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Sample records for acid synthase depends

  1. Expression of Fatty Acid Synthase Depends on NAC1 and Is Associated with Recurrent Ovarian Serous Carcinomas.

    Science.gov (United States)

    Ueda, Stefanie M; Yap, Kai Lee; Davidson, Ben; Tian, Yuan; Murthy, Vivek; Wang, Tian-Li; Visvanathan, Kala; Kuhajda, Francis P; Bristow, Robert E; Zhang, Hui; Shih, Ie-Ming

    2010-01-01

    Our previous reports demonstrated that NAC1, a BTB/POZ domain-containing nuclear protein, upregulates in recurrent ovarian serous carcinoma and participates in developing drug resistance in cancer cells. The current study applies quantitative proteomics to identify the proteins controlled by NAC1 by comparing the proteomes of SKOV3 cells with and without expression of a dominant negative NAC1 construct, N130. From the proteins that are downregulated by N130 (upregulated by NAC1), we chose to further characterize fatty acid synthase (FASN). Similar to change in protein level, the FASN transcript level in SKOV3 cells was significantly reduced by N130 induction or by NAC1 knockdown. Immunohistochemistry showed that NAC1 and FASN immunointensities in ovarian serous carcinoma tissues had a highly significant correlation (P 1 in serous carcinomas was associated with a worse overall survival time (P NAC1 is essential for FASN expression in ovarian serous carcinomas and the expression of FASN significantly correlates with tumor recurrence and disease aggressiveness. The dependence of drug resistant tumor cells on FASN suggests a potential application of FASN-based therapeutics for recurrent ovarian cancer patients.

  2. Expression of Fatty Acid Synthase Depends on NAC1 and Is Associated with Recurrent Ovarian Serous Carcinomas

    Directory of Open Access Journals (Sweden)

    Stefanie M. Ueda

    2010-01-01

    Full Text Available Our previous reports demonstrated that NAC1, a BTB/POZ domain-containing nuclear protein, upregulates in recurrent ovarian serous carcinoma and participates in developing drug resistance in cancer cells. The current study applies quantitative proteomics to identify the proteins controlled by NAC1 by comparing the proteomes of SKOV3 cells with and without expression of a dominant negative NAC1 construct, N130. From the proteins that are downregulated by N130 (upregulated by NAC1, we chose to further characterize fatty acid synthase (FASN. Similar to change in protein level, the FASN transcript level in SKOV3 cells was significantly reduced by N130 induction or by NAC1 knockdown. Immunohistochemistry showed that NAC1 and FASN immunointensities in ovarian serous carcinoma tissues had a highly significant correlation (P1 in serous carcinomas was associated with a worse overall survival time (P<.01. Finally, C93, a new FASN inhibitor, induced massive apoptosis in carboplatin/paclitaxel resistant ovarian cancer cells. In conclusion, we show that NAC1 is essential for FASN expression in ovarian serous carcinomas and the expression of FASN significantly correlates with tumor recurrence and disease aggressiveness. The dependence of drug resistant tumor cells on FASN suggests a potential application of FASN-based therapeutics for recurrent ovarian cancer patients.

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

    Science.gov (United States)

    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.

  4. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

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

    Science.gov (United States)

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

    2010-01-01

    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. PMID:20606731

  6. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  7. Fatty Acid Synthase Inhibitor C75 Ameliorates Experimental Colitis

    OpenAIRE

    Matsuo, Shingo; Yang, Weng-Lang; Aziz, Monowar; Kameoka, Shingo; Wang, Ping

    2013-01-01

    Abnormalities of lipid metabolism through overexpression of fatty acid synthase (FASN), which catalyzes the formation of long-chain fatty acids, are associated with the development of inflammatory bowel disease (IBD). C75 is a synthetic α-methylene-γ-butyrolactone compound that inhibits FASN activity. We hypothesized that C75 treatment could effectively reduce the severity of experimental colitis. Male C57BL/6 mice were fed 4% dextran sodium sulfate (DSS) for 7 d. C75 (5 mg/kg body weight) or...

  8. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC50 value of 10.3 μmol L-1. (author)

  9. Substrate channeling: alpha-ketobutyrate inhibition of acetohydroxy acid synthase in Salmonella typhimurium.

    OpenAIRE

    Shaw, K J; Berg, C M

    1980-01-01

    Excess alpha-ketobutyrate inhibited the growth of Salmonella typhimurium LT2 by inhibiting the acetohydroxy acid synthase-catalyzed synthesis of alpha-acetolactate (a valine precursor). As a result, cells were starved for valine, and both ilvB (encoding acetohydroxy acid synthase I) and ilvGEDA (ilvG encodes acetohydroxy acid synthase II) were derepressed. The addition of valine reversed the effects of alpha-ketobutyrate.

  10. Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

    OpenAIRE

    Shaw, K J; Berg, C M; Sobol, T J

    1980-01-01

    An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defect...

  11. Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

    OpenAIRE

    Elišáková, Veronika; Pátek, Miroslav; Holátko, Jiří; Nešvera, Jan; Leyval, Damien; Goergen, Jean-Louis; Delaunay, Stéphane

    2005-01-01

    Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesi...

  12. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  13. p63 promotes cell survival through fatty acid synthase.

    Directory of Open Access Journals (Sweden)

    Venkata Sabbisetti

    Full Text Available There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN, a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9 or immortalized prostate epithelial (iPrEC cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

  14. The effect of porphyrin and radiation on ferrochelatase and 5-aminolevulinic acid synthase in epidermal cells

    International Nuclear Information System (INIS)

    The effects of ultraviolet A (UVA) and blue light on ferrochelatase protein, and its mRNA level, in 5-aminolevulinic acid (ALA)-loaded A431 cells was evaluated. Western blot analysis of ferrochelatase protein showed a protein band of 43 kDA. There was a decrease in the protein concentration 24 h and 48 h after irradiation of these cells. In contrast, as judged by Northern blot analysis, there was no change in ferochelatase mRNA level. Measurement of ALA synthase activity showed an ALA dose-dependent but radiation-independent decrease of enzyme activity, suggesting an end-product feedback inhibition. Since reactive oxygen species generated by porphyrin-induced photochemical reaction may be involved in the decrease in ferrochelatase protein, the effect of scavengers of reactive oxygen species was evaluated by measuring porphyrin accumulation in irradiated, ALA-loaded A431 cells. Porphyrin accumulation was significantly decreased in the presence of singlet oxygen scavenger sodium azide (0.05 mM, 40.6% suppression) or hydroxyl radical scavenger mannitol (5.0 mM, 45% suppression). These data suggest that the photochemical reaction induced by porphyrin and irradiation resulted in a decrease in ferrochelatase protein content, but had no effect on ferrochelatase mRNA level nor on ALA synthase activity. The decrease in protein was partly mediated by the reactive oxygen species. (au)

  15. Isolation and partial characterization of the gene for goose fatty acid synthase.

    Science.gov (United States)

    Kameda, K; Goodridge, A G

    1991-01-01

    Fatty acid synthase is regulated by diet and hormones, with regulation being primarily transcriptional. In chick embryo hepatocytes in culture, triiodothyronine stimulates accumulation of enzyme and transcription of the gene. Since the 5'-flanking region of this gene is likely involved in hormonal regulation of its expression, we have isolated and partially characterized an avian fatty acid synthase gene. A genomic DNA library was constructed in a cosmid vector and screened with cDNA clones that contained sequence complementary to the 3' end of goose fatty acid synthase mRNA. A genomic clone (approximately 35 kilobase pairs (kb] was isolated, and a 6.5-kb EcoRI fragment thereof contained DNA complementary to the 3' noncoding region of fatty acid synthase mRNA. Additional cosmid libraries were screened with 5' fragments of previously isolated genomic clones, resulting in the isolation of five overlapping cosmid DNAs. The entire region of cloned DNA spans approximately 105 kb. Exon-containing fragments were identified by hybridization with end-labeled poly(A)+ RNA and by hybridization of labeled exon-containing genomic DNA fragments to fatty acid synthase mRNA. A new set of cDNA clones spanning approximately 3.2 kb was isolated from a lambda-ZAP goose liver cDNA library using the 5'-most exon-containing fragment of the 5'-most genomic DNA clone. This region of mRNA contains a 5'-untranslated sequence and a continuous open reading frame which includes a region that codes for the essential cysteine of the beta-ketoacyl synthase domain. The entire fatty acid synthase gene spans about 50 kb. The 5' 15 kb of the gene contain 7 exons. S1 nuclease and primer extension analyses were used to identify a single site for initiation of transcription, 174 nucleotides upstream from the putative translation initiation codon. Putative "TATA" and "CCAAT" boxes are located 28 and 60 base pairs (bp), respectively, upstream of the site of initiation of transcription. The 5'-flanking 597

  16. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny;

    2007-01-01

    triad. Three KASes with different substrate specificities participate in synthesis of the C(16) and C(18) products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its......Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding. Udgivelsesdato: 2007-Feb...

  17. Prostaglandin H synthase-mediated bioactivation of the amino acid pyrolysate product Trp P-2

    Energy Technology Data Exchange (ETDEWEB)

    Petry, T.W.; Krauss, R.S.; Eling, T.E.

    1986-08-01

    We report evidence that the mutagen and carcinogen 3-amino-1-methyl-5H pyrido(4,3b)indole (Trp P-2) is a substrate for co-oxidation by prostaglandin H synthase (PHS) in ram seminal vesicle (RSV) microsomes. Trp P-2 serves as a reducing cofactor for the hydroperoxidase activity of PHS as shown by the concentration-dependent inhibition of the hydroperoxidase catalyzed incorporation of molecular oxygen into phenylbutazone. Spectral data suggest that this metabolism results in disruption of the double bond conjugation within the nucleus of the molecule. A single metabolite peak which was dependent upon arachidonic acid and substrate concentration was separated from the parent compound by h.p.l.c. following incubation with RSV microsomes. Co-oxidation of Trp P-2 produced reactive intermediates which bound covalently to microsomal protein (9 nmol/mg) and to calf thymus DNA (475 pmol/mg). Binding was inhibited by indomethacin, and supported by substitution of hydrogen peroxide for arachidonic acid. These data suggest a possible role for PHS in the in situ activation of Trp P-2 to its ultimate carcinogenic form in tissues which contain PHS.

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

    Science.gov (United States)

    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. PMID:26617065

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

    Energy Technology Data Exchange (ETDEWEB)

    Dotson, G.D.; Woodard, R.W. [Univ. of Michigan, Ann Arbor, MI (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota [Neutron Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Shoyama, Yukihiro; Morimoto, Satoshi, E-mail: morimoto@phar.kyushu-u.ac.jp [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    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.

  1. Characterization of human glutathione-dependent microsomal prostaglandin E synthase-1

    OpenAIRE

    Thorén, Staffan

    2003-01-01

    Prostaglandins (PGs) are lipid mediators, which act as local hormones. PGs are formed in most calls and are synthesized de novo from membrane-released arachidonic acid (AA) upon cell activation. Prostaglandin H synthase (PGHS) -1 or 2, also referred to as COX-1 and COX-2, metabolize AA to PGH2, which is subsequently converted in a cell-specific manner by downstream enzymes to biologically active prostanoids, i.e. PGE2, PGD2, PGF2alpha, PGI2 or TXA2. PGHS-1 is constitutive...

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

    Science.gov (United States)

    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. PMID:22075919

  3. Determination of amino-acidic positions important for Ocimum basilicum geraniol synthase activity

    OpenAIRE

    Fischer, Marc; Meyer, Sophie; Claudel, Patricia; Steyer, Damien; Bergdoll, Marc; Hugueney, Philippe

    2013-01-01

    Terpenes are one of the largest and most diversified families of natural compounds. Although they have found numerous industrial applications, the molecular basis of their synthesis in plants has, until now, not been fully understood. Plant genomes have been shown to contain dozens of terpene synthase (TPS) genes, however knowledge of their amino-acidic protein sequence in not sufficient to predict which terpene(s) will be produced by a particular enzyme. In order to investigate the structura...

  4. Inhibitor of fatty acid synthase induced apoptosis in human colonic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Pei Lin Huang; Zhen Sheng Dai; Yue Lin Jin; Shi Neng Zhu; Shi Lun Lu

    2000-01-01

    @@INTRODUCTION The treatment of human epithelial malignancies is limited by drug resistance and toxic and side effects,which results in the failure in the treatment of majority of advanced cancer victims. To seek for a new, and specific antineoplastic therapy will provide hope for tumor treatment. Although disordered intermediary metabolism in cancer cells has been known for many years, much of the work focused on abnormal glucose catabolism. At the same time, little attention has been paid to fatty acid synthasis in tumor tissues, dispite of the significance of fatty acid synthase (FAS) in some clinical human ovarian[1], breast[2], colorectal[3],and prostatic cancers[4,5]. Tumor cells which express high levels of fatty acid synthesizing enzymes use endogeneously synthesized fatty acids for membrance biosynthesis and appear to export large amounts of lipid. In contrast, normal cells preferentially utilize diary lipid.

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

    Directory of Open Access Journals (Sweden)

    Damien Garrido

    2015-02-01

    Full Text Available 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

  6. Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

    Science.gov (United States)

    Walsh, Terence A; Bevan, Scott A; Gachotte, Daniel J; Larsen, Cory M; Moskal, William A; Merlo, P A Owens; Sidorenko, Lyudmila V; Hampton, Ronnie E; Stoltz, Virginia; Pareddy, Dayakar; Anthony, Geny I; Bhaskar, Pudota B; Marri, Pradeep R; Clark, Lauren M; Chen, Wei; Adu-Peasah, Patrick S; Wensing, Steven T; Zirkle, Ross; Metz, James G

    2016-08-01

    Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving. PMID:27398790

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

    Science.gov (United States)

    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.

  8. Fatty Acid Synthase Mediates the Epithelial-Mesenchymal Transition of Breast Cancer Cells

    OpenAIRE

    Li, Junqin; Dong, Lihua; Wei, Dapeng; Wang, Xiaodong; Zhang, Shuo; Li, Hua

    2014-01-01

    This study aimed to investigate the role of fatty acid synthase (FASN) in the epithelial-mesenchymal transition (EMT) of breast cancer cells. MCF-7 cells and MCF-7 cells overexpressing mitogen-activated protein kinase 5 (MCF-7-MEK5) were used in this study. MCF-7-MEK5 cells showed stable EMT characterized by increased vimentin and decreased E-cadherin expression. An In vivo animal model was established using the orthotopic injection of MCF-7 or MCF-7-MEK5 cells. Real-time quantitative PCR and...

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

    Science.gov (United States)

    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.

  10. Functional characterization of amyrin synthase involved in ursolic acid biosynthesis in Catharanthus roseus leaf epidermis.

    Science.gov (United States)

    Yu, Fang; Thamm, Antje M K; Reed, Darwin; Villa-Ruano, Nemesio; Quesada, Alfonso Lara; Gloria, Edmundo Lozoya; Covello, Patrick; De Luca, Vincenzo

    2013-07-01

    Catharanthus roseus accumulates high levels of the pentacyclic triterpene, ursolic acid, as a component of its wax exudate on the leaf surface. Bioinformatic analyses of transcripts derived from the leaf epidermis provide evidence for the specialized role of this tissue in the biosynthesis of ursolic acid. Cloning and functional expression in yeast of a triterpene synthase derived from this tissue showed it to be predominantly an α-amyrin synthase (CrAS), since the α-amyrin to β-amyrin reaction products accumulated in a 5:1 ratio. Expression analysis of CrAS showed that triterpene biosynthesis occurs predominantly in the youngest leaf tissues and in the earliest stages of seedling development. Further studies using laser capture microdissection to harvest RNA from epidermis, mesophyll, idioblasts, laticifers and vasculature of leaves showed the leaf epidermis to be the preferred sites of CrAS expression and provide conclusive evidence for the involvement of this tissue in the biosynthesis of ursolic acid in C. roseus. PMID:22652241

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

    Science.gov (United States)

    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. PMID:26864638

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

    Science.gov (United States)

    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. PMID:22224284

  13. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans.

    Directory of Open Access Journals (Sweden)

    Mai-Britt Mosbech

    Full Text Available Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2, and lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24, while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22. Here we show that functional loss of HYL-2 decreases lifespan, while loss of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also required for the observed lifespan extension, as well as the increased number of autophagosomes in hyl-1;lagr-1 animals. Both autophagic events and the transcription factors PHA-4/FOXA, DAF-16, and SKN-1 have previously been associated with dietary restriction-induced longevity. Accordingly, we find that hyl-1;lagr-1 animals display reduced feeding, increased resistance to heat, and reduced reproduction. Collectively, our data suggest that specific sphingolipids produced by different ceramide synthases have opposing roles in determination of C. elegans lifespan. We propose that loss of HYL-1 and LAGR-1 result in dietary restriction-induced autophagy and consequently prolonged longevity.

  14. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    Science.gov (United States)

    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. PMID:24594312

  15. Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

    Indian Academy of Sciences (India)

    Meng-Jun Li; Ai-Qin Li; Han Xia; Chuan-Zhi Zhao; Chang-Sheng Li; Shu-Bo Wan; Yu-Ping Bi; Xing-Jun Wang

    2009-06-01

    The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, -ketoacyl-ACP synthase (I, II, III), -ketoacyl-ACP reductase, -hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.

  16. Acute nitric oxide synthase inhibition and endothelin-1-dependent arterial pressure elevation

    Directory of Open Access Journals (Sweden)

    Robert eRapoport

    2014-04-01

    Full Text Available Key evidence that endogenous nitric oxide (NO inhibits the continuous, endothelin (ET-1-mediated drive to elevate arterial pressure includes demonstrations that ET-1 mediates a significant component of the pressure elevated by acute exposure to NO synthase (NOS inhibitors. This review examines the characteristics of this pressure elevation in order to elucidate potential mechanisms associated with the negative regulation of ET-1 by NO and, thereby, provide potential insight into the vascular pathophysiology underlying NO dysregulation. We surmise that the magnitude of the ET-1-dependent component of the NOS inhibitor-elevated pressure is 1 independent of underlying arterial pressure and other pressor pathways activated by the NOS inhibitors and 2 dependent on relatively higher NOS inhibitor dose, release of stored and de novo synthesized ET-1, and ETA receptor-mediated increased vascular resistance. Major implications of these conclusions include: 1 the marked variation of the ET-1-dependent component, i.e., from 0-100% of the pressure elevation, reflects the NO-ET-1 regulatory pathway. Thus, NOS inhibitor-mediated, ET-1-dependent pressure elevation in vascular pathophysiologies is an indicator of the level of compromised/enhanced function of this pathway; 2 NO is a more potent inhibitor of ET-1-mediated elevated arterial pressure than other pressor pathways, due in part to inhibition of intravascular pressure-independent release of ET-1. Thus, the ET-1-dependent component of pressure elevation in vascular pathophysiologies associated with NO dysregulation is of greater magnitude at higher levels of compromised NO.

  17. Functional interaction of endothelial nitric oxide synthase with a voltage-dependent anion channel

    Science.gov (United States)

    Sun, Jianxin; Liao, James K.

    2002-01-01

    Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS) whose function is modulated, in part, by specific protein interactions. By coimmunoprecipitation experiments followed by MS analyses, we identified a human voltage-dependent anion/cation channel or porin as a binding partner of eNOS. The interaction between porin and eNOS was demonstrated by coimmunoprecipitation studies in nontransfected human endothelial cells and Cos-7 cells transiently transfected with eNOS and porin cDNAs. In vitro binding studies with glutathione S-transferase–porin indicated that porin binds directly to eNOS and that this interaction augmented eNOS activity. The calcium ionophore, A23187, and bradykinin, which are known to activate eNOS, markedly increased porin–eNOS interaction, suggesting a potential role of intracellular Ca2+ in mediating this interaction. Theses results indicate that the interaction between a voltage-dependent membrane channel and eNOS may be important for regulating eNOS activity. PMID:12228731

  18. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    Science.gov (United States)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

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

    Directory of Open Access Journals (Sweden)

    Zhonglei Lu

    Full Text Available 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.

