neurite Outgrowth Via the Glycogen Synthase Kinase-3β Signaling Pathway in an Alzheimer's Disease Model.

    PubMed

    Long, Zhi-Min; Zhao, Lei; Jiang, Rong; Wang, Ke-Jian; Luo, Shi-Fang; Zheng, Min; Li, Xiao-Feng; He, Gui-Qiong

    2015-11-01

    Tau hyperphosphorylation and amyloid β-peptide overproduction, caused by altered localization or abnormal activation of glycogen synthase kinase-3β (GSK-3β), is a pathogenic mechanism in Alzheimer's disease (AD). Valproic acid (VPA) attenuates senile plaques and neuronal loss. Here, we confirmed that VPA treatment improved spatial memory in amyloid precursor protein (APP)/presenilin 1 (PS 1) double-transgenic mice and investigated the effect of VPA on synaptic structure and neurite outgrowth. We used ultrastructural analysis, immunocytochemistry, immunofluorescence staining, and Western blot analysis to assess the effect of VPA treatment in mice. VPA treatment thickened the postsynaptic density, increased the number of presynaptic vesicles, and upregulated the expression of synaptic markers PSD-95 and GAP43. VPA increased neurite length of hippocampal neurons in vivo and in vitro. In VPA-treated AD mouse brain, inactivated GSK-3β (pSer9-GSK-3β) was markedly increased, while hyperphosphorylation of tau at Ser396 and Ser262 was decreased; total tau levels remained similar. VPA treatment notably improved pSer133-cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels, which are associated with synaptic function and neurite outgrowth. VPA improves behavioral deficits in AD, modifies synaptic structure, and accelerates neurite outgrowth, by inhibiting the activity of GSK-3β, decreasing hyperphosphorylated tau, enhancing CREB and BDNF expression. © 2015 John Wiley & Sons Ltd.

  14. Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase kinase-3β in the striatum of rats treated with 3-nitropropionic acid.

    PubMed

    Duran-Vilaregut, Joaquim; Manich, Gemma; Del Valle, Jaume; Camins, Antoni; Pallàs, Mercè; Vilaplana, Jordi; Pelegrí, Carme

    2012-09-01

    3-Nitropropionic acid (3-NPA) is a mitochondrial toxin used in the laboratory to replicate neurodegenerative conditions that are accompanied by degeneration of the caudate-putamen. 3-NPA induces depletion in ATP production, reactive oxygen species production, and secondary excitotoxicity mediated by activation of N-methyl-D-aspartate receptors that culminates in the triggering of cell death mechanisms, including apoptosis. We here examined by immunohistochemical methods whether cellular expression of phospho(Ser1981) -ataxia telangiectasia mutated (ATM), phospho(Ser15) -p53, phospho(Ser473) -Akt, and phospho(Ser9) -glycogen synthase kinase-3β (GSK3β), which are key signal molecules that play a critical role in regulating cellular processes related to cell survival and demise, were involved in the striatal neurodegeneration in the brains of rats treated with 3-NPA. Our results indicate that the toxin induced the activation of ATM and p53 only in astrocytes, and a role for these proteins in neuronal degeneration was ruled out. On the other hand, striatal neurons lost the active form of Akt as soon as they began to appear pyknotic, indicating impairment of the PI3K/Akt/GSK3 pathway in their degenerative process. The inactive form of GSK3β was detected extensively, mainly in the rim of the striatal lesions around degenerating neurons, which could be attributed to a cell death or cell survival response.

  15. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties

    PubMed Central

    Ghasemzadeh, Ali; Jaafar, Hawa Z. E.; Karimi, Ehsan

    2012-01-01

    The effect of foliar application of salicylic acid (SA) at different concentrations (10−3 M and 10−5 M) was investigated on the production of secondary metabolites (flavonoids), chalcone synthase (CHS) activity, antioxidant activity and anticancer activity (against breast cancer cell lines MCF-7 and MDA-MB-231) in two varieties of Malaysian ginger, namely Halia Bentong and Halia Bara. The results of high performance liquid chromatography (HPLC) analysis showed that application of SA induced the synthesis of anthocyanin and fisetin in both varieties. Anthocyanin and fisetin were not detected in the control plants. Accordingly, the concentrations of some flavonoids (rutin and apigenin) decreased significantly in plants treated with different concentrations of SA. The present study showed that SA enhanced the chalcone synthase (CHS) enzyme activity (involving flavonoid synthesis) and recorded the highest activity value of 5.77 nkat /mg protein in Halia Bara with the 10−5 M SA treatment. As the SA concentration was decreased from 10−3 M to 10−5 M, the free radical scavenging power (FRAP) increased about 23% in Halia Bentong and 10.6% in Halia Bara. At a concentration of 350 μg mL−1, the DPPH antioxidant activity recorded the highest value of 58.30%–72.90% with the 10−5 M SA treatment followed by the 10−3 M SA (52.14%–63.66%) treatment. The lowest value was recorded in the untreated control plants (42.5%–46.7%). These results indicate that SA can act not only as an inducer but also as an inhibitor of secondary metabolites. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines was observed for H. Bara extracts treated with 10−5 M SA with values of 61.53 and 59.88%, respectively. The results suggest that the high anticancer activity in these varieties may be related to the high concentration of potent anticancer components including fisetin and anthocyanin. The results thus indicate that the synthesis of flavonoids in

  16. α-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions.