  20. Isolation and Molecular Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase Genes in Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Jia-Hong Zhu

    2015-02-01

    Full Text Available Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1–7 of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:27559295

  3. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  4. Fatty acid synthase inhibitors from plants: isolation, structure elucidation, and SAR studies.

    Science.gov (United States)

    Li, Xing-Cong; Joshi, Alpana S; ElSohly, Hala N; Khan, Shabana I; Jacob, Melissa R; Zhang, Zhizheng; Khan, Ikhlas A; Ferreira, Daneel; Walker, Larry A; Broedel, Sheldon E; Raulli, Robert E; Cihlar, Ronald L

    2002-12-01

    Fatty acid synthase (FAS) has been identified as a potential antifungal target. FAS prepared from Saccharomyces cerevisiae was employed for bioactivity-guided fractionation of Chlorophora tinctoria,Paspalum conjugatum, Symphonia globulifera, Buchenavia parviflora, and Miconia pilgeriana. Thirteen compounds (1-13), including three new natural products (1, 4, 12), were isolated and their structures identified by spectroscopic interpretation. They represented five chemotypes, namely, isoflavones, flavones, biflavonoids, hydrolyzable tannin-related derivatives, and triterpenoids. 3'-Formylgenistein (1) and ellagic acid 4-O-alpha-l-rhamnopyranoside (9) were the most potent compounds against FAS, with IC(50) values of 2.3 and 7.5 microg/mL, respectively. Furthermore, 43 (14-56) analogues of the five chemotypes from our natural product repository and commercial sources were tested for their FAS inhibitory activity. Structure-activity relationships for some chemotypes were investigated. All these compounds were further evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. Although there were several antifungal compounds in the set, correlation between the FAS inhibitory activity and antifungal activity could not be defined. PMID:12502337

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:26501439

  7. Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

    DEFF Research Database (Denmark)

    Prats, Clara; Cadefau, Joan A; Cussó, Roser;

    2005-01-01

    Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively...

  8. Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases

    DEFF Research Database (Denmark)

    Lundgren, S.; Gojkovic, Zoran; Piskur, Jure;

    2003-01-01

    beta-Alanine synthase (betaAS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, betaASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We...

  9. Apoptosis in rat gastric antrum: Evidence that regulation by food intake depends on nitric oxide synthase

    DEFF Research Database (Denmark)

    Cao, Bao-Hong; Mortensen, Kirsten; Tornehave, Ditte;

    2000-01-01

    of gastric endocrine cells. On starvation, the apoptotic index of the general epithelium and of the gastrin but not of the somatostatin, cells increased significantly. This was prevented by the nitric oxide synthase (NOS) inhibitor L-NAME but not by its inactive stereoisomer D-NAME. Immunoreactive neuronal...

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  11. Curcumin inhibits intracellular fatty acid synthase and induces apoptosis in human breast cancer MDA-MB-231 cells.

    Science.gov (United States)

    Fan, Huijin; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; Sun, Jia; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-05-01

    High levels of fatty acid synthase (FAS) expression have been found in many tumors, including prostate, breast, and ovarian cancers, and inhibition of FAS has been reported to obstruct tumor growth in vitro and in vivo. Curcumin is one of the major active ingredients of Curcuma longa, which has been proven to inhibit the growth of cancer cells. In the present study, we investigated the potential activity of curcumin as a FAS inhibitor for chemoprevention of breast cancer. As a result, curcumin induced human breast cancer MDA-MB-231 cell apoptosis with the half-inhibitory concentration value of 3.63 ± 0.26 µg/ml, and blocked FAS activity, expression and mRNA level in a dose-dependent manner. Curcumin also regulated B-cell lymphoma 2 (Bcl-2), Bax and p-Akt protein expression in MDA-MB-231 cells. Moreover, FAS knockdown showed similar effect as curcumin. All these results suggested that curcumin may induce cell apoptosis via inhibiting FAS. PMID:26985864

  12. Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

    Science.gov (United States)

    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

  13. Genome-wide changes accompanying knockdown of fatty acid synthase in breast cancer

    Directory of Open Access Journals (Sweden)

    Smith Jeffrey W

    2007-06-01

    Full Text Available Abstract Background The lipogenic enzyme fatty acid synthase (FAS is up-regulated in a wide variety of cancers, and is considered a potential metabolic oncogene by virtue of its ability to enhance tumor cell survival. Inhibition of tumor FAS causes both cell cycle arrest and apoptosis, indicating FAS is a promising target for cancer treatment. Results Here, we used gene expression profiling to conduct a global study of the cellular processes affected by siRNA mediated knockdown of FAS in MDA-MB-435 mammary carcinoma cells. The study identified 169 up-regulated genes (≥ 1.5 fold and 110 down-regulated genes (≤ 0.67 fold in response to knockdown of FAS. These genes regulate several aspects of tumor function, including metabolism, cell survival/proliferation, DNA replication/transcription, and protein degradation. Quantitative pathway analysis using Gene Set Enrichment Analysis software further revealed that the most pronounced effect of FAS knockdown was down-regulation in pathways that regulate lipid metabolism, glycolysis, the TCA cycle and oxidative phosphorylation. These changes were coupled with up-regulation in genes involved in cell cycle arrest and death receptor mediated apoptotic pathways. Conclusion Together these findings reveal a wide network of pathways that are influenced in response to FAS knockdown and provide new insight into the role of this enzyme in tumor cell survival and proliferation.

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

    Science.gov (United States)

    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.

  15. Electron microscope and small angle neutron scattering studies of chicken liver fatty acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Stoops, J.K.; Wakil, S.J.; Uberbacher, E.C.; Bunick, G.J.

    1986-05-01

    Electron microscopic studies of negatively stained chicken liver fatty acid synthase revealed images of various shapes and sizes. The dimeric structures could be related to each other as rod-life in open form and C-like in closed form. The rods measure 200A and 50A in their major and minor axis, respectively. The C-shaped structures have a diameter ranging from 70-100A, representing the degree to which they are closed. The model that most accurately represents the native enzyme was determined using small angle neutron scattering of the active enzyme in solution. These studies resulted in considerable refinement of the model obtained by electron microscopy. The enzyme has a radius of gyration of 58A and the scattering curves were best fit by a model in which the dimeric enzyme consisted of two side by side ellipsoidal cylinders with overall dimension of 150A X 136A X 60A. The molecule has a cleft extending the length of the major axis with a 5A overlap between the two cylinders. The ellipsoidal cross section of the subunit has a major and minor axis and 70 and 60A, respectively. This model is compatible with the linear functional model proposed earlier.

  16. Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

    OpenAIRE

    Patel, S.; Gaspers, L. D.; Boucherie, S.; Memin, E.; Stellato, K. A.; Guillon, G; Combettes, L; Thomas, A P

    2002-01-01

    Increases in both Ca2+ and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca2+ signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mim...

  17. Biophysical Investigation of the Mode of Inhibition of Tetramic Acids, the Allosteric Inhibitors of Undecaprenyl Pyrophosphate Synthase

    OpenAIRE

    Lee, Lac V.; Granda, Brian; Dean, Karl; Tao, Jianshi; Liu, Eugene; Zhang, Rui; Peukert, Stefan; Wattanasin, Sompong; XIE, XIAOLING; Ryder, Neil S.; Tommasi, Ruben; Deng, Gejing

    2010-01-01

    Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C55 undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was desi...

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

    Science.gov (United States)

    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 Å(2) are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA. PMID:27501775

  19. The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB.

    Science.gov (United States)

    Hamilton, Christopher S; Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2005-01-01

    All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.

  20. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    International Nuclear Information System (INIS)

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 310-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins

  1. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Geun [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of); Park, Chin-Ju [Gwangju Institute of Science and Technology, Division of Liberal Arts and Sciences and Department of Chemistry (Korea, Republic of); Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa, E-mail: joonhwa@gnu.ac.kr [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)

    2015-02-15

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3{sub 10}-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:23832368

  6. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Directory of Open Access Journals (Sweden)

    Micaela S Sordelli

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  7. Molecular cloning and regulation of murine fatty acid synthase mRNA

    International Nuclear Information System (INIS)

    Mouse liver mRNA that was enriched in sequences coding for fatty acid synthase (FAS) by sucrose-density gradient centrifugation was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into pBR322 and λgt10 and cloned. Clones containing putative cDNA sequences for FAS were identified by differential hybridization where 32P-cDNAs, synthesized from sucrose gradient purified liver mRNA from mice starved or starved and refed a fat-free diet, were used as probes. Two of these clones were further studied and found to contain sequences complementary to FAS mRNA by hybrid-selected translation and specific immunoprecipitation. Using these clones as probes, they selected 33 additional clones containing cDNA sequences for FAS. Partial DNA sequence data for these clones were obtained. Northern blot analysis revealed a single mRNA size of 9.3 kb when a cDNA clone with a 3.1 kb insert was used as a probe. This is in contrast to rat liver FAS which showed two mRNAs sizes of 9.2 and 10.0 kb. They also studied FAS mRNA level of 3T3-L1 preadipocytes during differentiation into adipocytes. An approximate 10-fold increase in FAS mRNA content was observed which corresponded with an increased rate of FAS synthesis indicating pretranslational regulation. The FAS cDNA probe was also employed to demonstrate that induction of FAS in the livers of previously starved mice that were fed a fat-free diet was controlled pretranslationally by a parallel modulation of the FAS mRNA concentration

  8. Effect of estrogen on gene expression of fatty acid synthase in periosteum

    Institute of Scientific and Technical Information of China (English)

    ZHENG Rui-min; LIN Shou-qing; LIU Yong; HUANG Man-ting; GONG Wei-yan; WU Zhi-hong

    2009-01-01

    Background Estrogen deficiency contributes to postmenopausal osteoporosis.Periosteum might be a potential target of estrogen,but the underlying mechanism at gene level is far from being elucidated.The objective of this study was to investigate the correlation between estrogen and fatty acid synthase(FAS)expression in periosteum.Methods Human periosteum cells were cultured in vitro.Expressed genes in the substrated cDNA library were verified using semi-quantitative PCR and real-time PCR.The expression of FAS in periosteum of ovarectomized(OVX)SD rats was investigated.Results FAS gene was most significantly expressed in the subtracted cDNA library of periosteal cells screened by semi-quantitative PCR.Low FAS expression was verified by real-time PCR in the estrogen exposed human periosteum rather than in the control.The estradiol levels were(20.81±12.62)pg/ml,(19.64±4.35)pg/ml and(13.47+1.84)pg/ml in the sham group,the control,and the OVX group,respectively.The estradiol levels in the OVX group was significantly lower(P=0.0386).The FAS gene expression in periosteum in the OVX group,sham group,and control group was 3.09±1.97,1.33±0.47 and 1.51±1.32,respectively.The gene expression in the OVX group was significantly higher (P=0.0372).Conclusion Estrogen modulates FAS gene expression in in vitro human perisoteum as well as in in vivo rat periosteum.

  9. Acid sphingomyelinase gene knockout ameliorates hyperhomocysteinemic glomerular injury in mice lacking cystathionine-β-synthase.

    Directory of Open Access Journals (Sweden)

    Krishna M Boini

    Full Text Available Acid sphingomyelinase (ASM has been implicated in the development of hyperhomocysteinemia (hHcys-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs and Asm mouse gene by cross breeding Cbs(+/- and Asm(+/- mice. Given that the homozygotes of Cbs(-/-/Asm(-/- mice could not survive for 3 weeks. Cbs(+/-/Asm(+/+, Cbs(+/-/Asm(+/- and Cbs(+/-/Asm(-/- as well as their Cbs wild type littermates were used to study the role of Asm(-/- under a background of Cbs(+/- with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs(+/- mice with different copies of Asm gene compared to Cbs(+/+ mice with different Asm gene copies. Cbs(+/-/Asm(+/+ mice had significantly increased renal Asm activity, ceramide production and O(2.(- level compared to Cbs(+/+/Asm(+/+, while Cbs(+/-/Asm(-/- mice showed significantly reduced renal Asm activity, ceramide production and O(2.(- level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs(+/-/Asm(-/- mice compared to Cbs(+/-/Asm(+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs(+/-/Asm(-/- mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs(+/-/Asm(-/- mice compared to Cbs(+/-/Asm(+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O(2.(- production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme

  10. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    International Nuclear Information System (INIS)

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the β-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution

  11. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Soo-Ik (Harvard Medical School, Boston, MA (USA)); Hammes, G.G. (Univ. of California, Santa Barbara (USA))

    1989-11-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.

  12. Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.

    Science.gov (United States)

    Bienert, Roland; Zimmermann, Boris; Rombach-Riegraf, Verena; Gräber, Peter

    2011-02-25

    H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

  13. Starvation for ilvB operon leader amino acids other than leucine or valine does not increase acetohydroxy acid synthase activity in Escherichia coli.

    OpenAIRE

    Tsui, P; Freundlich, M

    1985-01-01

    Eleven different amino acids are encoded in the ilvB leader mRNA. Starvation for leucine or valine, but not for any of the other nine amino acids, resulted in high levels of acetohydroxy acid synthase I. These results are discussed in terms of a report (C.A. Hauser and G.W. Hatfield, Proc. Natl. Acad. Sci. U.S.A. 81:76-79, 1984) which suggests that threonine and alanine, in addition to leucine and valine, are involved in the regulation of the ilvB operon.

  14. Aronia melanocarpa juice, a rich source of polyphenols, induces endothelium-dependent relaxations in porcine coronary arteries via the redox-sensitive activation of endothelial nitric oxide synthase.

    Science.gov (United States)

    Kim, Jong Hun; Auger, Cyril; Kurita, Ikuko; Anselm, Eric; Rivoarilala, Lalainasoa Odile; Lee, Hyong Joo; Lee, Ki Won; Schini-Kerth, Valérie B

    2013-11-30

    This study examined the ability of Aronia melanocarpa (chokeberry) juice, a rich source of polyphenols, to cause NO-mediated endothelium-dependent relaxations of isolated coronary arteries and, if so, to determine the underlying mechanism and the active polyphenols. A. melanocarpa juice caused potent endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to A. melanocarpa juice were minimally affected by inhibition of the formation of vasoactive prostanoids and endothelium-derived hyperpolarizing factor-mediated responses, and markedly reduced by N(ω)-nitro-l-arginine (endothelial NO synthase (eNOS) inhibitor), membrane permeant analogs of superoxide dismutase and catalase, PP2 (Src kinase inhibitor), and wortmannin (PI3-kinase inhibitor). In cultured endothelial cells, A. melanocarpa juice increased the formation of NO as assessed by electron paramagnetic resonance spectroscopy using the spin trap iron(II)diethyldithiocarbamate, and reactive oxygen species using dihydroethidium. These responses were associated with the redox-sensitive phosphorylation of Src, Akt and eNOS. A. melanocarpa juice-derived fractions containing conjugated cyanidins and chlorogenic acids induced the phosphorylation of Akt and eNOS. The present findings indicate that A. melanocarpa juice is a potent stimulator of the endothelial formation of NO in coronary arteries; this effect involves the phosphorylation of eNOS via the redox-sensitive activation of the Src/PI3-kinase/Akt pathway mostly by conjugated cyanidins and chlorogenic acids.

  15. Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

    Science.gov (United States)

    Feraud, Magali; Masclaux-Daubresse, Céline; Ferrario-Méry, Sylvie; Pageau, Karine; Lelandais, Maud; Ziegler, Christine; Leboeuf, Edouard; Jouglet, Tiphaine; Viret, Lauriane; Spampinato, Axelle; Paganelli, Vanina; Hammouda, Mounir Ben; Suzuki, Akira

    2005-11-01

    GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides

  16. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole;

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  17. Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

    Science.gov (United States)

    Patel, Sandip; Gaspers, Lawrence D; Boucherie, Sylviane; Memin, Elisabeth; Stellato, Kerri Anne; Guillon, Gilles; Combettes, Laurent; Thomas, Andrew P

    2002-09-13

    Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation. PMID:12097323

  18. Increased fatty acid synthase as a potential therapeutic target in multiple myeloma

    Institute of Scientific and Technical Information of China (English)

    Wei-qin WANG; Xiao-ying ZHAO; Hai-yan WANG; Yun LIANG

    2008-01-01

    Objective: To determine fatty acid synthase (FAS) expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma. Methods: FAS expression was determined by immunohistochemistry, reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma (MM patients) and peripheral blood mononuclear cells (PBMCs) obtained from 12 healthy donors. In parallel, additional analyses were performed on 2 human multiple myeloma cell lines, U266 and RPMI8226. U266 cells were treated with cerulenin at various concentrations (5 to 320μg/ml) for 24 h, and metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Apoptosis was evaluated by dual Annexin V/PI (propidium iodide) labeling and flow cytometry (FCM) in U266 cells treated with 20μg/ml cerulenin for 12 h or 24 h. Results: By immunohistochemistry, we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS. Similarly, by RT-PCR, 22 of 27 bone marrow samples obtained from MM patients, U266 and RPMI8226 showed FAS expression, whereas PBMC samples from 12 healthy donors did not express detectable level of FAS. FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients, U266 and RPMI8226 cell lines, and no FAS protein expression was detected in PBMC samples from 12 healthy donors. U266 cells were highly sensitive to cerulenin treatment, with a dosage-related effect on metabolic activity, as a measure for cell proliferation. U266 cells treated with20 μg/ml cerulenin for 12 and 24h also showed early sign of apoptosis with 56.9% and 69.3% Annexin V+/PI+ cells, and late apoptotic and necrotic cells with 3.2% and 17.6% Annexin V+/PI+ cells. Conclusion: Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines

  19. Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase.

    Science.gov (United States)

    Austin, Michael B; Saito, Tamao; Bowman, Marianne E; Haydock, Stephen; Kato, Atsushi; Moore, Bradley S; Kay, Robert R; Noel, Joseph P

    2006-09-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis. PMID:16906151

  20. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses.

    Science.gov (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity.

  1. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros ketoacyl-ACP synthase

    Directory of Open Access Journals (Sweden)

    Huiya eGu

    2016-05-01

    Full Text Available 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 is 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 (~2/3 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 lacking bacteria evolutionary control mechanisms could be used to improve MCFA production in this promising production strains. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to five fold. The level of increase is dependent on promoter strength and culturing conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Enderle, Mathias [Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main (Germany); Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); McCarthy, Andrew [EMBL Grenoble, 71 Avenue des Martyrs, 38042 Grenoble CEDEX 9 (France); Paithankar, Karthik Shivaji, E-mail: paithankar@em.uni-frankfurt.de [Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main (Germany); Grininger, Martin, E-mail: paithankar@em.uni-frankfurt.de [Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main (Germany); Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

  4. A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence

    Science.gov (United States)

    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. Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum

    OpenAIRE

    Bitencourt, Tamires Aparecida; Komoto, Tatiana Takahasi; Massaroto, Bruna Gabriele; Miranda, Carlos Eduardo Saraiva; Beleboni, Rene Oliveira; Marins, Mozart; Fachin, Ana Lúcia

    2013-01-01

    Background Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis. Methods The antifungal activity of th...

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

    Energy Technology Data Exchange (ETDEWEB)

    Hopperton, Kathryn E., E-mail: kathryn.hopperton@mail.utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Duncan, Robin E., E-mail: robin.duncan@uwaterloo.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Bazinet, Richard P., E-mail: richard.bazinet@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Archer, Michael C., E-mail: m.archer@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada)

    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

  7. The nopaline synthase (nos) promoter is inducible by UV-B radiation through a pathway dependent on reactive oxygen species

    International Nuclear Information System (INIS)

    The molecular mechanism of plant response to UV-B radiation was studied using the nopaline synthase (nos) promoter, which has been shown to be inducible by methyl jasmonate (MJ) and reactive oxygen species (ROS). In the leaves of transgenic tobacco (Nicotiana tabacum L.) plants that carried a fusion between the nos promoter and the chloramphenicol acetyltransferase (cat) gene, 2 h of UV-B treatment resulted in a transient increase in the level of cat mRNA, a maximum being reached at 6 h after the UV-B treatment. It was also found that MJ and UV-B enhance nos promoter expression via separate pathways. Diethyldithiocarbamic acid, a potent inhibitor of jasmonate production, had little effect on UV-B stimulation of the nos promoter. In contrast, antioxidants, such as dimethylthiourea, reduced glutathione, cysteine, N-acetylcysteine and DTT, blocked UV-B induction of the nos promoter, but did not affect MJ induction of the nos promoter. These results suggest that UV-B induction of the nos promoter is mediated via a pathway that requires reactive oxygen species and is distinct from the jasmonate or MJ mediating pathway. (author)

  8. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Kruse, Rikke; Harvald, Eva Bang;

    2013-01-01

    of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also......-1 result in dietary restriction-induced autophagy and consequently prolonged longevity....

  9. Role of Plant Fatty acid Elongase (3 keto acyl-CoA Synthase gene in Cuticular Wax Biosynthesis

    Directory of Open Access Journals (Sweden)

    Uppala Lokesh

    2013-12-01

    Full Text Available Plant surfaces are ensheathed by cuticular wax, amorphous intra-cuticular embedded in cutin polymer and crystalloid epi-cuticular that imparts a whitish appearance, confers drought resistance by reducing stomatal transpiration and also protects from U.V Radiation, phytophagous insects etc. Very long chain fatty acids acts as precursors for cuticular wax bio-synthesis. Wax bio-synthesis begins with fatty acid synthesis in the plastid (de novo synthesis of C16 and C18 and elongation of fatty acids in endoplasmic reticulum (C20 – C34 by four distinct enzymes 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase, 3-hydroxacyl-CoA dehydratase, trans-2,3-enoyl-CoA reductase (KCS, KCR, HCD, ECR. The KCS, a fatty acid elongase, determines the chain length and substrate specificity of the condensation reaction, a rate limiting step and the subsequent elongated products alkanes, aldehydes, primary alcohols, secondary alcohols, ketones and wax esters. 21 KCS genes were annotated in Arabidopsis thaliana Genome of which some KCSs were identified involved in cuticle formation (CER6 (CUT1, KCS1, KCS2, (DAISY, KCS20 and FDH.The current review will focus on the bio-chemical, genetic and molecular approaches on KCSs genes, predominantly KCS1 in plants particularly useful in identifying and characterizing gene products involved in wax bio-synthesis, secretion and function for developing transgenic crops that combat various stresses. INTRODUCTION

  10. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    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.

  11. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  12. Expression of nitric oxide synthase in T-cell-dependent liver injury initiated by ConA in Kunming mice

    Institute of Scientific and Technical Information of China (English)

    张修礼; 曲建慧; 万谟彬; 权启镇; 孙自勤; 王要军; 江学良; 李文波

    2004-01-01

    Objective: To investigate whether nitric oxide synthase (NOS) is expressed in T-cell-dependent liver injury initiated by concanavalin A (ConA) in Kunming mice and study the possible effect of nitric oxide(NO) on liver injury models. Methods: Liver injury in Kunming mice was induced by administration of ConA through tail vein. Expression of NOS in the liver was detected by NADPH diaphorase staining method. The possible effect of NO on liver injury models was obtained by L-NAME injection to suppress synthesis of NO. Results: NOS has a strong expression in hepatocytes after ConA injection, especially in those close to the central vein, while only a weak expression was found in the epithelial cells in control group. Liver injury became more serious when NO synthesis was inhibited by L-NAME, accompanied by great malondialdehyde(MDA) increase in serum and severe intrahepatic vascular thrombosis. Conclusion: NOS markedly expressed in ConAinduced liver injury, which may subsequently promote nitric oxide synthesis. Increasement of nitric oxide has a protective effect on ConA-induced liver injury.

  13. Effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthase inhibitor

    Institute of Scientific and Technical Information of China (English)

    Jun-lin JIANG; De-jian JIANG; Yu-hai TANG; Nian-sheng LI; Han-wu DENG; Yuan-jian LI

    2004-01-01

    AIM: To investigate the effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthesis inhibitor asymmetric dimethylarginine (ADMA) in rats and cultured ECV304 cells. METHODS: Endothelial injury was induced by a single injection of low density lipoprotein (LDL) (4 mg/kg, 48 h) in rats or incubation with LDL (300 mg/L) or oxidative-modified LDL (100 mg/L) in cultured ECV304 cells, and vasodilator responses to acetylcholine (ACh) in the aortic rings and the level of ADMA, nitrite/nitrate (NO) and tumor necrosis factoralpha (TNF-α) in the serum or cultured medium were determined. And the adhesion of the monocytes to endothelial cells and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the cultured ECV304 cells were measured. RESULTS: A single injection of LDL decreased endothelium-dependent relaxation to ACh, markedly increased the serum level of endogenous ADMA and TNF-α, and reduced serum level of NO. Pretreatment with simvastatin (30 or 60 mg/kg) markedly attenuated inhibition of vasodilator responses to ACh, the increased level of TNF-α and the decreased level of NO by LDL, but no effect on serum concentration of endogenous ADMA. In cultured ECV304 cells, LDL or ox-LDL markedly increased the level of ADMA and TNF-α and potentiated the adhesion of monocytes to endothelial cells, concomitantly with a significantly decrease in the activity of DDAH and serum level of NO. Pretreatment with simvastatin (0.1, 0.5, or 2.5 μmol/L) markedly decreased the level of TNFo and the adhesion of monocytes to endothelial cells, but did not affect the concentration of endogenous ADMA and the activity of DDAH. CONCLUSION: Simvastatin protect the vascular endothelium against the damages induced by LDL or ox-LDL in rats or cultured ECV304 cells, and the beneficial effects of simvastatin may be related to the reduction of inflammatory cytokine TNF-o level.

  14. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

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

    Directory of Open Access Journals (Sweden)

    Nataliya E Chorna

    Full Text Available 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.

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

    Science.gov (United States)

    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. PMID:27369551

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

    Science.gov (United States)

    Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

    2016-09-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

  18. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    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.

  20. SFH2 regulates fatty acid synthase activity in the yeast Saccharomyces cerevisiae and is critical to prevent saturated fatty acid accumulation in response to haem and oleic acid depletion.

    Science.gov (United States)

    Desfougères, Thomas; Ferreira, Thierry; Bergès, Thierry; Régnacq, Matthieu

    2008-01-01

    The yeast Saccharomyces cerevisiae is a facultative anaerobic organism. Under anaerobiosis, sustained growth relies on the presence of exogenously supplied unsaturated fatty acids and ergosterol that yeast is unable to synthesize in the absence of oxygen or upon haem depletion. In the absence of exogenous supplementation with unsaturated fatty acid, a net accumulation of SFA (saturated fatty acid) is observed that induces significant modification of phospholipid profile [Ferreira, Régnacq, Alimardani, Moreau-Vauzelle and Bergès (2004) Biochem. J. 378, 899-908]. In the present paper, we focus on the role of SFH2/CSR1, a hypoxic gene related to SEC14 and its involvement in lipid metabolism upon haem depletion in the absence of oleic acid supplementation. We observed that inactivation of SFH2 results in enhanced accumulation of SFA and phospholipid metabolism alterations. It results in premature growth arrest and leads to an exacerbated sensitivity to exogenous SFA. This phenotype is suppressed in the presence of exogenous oleic acid, or by a controlled expression of FAS1, one of the two genes encoding FAS. We present several lines of evidence to suggest that Sfh2p and oleic acid regulate SFA synthase in yeast at different levels: whereas oleic acid acts on FAS2 at the transcriptional level, we show that Sfh2p inhibits fatty acid synthase activity in response to haem depletion. PMID:17803462

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

    Directory of Open Access Journals (Sweden)

    Ahmed M. Naglah

    2016-04-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  3. Regulation of expression of citrate synthase by the retinoic acid receptor-related orphan receptor α (RORα.

    Directory of Open Access Journals (Sweden)

    Christine Crumbley

    Full Text Available The retinoic acid receptor-related orphan receptor α (RORα is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression.

  4. Increased expression of fatty acid synthase and acetyl-CoA carboxylase in the prefrontal cortex and cerebellum in the valproic acid model of autism

    Science.gov (United States)

    Chen, Jianling; Wu, Wei; Fu, Yingmei; Yu, Shunying; Cui, Donghong; Zhao, Min; Du, Yasong; Li, Jijun; Li, Xiaohong

    2016-01-01

    The primary aim of the present study was to investigate alterations in enzymes associated with fatty acid synthesis, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), in the prefrontal cortex and cerebellum of the valproic acid (VPA)-induced animal model of autism. In this model, pregnant rats were given a single intraperitoneal injection of VPA, and prefrontal cortex and cerebellum samples from their pups were analyzed. The results of western blotting and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that the protein and mRNA expression levels of FASN, ACC and phospho-ACC (pACC) were increased in the prefrontal cortex and cerebellum of the VPA model of autism. Furthermore, in the prefrontal cortex and cerebellum of the VPA model of autism, AMPK expression is increased, whereas PI3K and Akt expression are unchanged. This suggests that disorder of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/FASN and/or adenosine 5′-monophosphate-activated protein kinase (AMPK)/ACC pathway may be involved in the pathogenesis of autism. It is hypothesized that fatty acid synthesis participates in autism through PI3K/Akt/FASN and AMPK/ACC pathways. PMID:27602061

  5. Role of Nitric Oxide Dependence on Nitric Oxide Synthase-like Activity in the Water Stress Signaling of Maize Seedling

    Institute of Scientific and Technical Information of China (English)

    Gang-Ping Hao; Yu Xing; Jian-Hua Zhang

    2008-01-01

    Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-lika (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings. NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramathylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation Inhibited by NOS inhibitor L-NAME In dehydration-treated maize seedlings Indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-trested maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutsse (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity

  6. Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism

    Science.gov (United States)

    The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of m...

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    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.

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

    International Nuclear Information System (INIS)

    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

  10. Protection of INS-1 Cells from Free Fatty Acid-induced Apoptosis by Inhibiting the Glycogen Synthase Kinase-3

    Institute of Scientific and Technical Information of China (English)

    WU Wei; LUO Xiaoping

    2007-01-01

    To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 dia-betes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhib-ited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apop-tosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic β-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.

  11. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

    Directory of Open Access Journals (Sweden)

    Honghao Chen

    2013-12-01

    Full Text Available Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic. Based on previous research, these effects are mediated by a number of active ingredients, especially glycyrrhizic acid (GA. In the present study, a gene encoding β-amyrin synthase (β-AS involved in GA biosynthesis in G. uralensis has been cloned and expressed in Saccharomyces cerevisiae. The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene into β-amyrin. In fact the β-AS gene is particularly important in the GA biosynthetic pathway in G. uralensis. The complete sequence of the enzyme was determined and a phylogenetic tree based on the β-AS gene of G. uralensis and 20 other species was created. This showed that Glycyrrhiza glabra had the closest kinship with G. uralensis. The results of this work will be useful in determining how to improve the efficacy of G. uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

  12. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    Science.gov (United States)

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  13. Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

    Science.gov (United States)

    Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2010-06-01

    In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition. PMID:20127879

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

    OpenAIRE

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

    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 storag...

  15. Inhibition of nitric oxide synthase lowers fatty acid oxidation in preeclampsia-like mice at early gestational stage

    Institute of Scientific and Technical Information of China (English)

    MA Rui-qiong; SUN Min-na; YANG Zi

    2011-01-01

    Background Preeclampsia is one of hypertensive disorders in pregnancy. It is associated with abnormal lipid metabolism, including fatty acid oxidation metabolism. Long chain 3-hydroxyacyI-CoA dehydrogenase (LCHAD) plays an indispensable role in the oxidation of fatty acids. It has been reported that nitric oxide (NO) is one of the regulatory factors of the fatty acid oxidation pathway. The aim of this research was to investigate whether the nitric oxide synthase (NOS)inhibitor L-NAME may cause down-regulation of LCHAD in the pathogenesis of preeclampsia.Methods Pregnant wild-type (WT) mice were treated with L-NAME or normal saline (NS) during gestation days 7-18 (early group), days 11-18 (mid group) and days 16-18 (late group), and apoE-/- mice served as a control. Systolic blood pressure (SBP), urine protein, feto-placental outcome, plasma lipid levels and NO concentrations were measured, and the expression of mRNA and protein for LCHAD in placental tissue were determined by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively.Results In WT and apoE-/- mice, SBP and urinary protein increased following L-NAME injection. Fetal and placental weights and NO concentrations were reduced and total cholesterol, triglycerides and free fatty acid levels were increased in early and mid L-NAME groups in WT and apoE-/- mice, compared with the NS group. There was no significant difference between the late L-NAME group and NS group. RT-PCR and Western blotting analysis showed that the mRNA and protein levels of LCHAD expression were significantly down-regulated in the early and mid L-NAME groups but not in the late L-NAME group in the WT and apoE-/- mice compared with the corresponding NS groups.Conclusions Inhibition of NO in early and mid gestation in mice may cause hyperlipidemia and suppression of fatty acid oxidation, whereas preeclampsia-like conditions in late gestation may be a maternal vascular response to inhibition of NO.

  16. Crystal Structures of the Iron–Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate

    Energy Technology Data Exchange (ETDEWEB)

    Fenwick, Michael K. [Cornell Univ., Ithaca, NY (United States); Ealick, Steven E. [Cornell Univ., Ithaca, NY (United States)

    2016-07-12

    The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

  17. Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source.

    OpenAIRE

    Dailey, F E; Cronan, J E

    1986-01-01

    Escherichia coli K-12 has two acetohydroxy acid synthase (AHAS) isozymes (AHAS I and AHAS III). Both of these isozymes catalyze the synthesis of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are key intermediates of the isoleucine-valine biosynthetic pathway. Strains lacking either isozyme but not both activities have been previously shown to grow well in minimal media in the absence of isoleucine and valine on any of several commonly used carbon sources (e.g., glucose or su...

  18. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties

    OpenAIRE

    Ehsan Karimi; Jaafar, Hawa Z. E.; Ali Ghasemzadeh

    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 t...

  19. Retinoic acid triggers meiosis initiation via stra8-dependent pathway in Southern catfish, Silurus meridionalis.

    Science.gov (United States)

    Li, Minghui; Feng, Ruijuan; Ma, He; Dong, Ranran; Liu, Zhilong; Jiang, Wentao; Tao, Wenjing; Wang, Deshou

    2016-06-01

    Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts. PMID:26764212

  20. Comparative Amino Acids Studies on Phac Synthases and Proteases as Well as Establishing a New Trend in Experimental Design

    Directory of Open Access Journals (Sweden)

    Amro Abd al fattah Amara

    2012-04-01

    Full Text Available ABSTRACT: A question addressed in this study is: why similar enzymes are classified into different subclasses? As an example, PhaC synthases are classified according to four different classes (I, II, III and IV. To answer this question we proposed that besides the catalytic residues, the overall amino acids (AAs present are responsible for the differences observed. The AAs’ composition affects the structure/function/substrate specificity (SFS of these enzymes. The differences between the classes in various PhaC synthases and proteases were analysed to support our argument. Homology and phylogenic tree of some selected PhaC synthases of different strains (representing the four classes were demonstrated. The properties of a specific class of enzyme could not be changed into those of another by changing the catalytic residues. Moreover, these differences could not be detected from the proteins’ 3D structures, despite clear differences at the AAs level. Another question was also addressed: could we benefit from the various existing protein databases in the field of biotechnology? To answer this, we introduced a model for an Experimental Design based on the information in the protein database (for strains available in our lab regarding their ability to degrade castor oil. Two enzymes in the phenol degradation pathway, phenol 2-monooxygenase and catechol 1,2-dioxygenase, and a lipase enzyme were analysed. These enzymes were screened and analysed according to the BLAST-protein database and BRENDA. The comprehensive enzyme information system compared six strains against each other, including: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis, and Geobacillus stearothermophilus. Only P. aeruginosa proved to have the three required enzymes and was suitable for the production of lipases from castor oil (crude castor oil is usually contaminated with phenol as indicated by the databases. In

  1. Investigation of a 6-MSA Synthase Gene Cluster in Aspergillus aculeatus Reveals 6-MSA-derived Aculinic Acid, Aculins A-B and Epi-Aculin A

    DEFF Research Database (Denmark)

    Petersen, Lene Maj; Holm, Dorte Koefoed; Gotfredsen, Charlotte Held;

    2015-01-01

    . 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 13C-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....

  2. The role of ß-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

    DEFF Research Database (Denmark)

    González-Mellado, Damián; von Wettstein, Penny; Garcés, Rafael;

    2010-01-01

    The ß-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing...... proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons...

  3. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    Science.gov (United States)

    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. PMID:18682402

  4. Valproic acid-mediated transcriptional regulation of human GM3 synthase (hST3Gal V) in SK-N-BE(2)-C human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Haw-young KWON; Nam-young KANG; Hyun-mi DAE; Kyoung-sook KIM; Cheorl-ho KIM; Su-il DO; Young-choon LEE

    2008-01-01

    Aim:To investigate whether valproic acid (VPA) modulates human GM3 syn-thase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthe-sis, in human neuroblastoma cells. Methods: Using RT-PCR and immunofluo-rescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3-Gal V gene using luciferase constructs carrying 5'-deletions of the hST3Gal V promoter. Results: RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5'-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, site-directed mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-N-BE(2)-C cells. Conclusion: Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.

  5. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    Science.gov (United States)

    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. PMID:25788017

  6. 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.

    Science.gov (United States)

    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

  7. 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

    Science.gov (United States)

    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

  8. Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

    Directory of Open Access Journals (Sweden)

    Uldaeliz Trujillo

    Full Text Available The polyunsaturated fatty acid (PUFA synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect and in structural stabilization of the multidomain protein (synergistic effect. While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of

  9. Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

    KAUST Repository

    Trujillo, Uldaeliz

    2013-02-28

    The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP

  10. Palmitic acid exerts pro-inflammatory effects on vascular smooth muscle cells by inducing the expression of C-reactive protein, inducible nitric oxide synthase and tumor necrosis factor-α.

    Science.gov (United States)

    Wu, Di; Liu, Juntian; Pang, Xiaoming; Wang, Shuyue; Zhao, Jingjing; Zhang, Xiaolu; Feng, Liuxin

    2014-12-01

    Atherosclerosis is a chronic inflammatory disease in the vessel, and inflammatory cytokines play an important role in the inflammatory process of atherosclerosis. A high level of free fatty acids (FFAs) produced in lipid metabolism disorders are known to participate in the formation of atherosclerosis through multiple bioactivities. As the main saturated fatty acid in FFAs, palmitic acid stimulates the expression of inflammatory cytokines in macrophages. However, it is unclear whether palmitic acid exerts a pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe the effect of palmitic acid on the expression of C-reactive protein (CRP), tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) in VSMCs. Rat VSMCs were cultured, and palmitic acid was used as a stimulant for CRP, TNF-α and iNOS expression. mRNA expression was assayed with reverse transcription-polymerase chain reaction, and protein expression was detected with western blot analysis and immunocytochemistry. The results showed that palmitic acid significantly stimulated mRNA and protein expression of CRP, TNF-α and iNOS in VSMCs in time- and concentration-dependent manners, and therefore, palmitic acid is able to exert a pro-inflammatory effect on VSMCs via stimulating CRP, TNF-α and iNOS expression. The findings provide a novel explanation for the direct pro-inflammatory and atherogenic effects of palmitic acid, and for the association with metabolic syndrome, such as type 2 diabetes mellitus, obesity and atherosclerosis. Therefore, the intervention with anti-inflammatory agents may effectively delay the formation and progression of atherosclerosis in patients with metabolic syndrome.