    PubMed

    Zuljan, Federico A; Repizo, Guillermo D; Alarcon, Sergio H; Magni, Christian

    2014-10-01

    Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain. Copyright © 2014. Published by Elsevier B.V.

  17. Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.

    PubMed

    Huang, Wei; Booth, David M; Cane, Matthew C; Chvanov, Michael; Javed, Muhammad A; Elliott, Victoria L; Armstrong, Jane A; Dingsdale, Hayley; Cash, Nicole; Li, Yan; Greenhalf, William; Mukherjee, Rajarshi; Kaphalia, Bhupendra S; Jaffar, Mohammed; Petersen, Ole H; Tepikin, Alexei V; Sutton, Robert; Criddle, David N

    2014-08-01

    Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Intracellular calcium ([Ca(2+)](C)), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)](C) rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  18. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

    PubMed

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-11-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

  19. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius.

    PubMed

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-06-25

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT-PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.

  20. Associations of renal function with polymorphisms in the delta-aminolevulinic acid dehydratase, vitamin D receptor, and nitric oxide synthase genes in Korean lead workers.

    PubMed

    Weaver, Virginia M; Schwartz, Brian S; Ahn, Kyu-Dong; Stewart, Walter F; Kelsey, Karl T; Todd, Andrew C; Wen, Jiayu; Simon, David J; Lustberg, Mark E; Parsons, Patrick J; Silbergeld, Ellen K; Lee, Byung-Kook

    2003-10-01

    We analyzed data from 798 lead workers to determine whether polymorphisms in the genes encoding delta-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR) were associated with or modified relations of lead exposure and dose measures with renal outcomes. Lead exposure was assessed with job duration, blood lead, dimercaptosuccinic acid (DMSA)-chelatable lead, and tibia lead. Renal function was assessed with blood urea nitrogen (BUN), serum creatinine, measured creatinine clearance, calculated creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG), and retinol-binding protein. Mean (+/- SD) tibia lead, blood lead, and DMSA-chelatable lead levels were 37.2 +/- 40.4 microg/g bone mineral, 32.0 +/- 15.0 microg/dL, and 767.8 +/- 862.1 microg/g creatinine, respectively. After adjustment, participants with the ALAD(2) allele had lower mean serum creatinine and higher calculated creatinine clearance. We observed effect modification by ALAD on associations between blood lead and/or DMSA-chelatable lead and three renal outcomes. Among those with the ALAD(1-2) genotype, higher lead measures were associated with lower BUN and serum creatinine and higher calculated creatinine clearance. Participants with the eNOS variant allele were found to have higher measured creatinine clearance and BUN. In participants with the Asp allele, longer duration working with lead was associated with higher serum creatinine and lower calculated creatinine clearance and NAG; all were significantly different from relations in those with the Glu/Glu genotype except NAG (p = 0.08). No significant differences were seen in renal outcomes by VDR genotype, nor was consistent effect modification observed. The ALAD findings could be explained by lead-induced hyperfiltration.

  1. Effect modification by delta-aminolevulinic acid dehydratase, vitamin D receptor, and nitric oxide synthase gene polymorphisms on associations between patella lead and renal function in lead workers.

    PubMed

    Weaver, Virginia M; Lee, Byung-Kook; Todd, Andrew C; Ahn, Kyu-Dong; Shi, Weiping; Jaar, Bernard G; Kelsey, Karl T; Lustberg, Mark E; Silbergeld, Ellen K; Parsons, Patrick J; Wen, Jiayu; Schwartz, Brian S

    2006-09-01

    Genetic polymorphisms that affect lead toxicokinetics or toxicodynamics may be important modifiers of risk for adverse outcomes in lead-exposed populations. We recently reported associations between higher patella lead, which is hypothesized to represent a lead pool that is both bioavailable and cumulative, and adverse renal outcomes in current and former Korean lead workers. In the present study, we assessed effect modification by polymorphisms in the genes encoding for delta-aminolevulinic acid dehydratase (ALAD), the vitamin D receptor (VDR), and endothelial nitric oxide synthase on those associations. Similar analyses were conducted with three other lead biomarkers. Renal function was assessed via blood urea nitrogen, serum creatinine, measured and calculated creatinine clearances, urinary N-acetyl-beta-D-glucosaminidase, and retinol-binding protein. Mean (SD) blood, patella, tibia, and dimercaptosuccinic acid-chelatable lead values were 30.9 (16.7) microg/dl, 75.1 (101.1)and 33.6 (43.4) microg Pb/g bone mineral, and 0.63 (0.75) microg Pb/mg creatinine, respectively, in 647 lead workers. Little evidence of effect modification by genotype on associations between patella lead and renal outcomes was observed. The VDR polymorphism did modify associations between the other lead biomarkers and the serum creatinine and calculated creatinine clearance. Higher lead dose was associated with worse renal function in participants with the variant B allele. Models in two groups, dichotomized by median age, showed that this effect was present in the younger half of the population. Limited evidence of effect modification by ALAD genotype was observed; higher blood lead levels were associated with higher calculated creatinine clearance among participants with the ALAD(1-2) genotype. In conclusion, VDR and/or ALAD genotypes modified associations between all the lead biomarkers, except patella lead, and the renal outcomes.

  2. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    PubMed Central

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-01-01

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress. PMID:24865400

  3. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius

    PubMed Central

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-01-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance. PMID:24309561