  11. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

    OpenAIRE

    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

    2008-01-01

    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...

  12. 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

    Directory of Open Access Journals (Sweden)

    Richard Ventura

    2015-08-01

    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 selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

  13. Biosynthesis of Dictyostelium Discoideum Differentation-Inducing Factor by a Hybrid Type I Fatty Acid A-Type III polyketide synthase

    Energy Technology Data Exchange (ETDEWEB)

    Austin,M.; Saito, T.; Bowman, M.; Haydock, S.; Kato, A.; Moore, B.; Kay, R.; Noel, J.

    2006-01-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two {approx}3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.

  14. A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

    Science.gov (United States)

    Lassner, M W; Lardizabal, K; Metz, J G

    1996-02-01

    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.

  15. Disrupted short chain specific β-oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leber, Christopher; Choi, Jin Wook; Polson, Brian; Da Silva, Nancy A

    2016-04-01

    Biologically derived fatty acids have gained tremendous interest as an alternative to petroleum-derived fuels and chemical precursors. We previously demonstrated the synthesis of short chain fatty acids in Saccharomyces cerevisiae by introduction of the Homo sapiens fatty acid synthase (hFAS) with heterologous phosphopantetheine transferases and heterologous thioesterases. In this study, short chain fatty acid production was improved by combining a variety of novel enzyme and metabolic engineering strategies. The use of a H. sapiens-derived thioesterase and phosphopantetheine transferase were evaluated. In addition, strains were engineered to disrupt either the full β-oxidation (by deleting FAA2, PXA1, and POX1) or short chain-specific β-oxidation (by deleting FAA2, ANT1, and PEX11) pathways. Prohibiting full β-oxidation increased hexanoic and octanoic acid levels by 8- and 79-fold relative to the parent strain expressing hFAS. However, by targeting only short chain β-oxidation, hexanoic and octanoic acid levels increased further to 31- and 140-fold over the parent. In addition, an optimized hFAS gene increased hexanoic, octanoic, decanoic and total short chain fatty acid levels by 2.9-, 2.0-, 2.3-, and 2.2-fold, respectively, relative to the non-optimized counterpart. By combining these unique enzyme and metabolic engineering strategies, octanoic acid was increased more than 181-fold over the parent strain expressing hFAS. PMID:26388428

  16. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  17. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    Science.gov (United States)

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    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.

  18. First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

    Directory of Open Access Journals (Sweden)

    Patrick C Y Woo

    Full Text Available BACKGROUND: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. METHODOLOGY/PRINCIPAL FINDINGS: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05. There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05. CONCLUSIONS/SIGNIFICANCE: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid

  19. Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12.

    Science.gov (United States)

    Jackson, J H; Davis, E J; Madu, A C; Braxter, S E

    1981-01-01

    The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.

  20. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

    Science.gov (United States)

    Menendez, Javier A; Vellon, Luciano; Colomer, Ramon; Lupu, Ruth

    2005-05-20

    The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the

  2. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  3. Macrophage polarisation by fatty acids is PPARgamma-dependent.

    Science.gov (United States)

    Pararasa, Chatyan; Bailey, Clifford; Griffiths, Helen

    2014-10-01

    Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. We investigated the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Palmitate increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis. We have also explored whether specific dietary fatty acids drive monocyte to macrophage polarisation via metabolic pathways. Here we show that monocytes pre-incubated with the saturated fatty acid palmitate increase production of inflammatory cytokines such as TNFa and IL-6 in response to a phorbol myristate differentiation trigger. This increases mitochondrial superoxide production, reduces dependency on oxidative phosphorylation through ceramide-dependent inhibition of PPARgamma activity and increases TNFa production, again via a mechanism that requires ceramide production. PMID:26461339

  4. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jeongyeon; Ryoo, Sungwoo, E-mail: ryoosw08@kangwon.ac.kr

    2013-06-07

    Highlights: •Arginase inhibition suppressed proliferation of IL-1β-stimulated VSMCs in dose-dependent manner. •NO production from IL-1β-induced iNOS expression was augmented by arginase inhibition, reducing VSMC proliferation. •Incubation with cGMP analogues abolished IL-1β-dependent proliferation of VSMCs. -- Abstract: We investigated whether arginase inhibition suppressed interleukin (IL)-1β-stimulated proliferation in vascular smooth muscle cells (VSMCs) and the possible mechanisms involved. IL-1β stimulation increased VSMC proliferation, while the arginase inhibitor BEC and transfection of the antisense (AS) oligonucleotide against arginase I decreased VSMC proliferation and was associated with increased protein content of the cell cycle regulator p21Waf1/Cip1. IL-1β incubation induced inducible nitric oxide synthase (iNOS) mRNA expression and protein levels in a dose-dependent manner, but did not affect arginase I and II expression. Consistent with this data, IL-1β stimulation resulted in increase in NO production that was significantly augmented by arginase inhibition. The specific iNOS inhibitor 1400W abolished IL-1β-mediated NO production and further accentuated IL-1β-stimulated cell proliferation. Incubation with NO donors GSNO and DETA/NO in the presence of IL-1β abolished VSMCs proliferation and increased p21Waf1/Cip1 protein content. Furthermore, incubation with the cGMP analogue 8-Br-cGMP prevented IL-1β-induced VSMCs proliferation. In conclusion, arginase inhibition augmented iNOS-dependent NO production that resulted in suppression of IL-1β-induced VSMCs proliferation in a cGMP-dependent manner.

  5. CCR5-Dependent Activation of mTORC1 Regulates Translation of Inducible NO Synthase and COX-2 during Encephalomyocarditis Virus Infection.

    Science.gov (United States)

    Shaheen, Zachary R; Naatz, Aaron; Corbett, John A

    2015-11-01

    Encephalomyocarditis virus (EMCV) infection of macrophages results in the expression of a number of inflammatory and antiviral genes, including inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. EMCV-induced macrophage activation has been shown to require the presence of CCR5 and the activation of PI3K-dependent signaling cascades. The purpose of this study was to determine the role of PI3K in regulating the macrophage responses to EMCV. We show that PI3K regulates EMCV-stimulated iNOS and COX-2 expression by two independent mechanisms. In response to EMCV infection, Akt is activated and regulates the translation of iNOS and COX-2 through the mammalian target of rapamycin complex (mTORC)1. The activation of mTORC1 during EMCV infection is CCR5-dependent and appears to function in a manner that promotes the translation of iNOS and COX-2. CCR5-dependent mTORC1 activation functions as an antiviral response, as mTORC1 inhibition increases the expression of EMCV polymerase. PI3K also regulates the transcriptional induction of iNOS and COX-2 in response to EMCV infection by a mechanism that is independent of Akt and mTORC1 regulation. These findings indicate that macrophage expression of the inflammatory genes iNOS and COX-2 occurs via PI3K- and Akt-dependent translational control of mTORC1 and PI3K-dependent, Akt-independent transcriptional control.

  6. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    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.

  7. Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

    Science.gov (United States)

    Ploux, O; Breyne, O; Carillon, S; Marquet, A

    1999-01-01

    8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using

  8. High-fat feeding reduces endothelium-dependent vasodilation in rats: differential mechanisms for saturated and unsaturated fatty acids?

    Science.gov (United States)

    Song, Guang-Yao; Gao, Yu; Di, Yu-Wei; Pan, Li-Li; Zhou, Yu; Ye, Ji-Ming

    2006-08-01

    1. Chronic feeding with a high-fat diet can cause metabolic syndrome in rodents similar to humans, but the role of saturated versus unsaturated fats in vascular tension remains unclear. 2. The present study shows that rats on a diet rich in either saturated or unsaturated fat had higher blood pressure compared with chow-fed rats (approximately 130 vs 100 mmHg, respectively), along with hyperlipidaemia and insulin resistance. Compared with responses of phenylephrine-preconstricted artery segments from chow-fed rats, vasorelaxation of isolated renal arteries from high-fat fed rats was reduced substantially (> 50%) in response to acetylcholine (0.01-10 micromol/L) and moderately to nitroprusside (>or=1 micromol/L) at low concentrations. Acetylcholine-induced vasorelaxation of arteries from high-fat fed rats was also more sensitive to inhibition by the nitric oxide (NO) synthase inhibitors NG-nitro-L-arginine and methylene blue. 3. In human umbilical vein endothelial cells, the production of NO and endothelin-1 was significantly inhibited by unsaturated fatty acids. In comparison, saturated fatty acids stimulated endothelin-1 production without altering NO production. 4. The data indicate that both saturated and unsaturated high-fat feeding may result in an increase in blood pressure owing to reduced endothelium-dependent vasorelaxation in the arterial system. The impaired endothelium-dependent vasorelaxation induced by saturated and unsaturated fatty acids may involve different mechanisms.

  9. Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells

    NARCIS (Netherlands)

    Sampathkumar, A.; Gutierrez, R.; McFarlane, H.E.; Bringmann, M.; Lindeboom, J.J.; Emons, A.M.C.; Samuels, L.; Ketelaar, T.; Ehrhardt, D.W.; Persson, S.

    2013-01-01

    The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In

  10. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    Science.gov (United States)

    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.

  11. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    Directory of Open Access Journals (Sweden)

    Tao Zhou

    2015-01-01

    Full Text Available 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.

  12. 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.

    Science.gov (United States)

    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.

  13. The reductase domain in a Type I fatty acid synthase from the apicomplexan Cryptosporidium parvum: Restricted substrate preference towards very long chain fatty acyl thioesters

    Directory of Open Access Journals (Sweden)

    Shi Xiangyu

    2010-11-01

    Full Text Available Abstract Background The apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF that predicts a multifunctional Type I fatty acid synthase (CpFAS1 with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs, this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins. Results In the present study, the identity of CpFAS1-R as a reductase is confirmed by in silico analysis on sequence similarity and characteristic motifs. Phylogenetic analysis based on the R-domains supports a previous notion on the bacterial origin of apicomplexan Type I FAS/PKS genes. We also developed a novel assay using fatty acyl-CoAs as substrates, and determined that CpFAS1-R could only utilize very long chain fatty acyl-CoAs as substrates (i.e., with activity on C26 > C24 > C22 > C20, but no activity on C18 and C16. It was capable of using both NADPH and NADH as electron donors, but prefers NADPH to NADH. The activity of CpFAS1-R displayed allosteric kinetics towards C26 hexacosanoyl CoA as a substrate (h = 2.0; Vmax = 32.8 nmol min-1 mg-1 protein; and K50 = 0.91 mM. Conclusions We have confirmed the activity of CpFAS1-R by directly assaying its substrate preference and kinetic parameters, which is for the first time for a Type I FAS, PKS or non-ribosomal peptide

  14. The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

    DEFF Research Database (Denmark)

    von Malek, Bernadette; van der Graaff, Eric; Schneitz, Kay;

    2002-01-01

    exhibits a male-sterile phenotype. The dde2-2 phenotype can be rescued by application of methyl jasmonate, indicating that the mutant is affected in jasmonic acid biosynthesis. The combination of genetic mapping and a candidate-gene approach identified a frameshift mutation in the ALLENE OXIDE SYNTHASE...

  15. Folic acid induces salicylic acid-dependent immunity in Arabidopsis and enhances susceptibility to Alternaria brassicicola.

    Science.gov (United States)

    Wittek, Finni; Kanawati, Basem; Wenig, Marion; Hoffmann, Thomas; Franz-Oberdorf, Katrin; Schwab, Wilfried; Schmitt-Kopplin, Philippe; Vlot, A Corina

    2015-08-01

    Folates are essential for one-carbon transfer reactions in all organisms and contribute, for example, to de novo DNA synthesis. Here, we detected the folate precursors 7,8-dihydropteroate (DHP) and 4-amino-4-deoxychorismate (ADC) in extracts from Arabidopsis thaliana plants by Fourier transform ion cyclotron resonance-mass spectrometry. The accumulation of DHP, but not ADC, was induced after infection of plants with Pseudomonas syringae delivering the effector protein AvrRpm1. Application of folic acid or the DHP precursor 7,8-dihydroneopterin (DHN) enhanced resistance in Arabidopsis to P. syringae and elevated the transcript accumulation of the salicylic acid (SA) marker gene pathogenesis-related1 in both the treated and systemic untreated leaves. DHN- and folic acid-induced systemic resistance was dependent on SA biosynthesis and signalling. Similar to SA, folic acid application locally enhanced Arabidopsis susceptibility to the necrotrophic fungus Alternaria brassicicola. Together, the data associate the folic acid pathway with innate immunity in Arabidopsis, simultaneously activating local and systemic SA-dependent resistance to P. syringae and suppressing local resistance to A. brassicicola.

  16. Caenorhabditis elegans F09E10.3 Encodes a Putative 3-Oxoacyl-Thioester Reductase of Mitochondrial Type 2 Fatty Acid Synthase FASII that Is Functional in Yeast

    Directory of Open Access Journals (Sweden)

    Aner Gurvitz

    2009-01-01

    Full Text Available Caenorhabditis elegans F09E10.3 (dhs-25 was identified as encoding a 3-oxoacyl-thioester reductase, potentially of the mitochondrial type 2 fatty acid synthase (FASII system. Mitochondrial FASII is a relatively recent discovery in metazoans, and the relevance of this process to animal physiology has not been elucidated. A good animal model to study the role of FASII is the nematode C. elegans. However, the components of nematode mitochondrial FASII have hitherto evaded positive identification. The nematode F09E10.3 protein was ectopically expressed without an additional mitochondrial targeting sequence in Saccharomyces cerevisiae mutant cells lacking the homologous mitochondrial FASII enzyme 3-oxoacyl-ACP reductase Oar1p. These yeast oar1Δ mutants are unable to respire, grow on nonfermentable carbon sources, or synthesize sufficient levels of lipoic acid. Mutant yeast cells producing a full-length mitochondrial F09E10.3 protein contained NAD+-dependent 3-oxoacyl-thioester reductase activity and resembled the corresponding mutant overexpressing native Oar1p for the above-mentioned phenotype characteristics. This is the first identification of a metazoan 3-oxoacyl-thioester reductase (see Note Added in Proof.

  17. Chlorogenic acid protection of neuronal nitric oxide synthase-positive neurons in the hippocampus of mice with impaired learning and memory

    Institute of Scientific and Technical Information of China (English)

    Qiuyun Tu; Xiangqi Tang; Zhiping Hu

    2008-01-01

    BACKGROUND: Clinical practice and modern pharmacology have confirmed that ehlorogenic acid can ameliorate learning and memory impairments. OBJECTIVE: To observe the effects of chlorogenic acid on neuronal nitric oxide synthase (nNOS)-positive neurons in the mouse hippocampus, and to investigate the mechanisms underlying the beneficial effects of chlorogenic acid on learning and memory. DESIGN, TIME AND SETTING: The present randomized, controlled, neural cell morphological observation was performed at the Institute of Neurobiology, Central South University between January and May 2005.MATERIALS: Forty-eight female, healthy, adult, Kunming mice were included in this study. Learning and memory impairment was induced with an injection of 0.5 μL kainic acid (0.4 mg/mL) into the hippocampus.METHODS: The mice were randomized into three groups (n = 16): model, control, and chlorogenic acid-treated. At 2 days following learning and memory impairment induction, intragastric administration of physiological saline or chlorogenic acid was performed in the model and chlorogenic acid-treated groups, respectively. The control mice were administered 0.5 μ L physiological saline into the hippocampus, and 2 days later, they received an intragastric administration of physiological saline. Each mouse received two intragastric administrations (1 mL solution once) per day, for a total of 35 days. MAIN OUTCOME MEASURES: Detection of changes in hippocampal and cerebral cortical nNOS neurons by immunohistochemistry; determination of spatial learning and memory utilizing the Y-maze device.RESULTS: At day 7 and 35 after intervention, there was no significant difference in the number of nNOS-positive neurons in the cerebral cortex between the model, chlorogenic acid, and control groups (P > 0.05). Compared with the control group, the number of nNOS-positive neurons in the hippocampal CA1-4 region was significantly less in the model group (P 0.05). At day 7 following intervention, the number

  18. Cell density-dependent linoleic acid toxicity to Saccharomyces cerevisiae.

    Science.gov (United States)

    Ferreira, Túlio César; de Moraes, Lídia Maria Pepe; Campos, Elida Geralda

    2011-08-01

    Since the discovery of the apoptotic pathway in Saccharomyces cerevisiae, several compounds have been shown to cause apoptosis in this organism. While the toxicity of polyunsaturated fatty acids (PUFA) peroxides towards S. cerevisiae has been known for a long time, studies on the effect of nonoxidized PUFA are scarce. The present study deals specifically with linoleic acid (LA) in its nonoxidized form and investigates its toxicity to yeast. Saccharomyces cerevisiae is unable to synthesize PUFA, but can take up and incorporate them into its membranes. Reports from the literature indicate that LA is not toxic to yeast cells. However, we demonstrated that yeast cell growth decreased in cultures treated with 0.1 mM LA for 4 h, and 3-(4,5 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (a measure of respiratory activity) decreased by 47%. This toxicity was dependent on the number of cells used in the experiment. We show apoptosis induction by LA concomitant with increases in malondialdehyde, glutathione content, activities of catalase and cytochrome c peroxidase, and decreases in two metabolic enzyme activities. While the main purpose of this study was to show that LA causes cell death in yeast, our results indicate some of the molecular mechanisms of the cell toxicity of PUFA. PMID:21457450

  19. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

    Science.gov (United States)

    Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2014-09-01

    In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast. PMID:24752598

  20. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  1. Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated.

    Science.gov (United States)

    Voynova, Natalia S; Roubaty, Carole; Vazquez, Hector M; Mallela, Shamroop K; Ejsing, Christer S; Conzelmann, Andreas

    2015-12-01

    Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells under this condition. We established hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.

  2. Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

    Science.gov (United States)

    Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

    2016-01-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

  3. 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.

    Science.gov (United States)

    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.

  4. Uric acid attenuates nitric oxide production by decreasing the interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: a mechanism for uric acid-induced cardiovascular disease development.

    Science.gov (United States)

    Park, Jung-Hyun; Jin, Yoon Mi; Hwang, Soojin; Cho, Du-Hyong; Kang, Duk-Hee; Jo, Inho

    2013-08-01

    The elevated level of uric acid in the body is associated with increased risk of cardiovascular diseases, which is mediated by endothelial dysfunction. However, its underlying mechanism is not fully understood, although dysregulation of endothelial nitric oxide (NO) production is likely to be involved. Using human umbilical vascular endothelial cells (HUVEC), we explored the molecular mechanism of uric acid on endothelial NO synthase (eNOS) activity and NO production. Although high dose of uric acid (12mg/dl for 24h treatment) significantly decreased eNOS activity and NO production, it did not alter eNOS expression and phosphorylations at eNOS-Ser(1177), eNOS-Thr(495) and eNOS-Ser(114). Under this condition, we also found no alterations in the dimerization and acetylation of eNOS, compared with the control. Furthermore, uric acid did not change the activity of arginase II, an enzyme degrading l-arginine, a substrate of eNOS, and intracellular level of calcium, a cofactor for eNOS activation. We also found that uric acid did not alter xanthine oxidase activity, suggesting no involvement of xanthine oxidase-derived O2(-) production in the observed inhibitory effects. In vitro and in cell coimmunoprecipitation studies, however, revealed that uric acid significantly decreased the interaction between eNOS and calmodulin (CaM), an eNOS activator, although it did not change the intracellular CaM level. Like in HUVEC, uric acid also decreased eNOS-CaM interaction in bovine aortic EC. Finally, uric acid attenuated ionomycin-induced increase in the interaction between eNOS and CaM. This study suggests firstly that uric acid decreased eNOS activity and NO production through reducing the binding between eNOS and CaM in EC. Our result may provide molecular mechanism by which uric acid induces endothelial dysfunction.

  5. Salmonella enterica Serovar Typhimurium-Dependent Regulation of Inducible Nitric Oxide Synthase Expression in Macrophages by Invasins SipB, SipC, and SipD and Effector SopE2

    OpenAIRE

    Cherayil, Bobby J.; McCormick, Beth A.; Bosley, Jacob

    2000-01-01

    When Salmonella enterica invades mammalian cells, it activates signals leading to increased expression of inflammatory mediators. One such mediator is nitric oxide (NO), which is produced under control of the enzyme inducible NO synthase (iNOS). Induction of iNOS in response to Salmonella infection has been demonstrated, but the bacterial effector molecules that regulate expression of the enzyme have not been identified. In the study reported here, an analysis of Salmonella-dependent iNOS exp...

  6. Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart.

    Science.gov (United States)

    Vielma, Alejandra Z; León, Luisa; Fernández, Ignacio C; González, Daniel R; Boric, Mauricio P

    2016-01-01

    S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (-25-30%) and basal S-nitrosylation of total proteins (-25-60%), RyR2, SERCA2 and LTCC (-60-75%). NOS-1 inhibition reduced (-25-40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (-85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.

  7. The Formation of Pyrroline and Tetrahydropyridine Rings in Amino Acids Catalyzed by Pyrrolysine Synthase (PylD)

    KAUST Repository

    Quitterer, Felix

    2014-06-10

    The dehydrogenase PylD catalyzes the ultimate step of the pyrrolysine pathway by converting the isopeptide L-lysine-Nε-3R-methyl-D-ornithine to the 22nd proteinogenic amino acid. In this study, we demonstrate how PylD can be harnessed to oxidize various isopeptides to novel amino acids by combining chemical synthesis with enzyme kinetics and X-ray crystallography. The data enable a detailed description of the PylD reaction trajectory for the biosynthesis of pyrroline and tetrahydropyridine rings as constituents of pyrrolysine analogues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

    Directory of Open Access Journals (Sweden)

    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  9. The endothelium-dependent relaxation of rabbit aorta: effects of antioxidants and hydroxylated eicosatetraenoic acids.

    OpenAIRE

    Förstermann, U.; Neufang, B.

    1984-01-01

    Acetylcholine (ACh) induced concentration-dependent relaxations of rabbit aortic strips precontracted with noradrenaline. The relaxations were abolished if the endothelium of the strips was disrupted. Three different antioxidants (butylated hydroxytoluene, dithiothreitol and alpha-tocopherol) reversed the endothelium-dependent vasodilation in a concentration-dependent manner. However, the antioxidant ascorbic acid did not alter the vasodilatation. The hydroxylated eicosatetraenoic acids 5-HET...

  10. The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells

    Science.gov (United States)

    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.

  11. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: dhkim@amc.seoul.kr [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    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.

  12. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    Energy Technology Data Exchange (ETDEWEB)

    Kultti, Anne, E-mail: anne.kultti@uku.fi [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Pasonen-Seppaenen, Sanna [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Jauhiainen, Marjo [Department of Pharmaceutical Chemistry, University of Kuopio, FIN-70211 Kuopio (Finland); Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I. [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland)

    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.

  13. M-CSF from Cancer Cells Induces Fatty Acid Synthase and PPARβ/δ Activation in Tumor Myeloid Cells, Leading to Tumor Progression

    Directory of Open Access Journals (Sweden)

    Jonghanne Park

    2015-03-01

    Full Text Available We investigate crosstalk between cancer cells and stromal myeloid cells. We find that Lewis lung carcinoma cells significantly induce PPARβ/δ activity in myeloid cells in vitro and in vivo. Myeloid cell-specific knockout of PPARβ/δ results in impaired growth of implanted tumors, and this is restored by adoptive transfer of wild-type myeloid cells. We find that IL-10 is a downstream effector of PPARβ/δ and facilitates tumor cell invasion and angiogenesis. This observation is supported by the finding that the CD11blowIL-10+ pro-tumoral myeloid cell is scarcely detected in tumors from myeloid-cell-specific PPARβ/δ knockout mice, where vessel densities are also decreased. Fatty acid synthase (FASN is shown to be an upstream regulator of PPARβ/δ in myeloid cells and is induced by M-CSF secreted from tumor cells. Our study gives insight into how cancer cells influence myeloid stromal cells to get a pro-tumoral phenotype.

  14. Involvement of salicylic acid on antioxidant and anticancer properties, anthocyanin production and chalcone synthase activity in ginger (Zingiber officinale Roscoe) varieties.

    Science.gov (United States)

    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 ginger can be increased

  15. Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe Varieties

    Directory of Open Access Journals (Sweden)

    Ehsan Karimi

    2012-11-01

    Full Text Available 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

  16. 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.

    Science.gov (United States)

    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 (Ptranslation 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; Pexposure 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; Ptranslation as an important consequence of obesity. PMID:27532680

  17. Arginine-dependent acid-resistance pathway in Shigella boydii

    Science.gov (United States)

    Ability to survive the low pH of the human stomach is considered be an important virulent determinant. Acid tolerance of Shigella boydii 18 CDPH, the strain implicated in an outbreak may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arg...

  18. A Mutant of Hepatitis B Virus X Protein (HBxΔ127 Promotes Cell Growth through A Positive Feedback Loop Involving 5-Lipoxygenase and Fatty Acid Synthase

    Directory of Open Access Journals (Sweden)

    Qi Wang

    2010-02-01

    Full Text Available Hepatocellular carcinoma (HCC is one of the most common malignant tumors worldwide. Hepatitis B virus X protein (HBx contributes to the development of HCC, whereas HBx with COOH-terminal deletion is a frequent event in the HCC tissues. Previously, we identified a natural mutant of HBx-truncated 27 amino acids at the COOH-terminal (termed HBxΔ127, which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. Accordingly, fatty acid synthase (FAS plays a crucial role in cancer cell survival and proliferation; thus, we examined the signaling pathways involving FAS. Our data showed that HBxΔ127 strongly increased the transcriptional activities of FAS in human hepatoma HepG2 and H7402 cells. Moreover, we found that 5-lipoxygenase (5-LOX was responsible for the up-regulation of FAS by using MK886 (an inhibitor of 5-LOX and 5-LOX small interfering RNA. We observed that HBxΔ127 could upregulate 5-LOX through phosphorylated extracellular signal-regulated protein kinases 1/2 and thus resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX by ELISA. The additional LTB4 could upregulate the expression of FAS in the cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS. Collectively, HBxΔ127 promotes cell growth through a positive feedback loop involving 5-LOX and FAS, in which released LTB4 is involved in the up-regulation of FAS. Thus, our finding provides a new insight into the mechanism involving the promotion of cell growth mediated by HBxΔ127.

  19. Sevoflurane and nitric oxide synthase expression in rat cochlea

    Institute of Scientific and Technical Information of China (English)

    Yuantao Li; Qingzhong Hou; Mingguang Wu; Xiaolei Huang; Jun Cao; Yin Gu; Xiaofei Qi; Yawen Li

    2010-01-01

    Sevoflurane exhibits anesthetic action by inhibiting the auditory cortex,brain stem nitric oxide synthase activity,and reducing nitric oxide(NO),thereby interfering with the hearing process.However,the influence of sevoflurane on peripheric receptor(cochlea)NO remains poorly understood.Results from the present study showed that sevoflurane downregulated cochlear inducible NO synthase,endothelial NO synthase and neuronal NO synthase expression in a dose dependent manner.This suggests that sevoflurane can decrease cochlear NO synthase expression in a dose dependent manner.

  20. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  1. Conjugated Linoleic Acid (CLA) inhibits expression of the Spot 14 (THRSP) and fatty acid synthase genes and impairs the growth of human breast cancer and liposarcoma cells

    OpenAIRE

    Donnelly, Christina; Olsen, Arne M.; Lewis, Lionel D; Eisenberg, Burton L.; Eastman, Alan; Kinlaw, William B

    2009-01-01

    Spot 14 (THRSP, S14) is a nuclear protein involved in the regulation of genes required for fatty acid synthesis in normal and malignant mammary epithelial and adipose cells. Havartine and Bauman reported that conjugated linoleic acid (CLA) inhibits S14 gene expression in bovine mammary and mouse adipose tissues, and reduces milk fat production in cows. We hypothesized that CLA inhibits S14 gene expression in human breast cancer and liposarcoma cells, and that this will retard their growth. Ex...

  2. Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

    DEFF Research Database (Denmark)

    Mullaney, Brendan C; Blind, Raymond D; Lemieux, George A;

    2010-01-01

    Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3...... mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine...... program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans....

  3. Direct interaction with ACR11 is necessary for post-transcriptional control of GLU1-encoded ferredoxin-dependent glutamate synthase in leaves

    OpenAIRE

    Takabayashi, Atsushi; Niwata, Akihiro; Tanaka, Ayumi

    2016-01-01

    Because it plays an essential role in nitrogen (N) assimilation and photorespiration, the glutamine synthetase (GS)/glutamate synthase (GOGAT) system is widely accepted as occupying a central position in leaf N metabolism. However, the regulation of GOGAT at the post-transcriptional level is poorly understood. Here, we show that ACR11, an ACT (acronym for aspartate kinase, chorismate mutase, and TyrA) domain-containing family protein, interacts with Glu1-encoded ferredoxin (Fd)-GOGAT in Arabi...

  4. 脂肪酸合酶与冠心病的关系%Fatty Acid Synthase:Association with Coronary Heart Disease

    Institute of Scientific and Technical Information of China (English)

    杜建青; 赵婷婷

    2011-01-01

    脂肪酸合酶是催化内源性脂肪酸合成的关键酶,由其介导生成的饱和脂肪酸是动脉粥样斑块的构成成分之一.脂肪酸合酶还通过影响巨噬细胞对氧化低密度脂蛋白的摄取及胆固醇流出,参与粥样斑块的形成.此外,脂肪酸舍酶参与脂类代谢,抑制该酶活性具有减轻体重、增加胰岛素敏感性等作用,可使肥胖、糖尿病等冠心痛的危险因素逆转,因此,脂肪酸合酶与冠心病的发生发展密切相关.%Deregulation of fatty acid synthase (FASN) catalyzed de novo fatty acids biogenesis could play a central role in the pathogenesis of atherosclerosis. We reviewed pharmacological and genetic alterations of FASN activity that have been shown to significantly influence artherosclerosis and its risk factors including obesity, type 2 diabetes. First,the endogenous fatty acids which are catalyzed by the key enzyme FASN are one of atheroaclerotic plaque compositions.Secondly, FASN influences the oxidized low density lipoprotein intake and cholesterol efflux in macrophage, which would absolutely affect the plaque formation. Thirdly, FASN plays a key role in monocytes differentiation. Inhibitting FASN may reduce the formation of foam cells. In addition, FASN involved in lipid metabolism is also associated with metabolic diseases, such as obesity and diabetes which are the risk factors for coronary heart disease. We propose that the development or the progression of artherosclerosis can be prevented or reversed by the modulation of FASN status. The use of FASN inhibitors might be a valuable therapeutic approach for coronary disease.

  5. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4

    DEFF Research Database (Denmark)

    Brodersen, Klaus Peter; Petersen, Morten; Nielsen, Henrik Bjørn;

    2006-01-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective...

  6. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

    Science.gov (United States)

    Yu, Qin; Jalaludin, Adam; Han, Heping; Chen, Ming; Sammons, R Douglas; Powles, Stephen B

    2015-04-01

    Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

  7. Persistent induction of nitric oxide synthase in tumours from mice treated with the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid.

    Science.gov (United States)

    Moilanen, E; Thomsen, L L; Miles, D W; Happerfield, D W; Knowles, R G; Moncada, S

    1998-01-01

    An anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) induced nitric oxide synthase (NOS) in the tumour, spleen, thymus and small intestine, but not in the lung, liver, kidney, heart or skeletal muscle in B6D2F1 mice bearing subcutaneous colon 38 tumours. This pattern of induction is distinct from that caused by agents such as endotoxin, muramyl dipeptide or Corynebacterium parvum. The induction of NOS (iNOS) in the tumour was more persistent (maximal at 3 days) than in other tissues (maximal at 12 h). Immunohistochemical staining suggested that iNOS was located in macrophages and endothelial cells within and around the tumour. Treatment with 5,6-MeXAA also caused substantial increases in plasma nitrite and nitrate (NOx) concentrations that peaked at 8-12 h after 5,6-MeXAA. The increase in plasma NOx was prevented by a NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), indicating that it was due to enhanced production of NO. Tumour-bearing mice were more responsive than controls to 5,6-MeXAA both in their plasma NOx increase and in their lower maximally tolerated dose. L-NIO was unable to prevent the complete tumour necrosis and regression caused by 5,6-MeXAA at a dose that substantially inhibited the increase of plasma NOx. In conclusion, the experimental anti-tumour agent 5,6-MeXAA induced NO synthesis in tumour-associated macrophages and in immunologically active tissues in parallel with its effects on tumour growth. The experiments with a non-selective NOS inhibitor L-NIO, however, suggest that NO is not a significant component in the mechanism of the anti-tumour action of 5,6-MeXAA in this particular model. PMID:9472639

  8. Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase.

    Science.gov (United States)

    Algire, Carolyn; Amrein, Lilian; Zakikhani, Mahvash; Panasci, Lawrence; Pollak, Michael

    2010-06-01

    The molecular mechanisms responsible for the association of obesity with adverse colon cancer outcomes are poorly understood. We investigated the effects of a high-energy diet on growth of an in vivo colon cancer model. Seventeen days following the injection of 5x10(5) MC38 colon carcinoma cells, tumors from mice on the high-energy diet were approximately twice the volume of those of mice on the control diet. These findings were correlated with the observation that the high-energy diet led to elevated insulin levels, phosphorylated AKT, and increased expression of fatty acid synthase (FASN) by the tumor cells. Metformin, an antidiabetic drug, leads to the activation of AMPK and is currently under investigation for its antineoplastic activity. We observed that metformin blocked the effect of the high-energy diet on tumor growth, reduced insulin levels, and attenuated the effect of diet on phosphorylation of AKT and expression of FASN. Furthermore, the administration of metformin led to the activation of AMPK, the inhibitory phosphorylation of acetyl-CoA carboxylase, the upregulation of BNIP3 and increased apoptosis as estimated by poly (ADP-ribose) polymerase (PARP) cleavage. Prior work showed that activating mutations of PI3K are associated with increased AKT activation and adverse outcome in colon cancer; our results demonstrate that the aggressive tumor behavior associated with a high-energy diet has similar effects on this signaling pathway. Furthermore, metformin is demonstrated to reverse the effects of the high-energy diet, thus suggesting a potential role for this agent in the management of a metabolically defined subset of colon cancers. PMID:20228137

  9. Combined Phosphatase and Tensin Homolog (PTEN Loss and Fatty Acid Synthase (FAS Overexpression Worsens the Prognosis of Chinese Patients with Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Xuehua Zhu

    2012-08-01

    Full Text Available We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN, to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC. The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001. Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001. Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I–II versus grade III. Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014. Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049. Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011. Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS

  10. Fetal and neonatal exposure to nicotine leads to augmented hepatic and circulating triglycerides in adult male offspring due to increased expression of fatty acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Noelle [Department of Physiology and Pharmacology, The University of Western Ontario (Canada); Department of Obstetrics and Gynecology, The University of Western Ontario (Canada); The Lawson Health Research Institute, The University of Western Ontario (Canada); Nicholson, Catherine J. [Department of Obstetrics and Gynecology, McMaster University (Canada); Wong, Michael [Department of Physiology and Pharmacology, The University of Western Ontario (Canada); Department of Obstetrics and Gynecology, The University of Western Ontario (Canada); The Lawson Health Research Institute, The University of Western Ontario (Canada); Holloway, Alison C. [Department of Obstetrics and Gynecology, McMaster University (Canada); Hardy, Daniel B., E-mail: Daniel.Hardy@schulich.uwo.ca [Department of Physiology and Pharmacology, The University of Western Ontario (Canada); Department of Obstetrics and Gynecology, The University of Western Ontario (Canada); The Children' s Health Research Institute, The University of Western Ontario (Canada); The Lawson Health Research Institute, The University of Western Ontario (Canada)

    2014-02-15

    While nicotine replacement therapy is assumed to be a safer alternative to smoking during pregnancy, the long-term consequences for the offspring remain elusive. Animal studies now suggest that maternal nicotine exposure during perinatal life leads to a wide range of adverse outcomes for the offspring including increased adiposity. The focus of this study was to investigate if nicotine exposure during pregnancy and lactation leads to alterations in hepatic triglyceride synthesis. Female Wistar rats were randomly assigned to receive daily subcutaneous injections of saline (vehicle) or nicotine bitartrate (1 mg/kg/day) for two weeks prior to mating until weaning. At postnatal day 180 (PND 180), nicotine exposed offspring exhibited significantly elevated levels of circulating and hepatic triglycerides in the male offspring. This was concomitant with increased expression of fatty acid synthase (FAS), the critical hepatic enzyme in de novo triglyceride synthesis. Given that FAS is regulated by the nuclear receptor Liver X receptor (LXRα), we measured LXRα expression in both control and nicotine-exposed offspring. Nicotine exposure during pregnancy and lactation led to an increase in hepatic LXRα protein expression and enriched binding to the putative LXRE element on the FAS promoter in PND 180 male offspring. This was also associated with significantly enhanced acetylation of histone H3 [K9,14] surrounding the FAS promoter, a hallmark of chromatin activation. Collectively, these findings suggest that nicotine exposure during pregnancy and lactation leads to an increase in circulating and hepatic triglycerides long-term via changes in the transcriptional and epigenetic regulation of the hepatic lipogenic pathway. - Highlights: • Our data reveals the links nicotine exposure in utero and long-term hypertriglyceridemia. • It is due to nicotine-induced augmented expression of hepatic FAS and LXRα activity. • Moreover, this involves nicotine-induced enhanced

  11. Infection with HIV and HCV enhances the release of fatty acid synthase into circulation: evidence for a novel indicator of viral infection

    Directory of Open Access Journals (Sweden)

    Aragonès Gerard

    2010-08-01

    Full Text Available Abstract Background Fatty acid synthase (FASN is an enzyme synthesized by the liver and plays an important role in lipogenesis. The present study aimed to investigate whether serum FASN concentration may provide a direct link between HIV and/or HCV viral infections and lipid metabolic disorders commonly observed in HIV/HCV-infected patients. Methods We evaluated serum FASN concentration in 191 consecutive HIV-infected patients in the absence or presence of HCV co-infection. For comparison, 102 uninfected controls were included. Metabolic and inflammatory phenotype was also compared with respect to the presence of HCV co-infection. Results Serum FASN concentration was significantly higher in HIV-infected patients than in healthy participants and HCV co-infected patients showed higher levels than those without co-infection. Levels were also affected by treatment regimen, but marginally influenced by virological variables. Insulin concentration was the sole variable among metabolic parameters that demonstrated a significant correlation with serum FASN concentrations. Serum alanine aminotransferase (ALT values correlated significantly with serum FASN concentration and provided the best discrimination with respect to the presence or absence of HCV co-infection. In multivariate analysis, only ALT, monocyte chemoattractant protein-1 (MCP-1 and the presence of antiretroviral treatment regimen significantly contributed to explain serum FASN concentration in HIV/HCV co-infected patients. Conclusion Serum FASN concentration is significantly increased in HIV-infected individuals. The release of FASN into the circulation is further enhanced in patients who are co-infected with HCV. Subsequent studies should explore the usefulness of this indicator to monitor the effect of viral infections on disease progression and survival.

  12. Serous tubal intraepithelial carcinoma upregulates markers associated with high-grade serous carcinomas including Rsf-1 (HBXAP), cyclin E and fatty acid synthase.

    Science.gov (United States)

    Sehdev, Ann Smith; Kurman, Robert J; Kuhn, Elisabetta; Shih, Ie-Ming

    2010-06-01

    Serous tubal intraepithelial carcinoma (STIC) has been proposed as a precursor for many pelvic high-grade serous carcinomas. Our previous analysis of the ovarian cancer genome identified several genes with oncogenic potential that are amplified and/or overexpressed in the majority of high-grade serous carcinomas. Determining whether these genes are upregulated in STICs is important in further elucidating the relationship of STICs to high-grade serous carcinomas and is fundamental in understanding the molecular pathogenesis of high-grade serous carcinomas. In this study, 37 morphologically defined STICs were obtained from 23 patients with stage IIIC/IV high-grade serous carcinomas. Both STICs and the high-grade serous carcinomas were analyzed for expression of Rsf-1 (HBXAP), cyclin E, fatty acid synthase (FASN) and mucin-4. In addition, they were examined for expression of established markers including p53, Ki-67 and p16. We found that diffuse nuclear p53 and p16 immunoreactivity was observed in 27 (75%) of 36 and 18 (55%) of 33 STICs, respectively, whereas an elevated Ki-67 labeling index (>or=10%) was detected in 29 (78%) of 37 STICs. Cyclin E nuclear staining was seen in 24 (77%) of 35 STICs, whereas normal tubal epithelial cells were all negative. Increased Rsf-1 and FASN immunoreactivity occurred in 63%, and 62% of STICs, respectively, compared with adjacent normal-appearing tubal epithelium. Interestingly, only one STIC showed increased mucin-4 immunoreactivity. Carcinomas, when compared with STICs, overexpressed p16, Rsf-1, cyclin E and FASN in a higher proportion of cases. In conclusion, STICs express several markers including Rsf-1, cyclin E and FASN in high-grade serous carcinomas. In contrast, mucin-4 immunoreactivity either did not change or was reduced in most STICs. These results suggest that overexpression of Rsf-1, cyclin E and FASN occurs early in tumor progression.

  13. The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit.

    Science.gov (United States)

    Starrett, D A; Laties, G G

    1991-03-01

    When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A(+)) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.

  14. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V;

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  15. Fatty acid synthase is a key target in multiple essential tumor functions of prostate cancer: uptake of radiolabeled acetate as a predictor of the targeted therapy outcome.

    Directory of Open Access Journals (Sweden)

    Yukie Yoshii

    Full Text Available Fatty acid synthase (FASN expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In addition, how FASN inhibition affects tumor progression remains unclear. Here, we showed the method to predict FASN-targeted therapy outcome using radiolabeled acetate uptake and presented mechanisms of FASN inhibition with human prostate cancer cell lines, to provide the treatment strategy of FASN-targeted therapy. We revealed that tumor uptake of radiolabeled acetate reflected the FASN expression levels and sensitivity to FASN-targeted therapy with orlistat in vitro and in vivo. FASN-targeted therapy was noticeably effective against tumors with high FASN expression, which was indicated by high acetate uptake. To examine mechanisms, we established FASN knockdown prostate cancer cells by transduction of short-hairpin RNA against FASN and investigated the characteristics by analyses on morphology and cell behavior and microarray-based gene expression profiling. FASN inhibition not only suppressed cell proliferation but prevented pseudopodia formation and suppressed cell adhesion, migration, and invasion. FASN inhibition also suppressed genes involved in production of intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression. Collectively, our data demonstrated that uptake of radiolabeled acetate is a useful predictor of FASN-targeted therapy outcome. This suggests that [1-(11C]acetate positron emission tomography (PET could be a powerful tool to accomplish personalized FASN-targeted therapy by non

  16. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

    Science.gov (United States)

    Sydor, Tobias; von Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-03-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. PMID:23078612

  17. Novel type III polyketide synthases from Aloe arborescens.

    Science.gov (United States)

    Mizuuchi, Yuusuke; Shi, She-Po; Wanibuchi, Kiyofumi; Kojima, Akiko; Morita, Hiroyuki; Noguchi, Hiroshi; Abe, Ikuro

    2009-04-01

    Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85-96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl-CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild-type PKS obtained from A. arborescens. Interestingly, the aloesone-forming activity was maximum at 50 degrees C, and the novel heptaketide pyrone was non-enzymatically converted to aloesone. In PKS3, the active-site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site-directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone. PMID:19348024

  18. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

    Science.gov (United States)

    Wakuta, Shinji; Suzuki, Erika; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-06-17

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice. PMID:21619871

  19. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

    Science.gov (United States)

    Wakuta, Shinji; Suzuki, Erika; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-06-17

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice.

  20. Fatty Acid Synthase and Hormone-sensitive Lipase Expression in Liver Are Involved in Zinc-α2-glycoprotein-induced Body Fat Loss in Obese Mice

    Institute of Scientific and Technical Information of China (English)

    Feng-ying Gong; Jie-ying Deng; Hui-juan Zhu; Hui Pan; Lin-jie Wang; Hong-bo Yang

    2010-01-01

    Objective To explore the effects of zinc-a2-glycoprotein (ZAG) on body weight and body fat in high-fat-diet (HFD)-induced obesity in mice and the possible mechanism.Methods Thirty-six male mice were fed with standard food (SF) (n=9) and HFD (n=27), respec-tively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) in liver tissue were de-termined by reverse transcription-polymerase chain reaction.Results Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice (0.51±0.10 AU vs. 0.75±0.07 AU, P<0.01). Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r =-0.56, P<0.001), epididymal fat mass (r=-0. 67, P<0.001), percentage of epididymal fat (r=-0.65, P<0.001 ), and increased weight (r=-0.57, P<0.001) in simple SF-and HFD-fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididy-mal fat. Furthermore, FAS mRNA expression decreased (P<0.01) and HSL mRNA expression increased (P<0.001) in the liver in ZAG over-expressing mice.Conclusions ZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and in-creased HSL expression in the liver of obese mice.

  1. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...

  2. N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-γ (IFN-γ)

    OpenAIRE

    Platten, Michael; Wick, Wolfgang; Wischhusen, Jörg; WELLER, MICHAEL

    2001-01-01

    Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-γ (IFN-γ), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes.Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-γ (200 ...

  3. Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

    OpenAIRE

    Hoang, T.T.; Schweizer, H P

    1997-01-01

    The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemop...

  4. Surface Activity of Humic Acids Depending on Their Origin and Humification Degree

    OpenAIRE

    Klaviņš Māris; Purmalis Oskars

    2014-01-01

    Humic substances are able to reduce the surface tension of their solutions and thus can act as surface-active substances in the natural environment, which may have industrial application. The ability to influence the surface tension of humic acid solutions depends on the origin of the humic acids. The objective of this study was comparison of the ability of humic acids of different origin (soil, water, peat, lignite etc.) to influence the surface tension of their solutions, and identification...

  5. Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

    Science.gov (United States)

    Cary, Jeffrey W; Harris-Coward, Pamela Y; Ehrlich, Kenneth C; Di Mavungu, José Diana; Malysheva, Svetlana V; De Saeger, Sarah; Dowd, Patrick F; Shantappa, Sourabha; Martens, Stacey L; Calvo, Ana M

    2014-03-01

    The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival. PMID:24412484

  6. Surface Activity of Humic Acids Depending on Their Origin and Humification Degree

    Directory of Open Access Journals (Sweden)

    Klaviņš Māris

    2014-07-01

    Full Text Available Humic substances are able to reduce the surface tension of their solutions and thus can act as surface-active substances in the natural environment, which may have industrial application. The ability to influence the surface tension of humic acid solutions depends on the origin of the humic acids. The objective of this study was comparison of the ability of humic acids of different origin (soil, water, peat, lignite etc. to influence the surface tension of their solutions, and identification of the structural characteristics of peat humic acids that determine their surfactant properties. Industrially produced humic materials demonstrated no or insignificant impact on the surface tension of their solutions. However, humic acids isolated from peat had significant impact of the surface tension of their solutions, acting as weak surfactants. The surface tension of humic acid solutions decreased with increasing concentration, and depended on solution pH. Using a well-characterised bog profile, the ability to influence the surface tension of peat humic acids was shown to depend on age and humification degree. With increase of the humification degree and age, molecular complexity of humic acids and their ability to influence surface tension decreased; but nevertheless, the impact of the biological precursor (peat-forming bryophytes and plants could be identified.

  7. Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

    Science.gov (United States)

    Thumelin, S; Kohl, C; Girard, J; Pégorier, J P

    1999-06-01

    The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such

  8. Revisiting histidine-dependent acid phosphatases: a distinct group of tyrosine phosphatases

    OpenAIRE

    Veeramani, Suresh; Lee, Ming-Shyue; Lin, Ming-Fong

    2009-01-01

    Although classical protein tyrosine phosphatase (PTP) superfamily members are cysteine-dependent, emerging evidence shows that many acid phosphatases (AcPs) function as histidine-dependent PTPs in vivo. These AcPs dephosphorylate phospho-tyrosine substrates intracellularly and could have roles in development and disease. In contrast to cysteine-dependent PTPs, they utilize histidine, rather than cysteine, for substrate dephosphorylation. Structural analyses reveal that active site histidine, ...

  9. OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling

    OpenAIRE

    Wakuta, Shinji; SUZUKI, ERIKA; Saburi, Wataru; Matsuura, Hideyuki; Nabeta, Kensuke; Imai, Ryozo; Matsui, Hirokazu

    2011-01-01

    The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our...

  10. Muscle fibre-type dependence of neuronal nitric oxide synthase-mediated vascular control in the rat during high speed treadmill running.

    Science.gov (United States)

    Copp, Steven W; Holdsworth, Clark T; Ferguson, Scott K; Hirai, Daniel M; Poole, David C; Musch, Timothy I

    2013-06-01

    We have recently shown that nitric oxide (NO) derived from neuronal NO synthase (nNOS) does not contribute to the hyperaemic response within rat hindlimb skeletal muscle during low-speed treadmill running. This may be attributed to low exercise intensities recruiting primarily oxidative muscle and that vascular effects of nNOS-derived NO are manifest principally within glycolytic muscle. We tested the hypothesis that selective nNOS inhibition via S-methyl-l-thiocitrulline (SMTC) would reduce rat hindlimb skeletal muscle blood flow and vascular conductance (VC) during high-speed treadmill running above critical speed (asymptote of the hyperbolic speed versus time-to-exhaustion relationship for high-speed running and an important glycolytic fast-twitch fibre recruitment boundary in the rat) principally within glycolytic fast-twitch muscle. Six rats performed three high-speed treadmill runs to exhaustion to determine critical speed. Subsequently, hindlimb skeletal muscle blood flow (radiolabelled microspheres) and VC (blood flow/mean arterial pressure) were determined during supra-critical speed treadmill running (critical speed + 15%, 52.5 ± 1.3 m min(-1)) before (control) and after selective nNOS inhibition with 0.56 mg kg(-1) SMTC. SMTC reduced total hindlimb skeletal muscle blood flow (control: 241 ± 23, SMTC: 204 ± 13 ml min(-1) (100 g)(-1), P exercise by identifying fibre-type-selective peripheral vascular effects of nNOS-derived NO during high-speed treadmill running. PMID:23507879

  11. Direct interaction with ACR11 is necessary for post-transcriptional control of GLU1-encoded ferredoxin-dependent glutamate synthase in leaves.

    Science.gov (United States)

    Takabayashi, Atsushi; Niwata, Akihiro; Tanaka, Ayumi

    2016-01-01

    Because it plays an essential role in nitrogen (N) assimilation and photorespiration, the glutamine synthetase (GS)/glutamate synthase (GOGAT) system is widely accepted as occupying a central position in leaf N metabolism. However, the regulation of GOGAT at the post-transcriptional level is poorly understood. Here, we show that ACR11, an ACT (acronym for aspartate kinase, chorismate mutase, and TyrA) domain-containing family protein, interacts with Glu1-encoded ferredoxin (Fd)-GOGAT in Arabidopsis chloroplasts. In addition, Arabidopsis acr11 mutants have lost the capability to control Fd-GOGAT levels in response to light/dark diurnal cycles, nitrogen inputs, and changes in photorespiratory activity. Considering that ACR11 has putative glutamine-binding domains, our results indicate that ACR11 is necessary for post-transcriptional control of leaf Glu1-encoded Fd-GOGAT. This regulation takes place through direct interaction of ACR11 and Fd-GOGAT, possibly in an allosteric manner. PMID:27411448

  12. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  13. Hydroxyquinolines inhibit ribonucleic acid-dependent deoxyribonucleic acid polymerase and inactivate Rous sarcoma virus and herpes simplex virus.

    Science.gov (United States)

    Rohde, W; Mikelens, P; Jackson, J; Blackman, J; Whitcher, J; Levinson, W

    1976-08-01

    8-Hydroxyquinoline and several of its derivatives inactivate the transforming ability of Rous sarcoma virus and inhibit its ribonucleic acid-dependent deoxyribonucleic acid polymerase activity. The copper complex of these metal-binding ligands is as active as the free ligand. The activity of the 8-hydroxyquinolines is approximately 50-fold more effective than another group of metal-binding compounds that we have tested, the thiosemicarbazones. In contrast to the potency of the 8-hydroxyquinolines to inactivate Rous sarcoma virus, no intracellular inhibition of transformation could be demonstrated at a concentration that did not affect the growth and appearance of the cells. Cellular deoxyribonucleic acid synthesis was inhibited to a greater extent than was ribonucleic acid or protein synthesis. The phenomenon of "concentration quenching" was observed with high concentrations of drug, causing less inhibition of deoxyribonucleic acid synthesis than was observed with lower concentrations. Herpes simplex virus type 1 was inactivated also by the 8-hydroxyquinolines and their copper complexes. No intracellular inhibition of plaque formation was observed. Treatment with 8-hydroxyquinoline sulfate had no effect on the resolution of herpetic keratitis in rabbits. Some 8-hydroxyquinolines bind to deoxyribonucleic acid in the presence of copper, a phenomenon that may be important in their antiviral activity. PMID:185949

  14. Study on structure-activity relationship of mutation-dependent herbicide resistance acetohydroxyacid synthase through 3D-QSAR and mutation

    Institute of Scientific and Technical Information of China (English)

    YU ZhiHong; NIU CongWei; BAN ShuRong; WEN Xin; XI Zhen

    2007-01-01

    Seventy-four sulfonylureas were synthesized and tested for their inhibitory activity against the whole enzyme of E. Coli acetohydroxyacid synthase (AHAS, EC 2.2.1.6) isoenzyme Ⅱ, and 3D-QSAR analyses were performed based on these inhibitory activities. The binding conformation of chlorimuron-ethyl, a commercial herbicide of AHAS, in the crystal structure of AHAS complex was extracted and used as template to build the initial three-dimensional structure of other sulfonylureas, and then all structures were fully geometry optimized. After systematic optimization of the alignment rule, molecular orientation, grid space and attenuation factor, two satisfactory models with excellent performances (CoMFA: q2 = 0.735, r2 = 0.954, n = 7, r 2pred = 0.832; CoMSIA: q2 = 0.721, r2 = 0.913, n = 8, r 2pred = 0.844) were established. By mapping the 3D contour maps of CoMFA and CoMSIA models into the possible inhibitory active site in the crystal structure of catalytic subunit of yeast AHAS, a plausible binding model for AHAS, with best fit QSAR in the literature so far, was proposed. Moreover, the results of 3D-QSAR were further utilized to interpret resistance of site-directed mutants. A relative activity index (RAI) for AHAS enzyme mutant was defined for the first time to relate the 3D-QSAR and resistance of mutants. This study, for the first time, demonstrated that combination of 3D-QSAR and enzyme mutation can be used to decipher the molecular basis of ligand-receptor interaction mechanism. This study refined our understanding of the ligand-receptor interaction and resistance mechanism in AHAS-sulfonylurea system, and provided basis for designing new potent herbicides to combat the herbicide resistance.

  15. Proton-dependent glutamine uptake by aphid bacteriocyte amino acid transporter ApGLNT1.

    Science.gov (United States)

    Price, Daniel R G; Wilson, Alex C C; Luetje, Charles W

    2015-10-01

    Aphids house large populations of the gammaproteobacterial symbiont Buchnera aphidicola in specialized bacteriocyte cells. The combined biosynthetic capability of the holobiont (Acyrthosiphon pisum and Buchnera) is sufficient for biosynthesis of all twenty protein coding amino acids, including amino acids that animals alone cannot synthesize; and that are present at low concentrations in A. pisum's plant phloem sap diet. Collaborative holobiont amino acid biosynthesis depends on glutamine import into bacteriocytes, which serves as a nitrogen-rich amino donor for biosynthesis of other amino acids. Recently, we characterized A. pisum glutamine transporter 1 (ApGLNT1), a member of the amino acid/auxin permease family, as the dominant bacteriocyte plasma membrane glutamine transporter. Here we show ApGLNT1 to be structurally and functionally related to mammalian proton-dependent amino acid transporters (PATs 1-4). Using functional expression in Xenopus laevis oocytes, combined with two-electrode voltage clamp electrophysiology we demonstrate that ApGLNT1 is electrogenic and that glutamine induces large inward currents. ApGLNT1 glutamine induced currents are dependent on external glutamine concentration, proton (H+) gradient across the membrane, and membrane potential. Based on these transport properties, ApGLNT1-mediated glutamine uptake into A. pisum bacteriocytes can be regulated by changes in either proton gradients across the plasma membrane or membrane potential. PMID:26028424

  16. The ent-15α-Acetoxykaur-16-en-19-oic Acid Relaxes Rat Artery Mesenteric Superior via Endothelium-Dependent and Endothelium-Independent Mechanisms

    Science.gov (United States)

    Ribeiro, Êurica Adélia Nogueira; Herculano, Edla de Azevedo; da Costa, Cintia Danieli Ferreira; Furtado, Fabiola Fialho; da-Cunha, Emídio Vasconcelos Leitão; Barbosa-Filho, José Maria; da Silva, Marcelo Sobral; de Medeiros, Isac Almeida

    2012-01-01

    The objective of the study was to investigate the mechanism of the relaxant activity of the ent-15α-acetoxykaur-16-en-19-oic acid (KA-acetoxy). In rat mesenteric artery rings, KA-acetoxy induced a concentration-dependent relaxation in vessels precontracted with phenylephrine. In the absence of endothelium, the vasorelaxation was significantly shifted to the right without reduction of the maximum effect. Endothelium-dependent relaxation was significantly attenuated by pretreatment with L-NAME, an inhibitor of the NO-synthase (NOS), indomethacin, an inhibitor of the cyclooxygenase, L-NAME + indomethacin, atropine, a nonselective antagonist of the muscarinic receptors, ODQ, selective inhibitor of the guanylyl cyclase enzyme, or hydroxocobalamin, a nitric oxide scavenger. The relaxation was completely reversed in the presence of L-NAME + 1 mM L-arginine or L-arginine, an NO precursor. Diterpene-induced relaxation was not affected by TEA, a nonselective inhibitor of K+ channels. The KA-acetoxy antagonized CaCl2-induced contractions in a concentration-dependent manner and also inhibited an 80 mM KCl-induced contraction. The KA-acetoxy did not interfere with Ca2+ release from intracellular stores. The vasorelaxant induced by KA-acetoxy seems to involve the inhibition of the Ca2+ influx and also, at least in part, by endothelial muscarinic receptors activation, NO and PGI2 release. PMID:23346202

  17. The ent-15α-Acetoxykaur-16-en-19-oic Acid Relaxes Rat Artery Mesenteric Superior via Endothelium-Dependent and Endothelium-Independent Mechanisms

    Directory of Open Access Journals (Sweden)

    Êurica Adélia Nogueira Ribeiro

    2012-01-01

    Full Text Available The objective of the study was to investigate the mechanism of the relaxant activity of the ent-15α-acetoxykaur-16-en-19-oic acid (KA-acetoxy. In rat mesenteric artery rings, KA-acetoxy induced a concentration-dependent relaxation in vessels precontracted with phenylephrine. In the absence of endothelium, the vasorelaxation was significantly shifted to the right without reduction of the maximum effect. Endothelium-dependent relaxation was significantly attenuated by pretreatment with L-NAME, an inhibitor of the NO-synthase (NOS, indomethacin, an inhibitor of the cyclooxygenase, L-NAME + indomethacin, atropine, a nonselective antagonist of the muscarinic receptors, ODQ, selective inhibitor of the guanylyl cyclase enzyme, or hydroxocobalamin, a nitric oxide scavenger. The relaxation was completely reversed in the presence of L-NAME + 1 mM L-arginine or L-arginine, an NO precursor. Diterpene-induced relaxation was not affected by TEA, a nonselective inhibitor of K+ channels. The KA-acetoxy antagonized CaCl2-induced contractions in a concentration-dependent manner and also inhibited an 80 mM KCl-induced contraction. The KA-acetoxy did not interfere with Ca2+ release from intracellular stores. The vasorelaxant induced by KA-acetoxy seems to involve the inhibition of the Ca2+ influx and also, at least in part, by endothelial muscarinic receptors activation, NO and PGI2 release.

  18. Hepatic Fasting-Induced PPARα Activity Does Not Depend on Essential Fatty Acids.

    Science.gov (United States)

    Polizzi, Arnaud; Fouché, Edwin; Ducheix, Simon; Lasserre, Frédéric; Marmugi, Alice P; Mselli-Lakhal, Laila; Loiseau, Nicolas; Wahli, Walter; Guillou, Hervé; Montagner, Alexandra

    2016-01-01

    The liver plays a central role in the regulation of fatty acid metabolism, which is highly sensitive to transcriptional responses to nutrients and hormones. Transcription factors involved in this process include nuclear hormone receptors. One such receptor, PPARα, which is highly expressed in the liver and activated by a variety of fatty acids, is a critical regulator of hepatic fatty acid catabolism during fasting. The present study compared the influence of dietary fatty acids and fasting on hepatic PPARα-dependent responses. Pparα(-/-) male mice and their wild-type controls were fed diets containing different fatty acids for 10 weeks prior to being subjected to fasting or normal feeding. In line with the role of PPARα in sensing dietary fatty acids, changes in chronic dietary fat consumption influenced liver damage during fasting. The changes were particularly marked in mice fed diets lacking essential fatty acids. However, fasting, rather than specific dietary fatty acids, induced acute PPARα activity in the liver. Taken together, the data imply that the potent signalling involved in triggering PPARα activity during fasting does not rely on essential fatty acid-derived ligand. PMID:27669233

  19. Developmentally dependent and different roles of fatty acids OMEGA-6 and OMEGA-3

    DEFF Research Database (Denmark)

    Mourek, J

    2011-01-01

    The developmentally-dependent differences in the biological significances and effects of PUFA-OMEGA-6 (namely of arachidonic acid) and PUFA-OMEGA-3 (namely of docosahexaenoic acid) are discussed. The clinical results as well as developmental experiences are indicating a hypothesis of the evolution...... that created mutual relationship between those two substances (with immunological basis and following recuperation). The anti-inflammatory actions of PUFA-OMEGA-3 are the most visible (and significant) contrasts as compared with the large affects of namely arachidonic acid and its metabolites....

  20. Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.

    Science.gov (United States)

    Avery, S V; Howlett, N G; Radice, S

    1996-01-01

    One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditions contained only saturated and monounsaturated fatty acids, considerable incorporation of the diunsaturated fatty acid linoleate (18:2) (to more than 65% of the total fatty acids) was observed in both whole-cell homogenates and plasma membrane-enriched fractions from cells grown in linoleate-supplemented medium. Linoleate enrichment had no discernible effect on the growth of S. cerevisiae. However, linoleate-enriched cells were markedly more susceptible to copper-induced plasma membrane permeabilization. Thus, after addition of Cu(NO3)2, rates of cellular K+ release (loss of membrane integrity) were at least twofold higher from linoleate-supplemented cells than from unsupplemented cells; this difference increased with reductions in the Cu2+ concentration supplied. Levels of cellular Cu accumulation were also higher in linoleate-supplemented cells. These results were correlated with a very marked dependence of whole-cell Cu2+ toxicity on cellular fatty acid unsaturation. For example, within 10 min of exposure to 5 microM Cu2+, only 3% of linoleate-enriched cells remained viable (capable of colony formation). In contrast, 100% viability was maintained in cells previously grown in the absence of a fatty acid supplement. Cells displaying intermediate levels of linoleate incorporation showed intermediate Cu2+ sensitivity, while cells enriched with the triunsaturated fatty acid linolenate (18:3) were most sensitive to Cu2+. These results demonstrate for the first time that changes

  1. Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells

    OpenAIRE

    Aragonès Gemma; Saavedra Paula; Heras Mercedes; Cabré Anna; Girona Josefa; Masana Lluís

    2012-01-01

    Abstract Background Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. Methods In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation a...

  2. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway

    Science.gov (United States)

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-01

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by Nω-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension. PMID:26739766

  3. The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

    NARCIS (Netherlands)

    Vain, T.; Crowell, E.F.; Timpano, H.; Biot, E.; Desprez, T.; Mansoori Zangir, N.; Trindade, L.M.; Pagant, S.; Robert, S.; Hofte, H.; Gonneau, M.; Vernhettes, S.

    2014-01-01

    Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis

  4. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...

  5. Identification of the Leishmania major Proteins LmjF07.0430, LmjF07.0440, and LmjF27.2440 as Components of Fatty Acid Synthase II

    Directory of Open Access Journals (Sweden)

    Aner Gurvitz

    2009-01-01

    Full Text Available Leishmania major causes leishmaniasis and is grouped within the Trypanosomatidae family, which also includes the etiologic agent for African sleeping sickness, Trypanosoma brucei. Previous studies on T. brucei showed that acyl carrier protein (ACP of mitochondrial fatty acid synthase type 2 (FASII plays a crucial role in parasite survival. Additionally, 3-oxoacyl-ACP synthase TbKASIII as well as TbHTD2 representing 3-hydroxyacyl-ACP dehydratase were also identified; however, 3-oxoacyl-ACP reductase TbKAR1 has hitherto evaded positive identification. Here, potential Leishmania FASII components LmjF07.0440 and LmjF07.0430 were revealed as 3-hydroxyacyl-ACP dehydratases LmHTD2-1 and LmHTD2-2, respectively, whereas LmjF27.2440 was identified as LmKAR1. These Leishmania proteins were ectopically expressed in Saccharomyces cerevisiae htd2Δ or oar1Δ respiratory deficient cells lacking the corresponding mitochondrial FASII enzymes Htd2p and Oar1p. Yeast mutants producing mitochondrially targeted versions of the parasite proteins resembled the self-complemented cells for respiratory growth. This is the first identification of a FASII-like 3-oxoacyl-ACP reductase from a kinetoplastid parasite.

  6. Prebiotic Amino Acid Thioester Synthesis: Thiol-Dependent Amino Acid Synthesis from Formose substrates (Formaldehyde and Glycolaldehyde) and Ammonia

    Science.gov (United States)

    Weber, Arthur L.

    1998-01-01

    Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters, the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters-intermediates capable of forming peptides. A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.

  7. Sulfuric acid nucleation: power dependencies, variation with relative humidity, and effect of bases

    Directory of Open Access Journals (Sweden)

    J. H. Zollner

    2012-01-01

    Full Text Available Nucleation of particles composed of sulfuric acid, water, and nitrogen base molecules was studied using a continuous flow reactor. The particles formed from these vapors were detected with an ultrafine condensation particle counter, while vapors of sulfuric acid and nitrogen bases were detected by chemical ionization mass spectrometry. Variation of particle numbers with sulfuric acid concentration yielded a power dependency on sulfuric acid of 5 ± 1 for relative humidities of 14–68% at 296 K; similar experiments with varying water content yielded power dependencies on H2O of ~7. The critical cluster contains about 5 H2SO4 molecules and a new treatment of the power dependency for H2O suggests about 12 H2O molecules for these conditions. Addition of 2-to-45 pptv of ammonia or methyl amine resulted in up to millions of times more particles than in the absence of these compounds. Particle detection efficiencies, sulfuric acid and nitrogen base detection, wall losses, and the extent of particle growth are discussed with the help of a recent computational fluid dynamics study that simulated the flow and chemistry in the flow reactor. Results are compared to previous laboratory nucleation studies and they are also discussed in terms of atmospheric nucleation scenarios.

  8. Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces

    International Nuclear Information System (INIS)

    Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with [3H]arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of [3H]free fatty acids. These effects were attenuated in Ca2+-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca2+ with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of [3H]free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca2+ influx and that at least 80% of the [3H]free fatty acid accumulation required calcium

  9. Calcium-dependent phospholipid catabolism and arachidonic acid mobilization in cerebral minces

    Energy Technology Data Exchange (ETDEWEB)

    Damron, D.S.; Dorman, R.V. (Kent State Univ., OH (USA))

    1990-06-01

    Cerebral minces were used to investigate the role of calcium influx on trauma-induced alterations of brain lipid metabolism. Cerebral phospholipids, nonpolar lipids, and free fatty acids were radiolabeled in vivo with ({sup 3}H)arachidonic acid. Tissue incubation stimulated the time-dependent catabolism of choline and inositol glycerophospholipids, and resulted in the accumulation of ({sup 3}H)free fatty acids. These effects were attenuated in Ca{sup 2}{sup +}-free incubations, and when EGTA or verapamil were present. The inhibition of calcium influx also reduced the labeling of diglycerides, whereas ethanolamine and serine glycerophospholipids were not affected by incubation or treatments. Replacing Ca{sup 2}{sup +} with other cations also attenuated the incubation-dependent alterations in lipid metabolism. However, only cadmium was able to compete with calcium and reduce the accumulation of ({sup 3}H)free fatty acids. It appeared that about half of the observed phospholipid catabolism was dependent on Ca{sup 2}{sup +} influx and that at least 80% of the ({sup 3}H)free fatty acid accumulation required calcium.

  10. SFH2 regulates fatty acid synthase activity in the yeast Saccharomyces cerevisiae and is critical to prevent saturated fatty acid accumulation in response to haem and oleic acid depletion

    OpenAIRE

    Desfougères, Thomas; Ferreira, Thierry; Bergès, Thierry; Régnacq, Matthieu

    2007-01-01

    Abstract The yeast Saccharomyces cerevisiae is a facultative anaerobic organism. In anaerobiosis, sustained growth relies on the presence of exogenously supplied unsaturated fatty acids and ergosterol that yeast is unable to synthesize in the absence of oxygen or upon haem depletion. In the absence of exogenous supplementation with unsaturated fatty acid, a net accumulation of saturated fatty acid (SFA) is observed that induces significant modification of phospholipid profile [1]. ...

  11. 5-Aminosalicylic acid dependency in Crohn's disease: A Danish Crohn Colitis Database study

    DEFF Research Database (Denmark)

    Duricova, D.; Pedersen, N.; Elkjaer, M.;

    2010-01-01

    Background and aims The role of 5 aminosalicylic acid (5 ASA) in Crohns disease is unclear The outcome of the first course of 5 ASA monotherapy with emphasis on 5 ASA dependency was retrospectively assessed in consecutive cohort of 537 Crohn s disease patients diagnosed 1953-2007 Methods Following...

  12. Time-dependent clearance of mycophenolic acid in renal transplant recipients

    NARCIS (Netherlands)

    R.M. van Hest (Reinier); T. van Gelder (Teun); R. Bouw (René); T. Goggin (Timothy); R. Gordon (Robert); R. Mamelok (Richard); R.A. Mathot (Ron)

    2007-01-01

    textabstractAims: Pharmacokinetic studies of the immunosuppressive compound mycophenolic acid (MPA) have shown a structural decrease in clearance (CL) over time after renal transplantation. The aim of this study was to characterize the time-dependent CL of MPA by means of a population pharmacokineti

  13. Highly Efficient Synthesis of Two Hyaluronan Trisaccharide Analogues for Potential Hyaluronic Acid Synthases Inhibitors%透明质酸三糖模拟物的高效合成

    Institute of Scientific and Technical Information of China (English)

    魏国华; 杜宇国; Khushi L. Matta

    2009-01-01

    The syntheses of two hyaluronan trisaccharide analogues, naphthyl 0-(3-methoxy-B-D-glucopy-ranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-B-D-glucopyranosyl)-(1,4)-0-B-D-glucopyranosyluronic acid and naphthyl O-(3-methoxy-2-acetamido-2-deoxy-B-D-glucopyranosyl)-(1,4)-O-(B-D-glucopyranosylu-ronic acid)-(1,3 )-O-2-acetamido-2-deoxy-B-D-glucopyranoside, were described. Construction of the target molecules was achieved through a combination of BF_3·Et_2O/toluene system and trichloroacetimidate glycosyia-tion methodology. This is the first report on the synthesis of the 3-methoxyl derivatives, which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan and can be used for potential hyaluronic acid synthases inhibitors.%设计合成了2个透明质酸(HA)模拟物1和2, 通过最小基团MeO的引入修饰, 模拟天然HA片段的特性, 用于透明质酸合成酶(HAS)催化机理与抑制剂的研究.

  14. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  15. Influence of ellagic acid on prostate cancer cell proliferation:A caspase-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    Arshi Malik; Sarah Afaq; Mohammad Shahid; Kafil Akhtar; Abdullah Assiri

    2011-01-01

    Objective:To evaluate the effect of allagic acid treatment on the cell viability of human prostate cancer cells.Methods: Ellagic acid (10-100mol/L) treatment (48 h) of human prostate carcinomaPC3 cells was found to result in a dose-dependent inhibition of cell growth and apoptosis ofPC3 cells as assessed by MTTassay, western blotting, flow cytometry and confocal microscopy.Results: We observed that ellagic acid treatment ofPC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. This ellagic acid caused cell growth inhibition was found to be accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase(PARP) and morphological changes. Further, induction of apoptosis accompanied a decrease in the levels of antiapoptotic protein Bcl-2 and increase in proapoptotic protein Bax, thus shifting the Bax: Bcl-2 ratio in favor of apoptosis. Ellagic acid treatment of PC3 cells was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase-3, -6, -8 and-9. This ellagic acid-mediated induction of apoptosis was significantly (80%-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone(Z-VAD-FMK). Thus these data suggested an essential role of caspases in ellagic acid-mediated apoptosis ofPC3 cells.Conclusions:It is tempting to suggest that consumption of tropical pigmented fruits and vegetables could be an effective strategy to combat prostate cancer.

  16. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    International Nuclear Information System (INIS)

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  17. Cloning and Expression of Poly-glutamic Acid Synthase Gene in Escherichia coli%γ-PGA合成酶基因在大肠杆菌中的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    乔广军; 汪晨; 周志蕙; 张凯; 蔡恒

    2013-01-01

      研究了γ-PGA合成酶基因pgsBCA在大肠杆菌中的克隆和表达,以pET28a(+)为载体,构建表达载体pET28a (+)-pgsBCA,导入宿主Escherichia coli Rosetta中,诱导使之表达.将发酵液离心去除菌体,得到上清液用旋转蒸发仪浓缩后,采用SDS-PAGE电泳检测重组菌E.coli Rosetta/pET28a-pgsBCA产生的γ-PGA分子量在200-300kDa之间,将产物水解,采用薄层层析法鉴定产物由单一的谷氨酸组成,表明γ-PGA合成酶基因pgsBCA在大肠杆菌中成功表达.%Studied poly-glutamic acid synthase gene pgsBCA cloned and expressed in the the E.coli, pET28a (+) was selected as the carrier to construct the expression vector pET28a (+)-pgsBCA and to be imported into host E. coli Rosetta, and induced it to express. Dealing with fermentation broth, centrifuged to remove bacteria body and obtained supernatant, using SDS-PAGE electrophores to detect the PGA molacular weight between 200-300kDa pro-duced by recombinant bacteria, hydrolysised the product, using the thin-layer chromatography identification, we found that the product was composed by a single glutamic acid, which showed that-PGA synthase gene pgsBCA was successfully expressed in E.coli.

  18. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    OpenAIRE

    Igamberdiev, Abir U.; Kleczkowski, Leszek A.

    2015-01-01

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, ...

  19. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

    Science.gov (United States)

    Sorigué, Damien; Légeret, Bertrand; Cuiné, Stéphan; Morales, Pablo; Mirabella, Boris; Guédeney, Geneviève; Li-Beisson, Yonghua; Jetter, Reinhard; Peltier, Gilles; Beisson, Fred

    2016-08-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

  20. Virulent but not Avirulent Mycobacterium tuberculosis Can Evade the Growth Inhibitory Action of a T Helper 1–dependent, Nitric Oxide Synthase 2–independent Defense in Mice

    Science.gov (United States)

    Jung, Yu-Jin; LaCourse, Ronald; Ryan, Lynn; North, Robert J.

    2002-01-01

    Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)-γ and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to understanding the basis of Mtb virulence, the ability of gene-deleted mice incapable of making NOS2 (NOS2−/−), gp91Phox subunit of the respiratory burst NADPH-oxidase complex (Phox−/−), or either enzyme (NOS2/Phox−/−), to control airborne infection with the avirulent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with the virulent H37Rv strain. NOS2−/−, Phox−/−, and NOS2/Phox−/− mice showed no deficiency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2−/− mice, but not Phox−/− mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as virulent Mtb, depended on the synthesis of IFN-γ, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN-γ and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1–dependent, NOS2-independent mechanism of defense. PMID:12370260

  1. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan); Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp [Department of Nutritional Sciences, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 (Japan)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  2. Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.

    Science.gov (United States)

    Brodersen, Peter; Petersen, Morten; Bjørn Nielsen, Henrik; Zhu, Shijiang; Newman, Mari-Anne; Shokat, Kevan M; Rietz, Steffen; Parker, Jane; Mundy, John

    2006-08-01

    Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA-ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4-EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.

  3. Structural organization of films based on polyaniline/polysulfonic acid complexes depending on the synthesis method

    International Nuclear Information System (INIS)

    The optical properties and morphology of complexes based on polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), depending on their synthesis conditions, have been characterized by UV-visible spectroscopy and atomic force microscopy. The dependence of the electron absorption spectra of PANI/PAMPS complexes and the surface topography of their films on the initiation way of PANI formation (chemical and enzymatic) and the use of promoters of aniline polymerization has been investigated. The aniline polymerization kinetics with and without polymerization promoters has been studied. All PANI/PAMPS complexes are found to have a nanocomposite time-stable structure.

  4. PKCzeta protects against UV-C-induced apoptosis by inhibiting acid sphingomyelinase-dependent ceramide production

    OpenAIRE

    Charruyer, Alexandra; Jean, Christine; Colomba, Audrey; Jaffrézou, Jean-Pierre; Quillet-Mary, Anne; Laurent, Guy; Bezombes, Christine

    2007-01-01

    Abstract In a recent study, we described that UV-C irradiation resulted in redox-dependent activation and relocalization of acid sphingomyelinase (A-SMase) to the external surface of raft membrane microdomains, hydrolysis of sphingomyelin (SM) associated to the plasma membrane outer leaflet, ceramide (CER) generation and apoptosis. In the present study, we have investigated the influence of PKC? , an atypical form of PKC on this pathway. This study shows that PKC? overexpression r...

  5. Viscosity controls humidity dependence of N2O5 uptake to citric acid aerosol

    Directory of Open Access Journals (Sweden)

    G. Gržinić

    2015-08-01

    Full Text Available The heterogeneous loss of dinitrogen pentoxide (N2O5 to aerosol particles has a significant impact on the night time nitrogen oxide cycle and therefore the oxidative capacity in the troposphere. Using a 13N short lived radioactive tracer method we studied the uptake kinetics of N2O5 on citric acid aerosol particles as a function of relative humidity (RH. The results show that citric acid exhibits lower reactivity than similar di- and polycarboxylic acids, with uptake coefficients between ~ 3 × 10−4–~ 3 × 10−3 depending on humidity (17–70 % RH. This humidity dependence can be explained by a changing viscosity and, hence, diffusivity in the organic matrix. Since the viscosity of highly concentrated citric acid solutions is not well established, we present four different parameterizations of N2O5 diffusivity based on the available literature data or estimates for viscosity and diffusivity. Above 50 % RH, uptake is consistent with the reacto-diffusive kinetic regime whereas below 50 % RH, the uptake coefficient is higher than expected from hydrolysis of N2O5 within the bulk of the particles, and the uptake kinetics may be limited by loss on the surface only. This study demonstrates the impact of viscosity in highly oxidized and highly functionalized secondary organic aerosol material on the heterogeneous chemistry of N2O5 and may explain some of the unexpectedly low loss rates to aerosol derived from field studies.

  6. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner.

    Science.gov (United States)

    Kottyan, Leah C; Collier, Ann R; Cao, Khanh H; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G; Witte, Owen N; Khurana Hershey, Gurjit K; Rothenberg, Marc E; Zimmermann, Nives

    2009-09-24

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5'-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase-dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65(-/-) mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner. PMID:19641187

  7. Systemic injection of kainic acid differently affects LTP magnitude depending on its epileptogenic efficiency.

    Directory of Open Access Journals (Sweden)

    Luz M Suárez

    Full Text Available Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP. Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p. to develop SE in a majority of animals (72-85% rats. A group of rats were resistant to develop SE (15-28% after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals' performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.

  8. Viscosity controls humidity dependence of N2O5 uptake to citric acid aerosol

    Science.gov (United States)

    Gržinić, G.; Bartels-Rausch, T.; Berkemeier, T.; Türler, A.; Ammann, M.

    2015-12-01

    The heterogeneous loss of dinitrogen pentoxide (N2O5) to aerosol particles has a significant impact on the night-time nitrogen oxide cycle and therefore the oxidative capacity in the troposphere. Using a 13N short-lived radioactive tracer method, we studied the uptake kinetics of N2O5 on citric acid aerosol particles as a function of relative humidity (RH). The results show that citric acid exhibits lower reactivity than similar dicarboxylic and polycarboxylic acids, with uptake coefficients between ∼ 3 × 10-4-∼ 3 × 10-3 depending on humidity (17-70 % RH). At RH above 50 %, the magnitude and the humidity dependence can be best explained by the viscosity of citric acid as compared to aqueous solutions of simpler organic and inorganic solutes and the variation of viscosity with RH and, hence, diffusivity in the organic matrix. Since the diffusion rates of N2O5 in highly concentrated citric acid solutions are not well established, we present four different parameterizations of N2O5 diffusivity based on the available literature data or estimates for viscosity and diffusivity of H2O. Above 50 % RH, uptake is consistent with the reacto-diffusive kinetic regime whereas below 50 % RH, the uptake coefficient is higher than expected from hydrolysis of N2O5 within the bulk of the particles, and the uptake kinetics is most likely limited by loss on the surface only. This study demonstrates the impact of viscosity in highly oxidized and highly functionalized secondary organic aerosol material on the heterogeneous chemistry of N2O5 and may explain some of the unexpectedly low loss rates to aerosol derived from field studies.

  9. Temperature dependence of amino acid side chain IR absorptions in the amide I' region.

    Science.gov (United States)

    Anderson, Benjamin A; Literati, Alex; Ball, Borden; Kubelka, Jan

    2014-05-01

    Amide I' IR spectra are widely used for studies of structural changes in peptides and proteins as a function of temperature. Temperature dependent absorptions of amino acid side-chains that overlap the amide I' may significantly complicate the structural analyses. While the side-chain IR spectra have been investigated previously, thus far their dependence on temperature has not been reported. Here we present the study of the changes in the IR spectra with temperature for side-chain groups of aspartate, glutamate, asparagine, glutamine, arginine, and tyrosine in the amide I' region (in D2O). Band fitting analysis was employed to extract the temperature dependence of the individual spectral parameters, such as peak frequency, integrated intensity, band width, and shape. As expected, the side-chain IR bands exhibit significant changes with temperature. The majority of the spectral parameters, particularly the frequency and intensity, show linear dependence on temperature, but the direction and magnitude vary depending on the particular side-chain group. The exception is arginine, which exhibits a distinctly nonlinear frequency shift with temperature for its asymmetric CN3H5(+) bending signal, although a linear fit can account for this change to within ~1/3 cm(-1). The applicability of the determined spectral parameters for estimations of temperature-dependent side-chain absorptions in peptides and proteins are discussed.

  10. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

    Science.gov (United States)

    Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

    2015-01-01

    Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

  11. The effects of trans-fatty acids on TAG regulation in mice depend on dietary unsaturated fatty acids.

    Science.gov (United States)

    Saín, Juliana; González, Marcela Aída; Lavandera, Jimena Verónica; Scalerandi, María Victoria; Bernal, Claudio Adrián

    2016-08-01

    The aim of this study was to investigate the effects of trans-fatty acids (TFA) on liver and serum TAG regulation in mice fed diets containing different proportions of n-3, n-6 and n-9 unsaturated fatty acids (UFA) from olive (O), maize (C) or rapeseed (R) oils partially substituted or not with TFA (Ot, Ct and Rt, respectively). Male CF1 mice were fed (30 d) one of these diets. The effects of the partial substitution (1 %, w/w) of different UFA with TFA on the activity and expression of hepatic enzymes involved in lipogenesis and fatty acids oxidation were evaluated, as well as their transcription factor expressions. Some of the mechanisms involved in the serum TAG regulation, hepatic VLDL rich in TAG (VLDL-TAG) secretion rate and lipoprotein lipase (LPL) activity were assessed. In liver, TFA induced an increase in TAG content in the Ot and Rt groups, and this effect was associated with an imbalance between lipogenesis and β-oxidation. In the Ot group, exacerbated lipogenesis may be one of the mechanisms responsible for the liver steatosis induced by TFA, whereas in Rt it has been related to a decreased β-oxidation, compared with their respective controls. The enhanced hepatic VLDL-TAG secretion in the Ot and Rt groups was compensated with a differential removal of TAG by LPL enzyme in extrahepatic tissues, leading to unchanged serum TAG levels. In brief, the effects of low levels of TFA on liver and serum TAG regulation in mice depend on the dietary proportions of n-3, n-6 and n-9 UFA. PMID:27464460

  12. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    NARCIS (Netherlands)

    Campa, Daniele; McKay, James; Sinilnikova, Olga; Huesing, Anika; Vogel, Ulla; Hansen, Rikke Dalgaard; Overvad, Kim; Witt, Petra Mariann; Clavel-Chapelon, Francoise; Boutron-Ruault, Marie-Christine; Chajes, Veronique; Rohrmann, Sabine; Chang-Claude, Jenny; Boeing, Heiner; Fisher, Eva; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Palli, Domenico; Villarini, Anna; Sacerdote, Carlotta; Mattiello, Amalia; Tumino, Rosario; Peeters, Petra H. M.; van Gils, Carla H.; Bueno-de-Mesquita, H. Bas; Lund, Eiliv; Dolores Chirlaque, Maria; Sala, Nuria; Rodriguez Suarez, Laudina; Barricarte, Aurelio; Dorronsoro, Miren; Sanchez, Maria-Jose; Lenner, Per; Hallmans, Goeran; Tsilidis, Kostas; Bingham, Sheila; Khaw, Kay-Tee; Gallo, Valentina; Norat, Teresa; Riboli, Elio; Rinaldi, Sabina; Lenoir, Gilbert; Tavtigian, Sean V.; Canzian, Federico; Kaaks, Rudolf

    2009-01-01

    Fatty acid synthase (FAS) is the major enzyme of lipogenesis. It catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce palmitic acid. Transcription of the FAS gene is controlled synergistically by the transcription factors ChREBP (carbohydrate response element-binding p

  13. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    DEFF Research Database (Denmark)

    Campa, Daniele; McKay, James; Sinilnikova, Olga;

    2009-01-01

    Fatty acid synthase (FAS) is the major enzyme of lipogenesis. It catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce palmitic acid. Transcription of the FAS gene is controlled synergistically by the transcription factors ChREBP (carbohydrate response element-bindin...

  14. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  15. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    Science.gov (United States)

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems.

  16. Relating physical state and reactivity: humidity dependent ozone uptake on tannic and shikimic acid

    Science.gov (United States)

    Steimer, S.; Huisman, A.; Krieger, U.; Lampimäki, M.; Marcolli, C.; Peter, T.; Ammann, M.

    2012-04-01

    Atmospheric aerosols are an important focus of environmental research due to their effect on climate and human health. Recent findings show that organic aerosol particles are capable of forming amorphous solids and semi-solids under atmospheric conditions [1]. Such particles should be highly viscous, leading to low diffusivity within the bulk. This would then slow down eventual chemical reactions in the bulk, thereby increasing the lifetime of the organic compounds involved. First indications of such behavior were recently shown for the reaction of thin protein films with ozone [2]. To investigate the influence of the physical state on the reactivity of atmospherically relevant compounds, the uptake of ozone on two different organics was measured using a coated wall flow tube system. The investigated organic compounds are tannic acid, which is a proxy for atmospheric polyphenolic materials, and shikimic acid, a constituent of biomass burning aerosols. The viscosity of the organic film was adjusted by varying the humidity of the system, assuming a correlation between the two parameters due to water acting as a plasticizer. The investigated humidity range was 0% - 95% RH for tannic and 0% - 92% RH for shikimic acid. It was found that both of the compounds show a long term uptake of ozone which lasts for more than 20 h. The uptake coefficient is clearly humidity dependent and increases by close to two orders of magnitude between driest and wettest conditions. At a given humidity, shikimic acid is the more reactive compound. The measured humidity dependence supports the hypothesis that the uptake coefficient is a strong function of the diffusion coefficient of ozone in the organic film. 1. Virtanen, A., et al., An amorphous solid state of biogenic secondary organic aerosol particles. Nature, 2010. 467(7317): p. 824-827. 2. Shiraiwa, M., et al., Gas uptake and chemical aging of semisolid organic aerosol particles. Proceedings of the National Academy of Sciences of the

  17. Regulation of phenylacetic acid uptake is sigma54 dependent in Pseudomonas putida CA-3.

    LENUS (Irish Health Repository)

    O' Leary, Niall D

    2011-10-13

    primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. Conclusions In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.

  18. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner

    OpenAIRE

    Kottyan, Leah C.; Collier, Ann R.; Cao, Khanh H.; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G.; Owen N Witte; Gurjit K Khurana Hershey; Rothenberg, Marc E.; Zimmermann, Nives

    2009-01-01

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5′-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase–dependent manner. Furthermore, we identify G protein-coupled receptor 65 (...

  19. Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Hallström, Björn M.; Blicher, Thomas H.;

    2014-01-01

    , preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5.Genome sequencing followed by functional analysis identified the causal mutation in SFA1 gene encoding S-(hydroxymethyl)glutathione dehydrogenase. Based on our findings, we...... propose that 3HP toxicity is mediated by3-hydroxypropionic aldehyde (reuterin) and that glutathione-dependent reactions are used for reuterin detoxification. The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acid and aldehydes by...

  20. The mycosubtilin synthetase of Bacillus subtilis ATCC6633 : A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

    NARCIS (Netherlands)

    Duitman, EH; Hamoen, LW; Rembold, M; Venema, G; Seitz, H; Saenger, W; Bernhard, F; Reinhardt, R; Schmidt, M; Ullrich, C; Stein, T; Leenders, F; Vater, J

    1999-01-01

    Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a p-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Cl

  1. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  2. Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

    International Nuclear Information System (INIS)

    The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca2+ pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca2+ entrance was induced by A23187 in HepG2. Cell death, Ca2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

  3. Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

    Science.gov (United States)

    Lederer, Florence; Vignaud, Caroline; North, Paul; Bodevin, Sabrina

    2016-09-01

    A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes. PMID:27155230

  4. CHANGES IN THE ANTIOXIDANT CAPACITY OF POTATOES DEPENDING ON THE CULTIVAR, CONTENTS OF POLYPHENOLS, CHLOROGENIC ACID AND ASCORBIC ACID

    Directory of Open Access Journals (Sweden)

    Janette Musilova

    2015-02-01

    Full Text Available Effect of cultivar is known as the most important factor determining the qualitative and quantitative characteristics of potatoes. In the study the influence of this factor on the content of chlorogenic acid (CGA, ascorbic acid (AA, total polyphenols (TPC and antioxidant capacity (TAC in six potato cultivars (Viola, Malvina, Evelin, Arlet, Megan, Timea was investigated. Potatoes were grown in vitro on peat substrate in a greenhouse and were harvested in the stage of physiological ripeness. The content of CGA, AA and the TAC were determined in fresh matter of potato tubers. CGA content was determined using standard HPLC gradient method. The lowest CGA amount was determined in cv. Viola (18.49 mg/kg FM and the highest one in cv. Megan (46.73 mg/kg FM. The determined AA content was in interval 5.40 mg/100 g FM (cv. Evelin – 20.10 mg/100 g FM (cv. Viola. Total antioxidant capacity expressed as mg eqv. Trolox/kg FM was the lowest in cv. Megan (41.06 mg TE/kg FM and the highest in cv. Arlet (56.16 mg TE/kg FM. For TP content determination lyophilised potato samples were used. The determined values ranged from 256.44 until 425.37 mg.kg-1 DM in followed order: Evelin < Arlet < Megan < Timea < Viola < Malvina. The obtained results were evaluated using one-factorial analysis ANOVA (LSD-test, statistical software Statgraphic. Mutual correlations between the contents of CGA, AA, TP and TAC were evaluated using regression and correlation analysis (Microsoft Excel. Statistically significant dependence (P-value < 0.05 between observed factors was confirmed.

  5. Conductivity-Dependent Flow Field-Flow Fractionation of Fulvic and Humic Acid Aggregates

    Directory of Open Access Journals (Sweden)

    Martha J. M. Wells

    2015-09-01

    Full Text Available Fulvic (FAs and humic acids (HAs are chemically fascinating. In water, they have a strong propensity to aggregate, but this research reveals that tendency is regulated by ionic strength. In the environment, conductivity extremes occur naturally—freshwater to seawater—warranting consideration at low and high values. The flow field flow fractionation (flow FFF of FAs and HAs is observed to be concentration dependent in low ionic strength solutions whereas the corresponding flow FFF fractograms in high ionic strength solutions are concentration independent. Dynamic light scattering (DLS also reveals insight into the conductivity-dependent behavior of humic substances (HSs. Four particle size ranges for FAs and humic acid aggregates are examined: (1 <10 nm; (2 10 nm–6 µm; (3 6–100 µm; and (4 >100 µm. Representative components of the different size ranges are observed to dynamically coexist in solution. The character of the various aggregates observed—such as random-extended-coiled macromolecules, hydrogels, supramolecular, and micellar—as influenced by electrolytic conductivity, is discussed. The disaggregation/aggregation of HSs is proposed to be a dynamic equilibrium process for which the rate of aggregate formation is controlled by the electrolytic conductivity of the solution.

  6. Myogenic and metabolic feedback in cerebral autoregulation: Putative involvement of arachidonic acid-dependent pathways.

    Science.gov (United States)

    Berg, Ronan M G

    2016-07-01

    The present paper presents a mechanistic model of cerebral autoregulation, in which the dual effects of the arachidonic acid metabolites 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) on vascular smooth muscle mediate the cerebrovascular adjustments to a change in cerebral perfusion pressure (CPP). 20-HETE signalling in vascular smooth muscle mediates myogenic feedback to changes in vessel wall stretch, which may be modulated by metabolic feedback through EETs released from astrocytes and endothelial cells in response to changes in brain tissue oxygen tension. The metabolic feedback pathway is much faster than 20-HETE-dependent myogenic feedback, and the former thus initiates the cerebral autoregulatory response, while myogenic feedback comprises a relatively slower mechanism that functions to set the basal cerebrovascular tone. Therefore, assessments of dynamic cerebral autoregulation, which may provide information on the response time of the cerebrovasculature, may specifically be used to yield information on metabolic feedback mechanisms, while data based on assessments of static cerebral autoregulation represent the integrated functionality of myogenic and metabolic feedback. PMID:27241246

  7. Modulation of the voltage-dependent anion channel of mitochondria by elaidic acid.

    Science.gov (United States)

    Tewari, Debanjan; Bera, Amal Kanti

    2016-08-26

    Dietary trans fatty acids (TFAs) are known to increase the risk of cardiovascular diseases by altering plasma lipid profile and activating various inflammatory signaling pathways. Here we show that elaidic acid (EA), the most abundant TFA in diet, alters the electrophysiological properties of voltage-dependent anion channel (VDAC) of mitochondria. Purified bovine brain VDAC, when incorporated in the planar lipid bilayer (PLB) composed of 1,2-diphytanoyl-sn-glycero-3 phosphatidyl choline (DPhPC) and EA in a 9 to 1 ratio (wt/wt), exhibited complete closing events at different voltages. The closing events were observed at even -10 mV, a voltage at which VDAC usually remains fully open all the time. Additionally, the voltage sensitivity of VDAC was lost in presence of EA; the channel conductance did not decrease with increasing voltages. In identical experimental conditions, membrane containing oleic acid (OA), the cis isomer of EA did not produce any such effect. We propose that EA possibly exerts its adverse effect by modulating VDAC. PMID:27318085

  8. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  9. 结直肠癌患者血清脂肪酸合成酶水平的检测及其临床意义%Serum levels of fatty acid synthase in colorectal cancer patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    闾晨涛; 韩潞; 江勇; 韩东兴

    2014-01-01

    背景与目的:脂肪酸合成酶(fatty acid synthase, FAS)是唯一有能力在细胞内合成长链脂肪酸的蛋白。由于肿瘤组织对脂肪酸的需求旺盛,包括结直肠癌在内的多种恶性肿瘤组织中常见FAS过表达。本实验研究结直肠癌患者血清FAS水平与肿瘤病理特征的关系。方法:选择2013年3月-2014年3月接受根治性手术治疗的60例结直肠癌患者为研究组,另选20名健康志愿者为对照组。采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清FAS水平,分析结直肠癌患者血清FAS水平与其临床病理特征的关系。结果:研究组FAS平均为20.77±10.56 mg/L,对照组FAS水平为10.33±5.65 mg/L,差异有统计学意义(P0.05)。结论:血清FAS水平可能与结直肠癌的发生、发展有一定关系,可以对其作为评估肿瘤进展情况的标志物的可行性作进一步研究。%Background and purpose:Fatty acid synthase (FAS) is the sole protein in the human genome capable of intracellular synthesis of long-chain fatty acids. FAS overexpression is detected in various cancer tissues including colorectal cancer because of the increasing requirement of tumor for long-chain fatty acid. This study was to investigate the association between serum levels of FAS in patients with colorectal cancer and clinicopathological characteristics of colorectal cancer.Methods:A total of 60 patients who underwent radical surgical resection for colorectal cancer from Mar. 2013 to Mar. 2014 were selected as the study group, while 20 healthy volunteers were selected as the control group. The serum levels of FAS were measured by enzyme-linked immunosorbent assay (ELISA) methods. Differences of serum levels of FAS in patients with various clinicopathological characteristics of colorectal cancer were analyzed.Results:The serum levels of FAS in the study group were signiifcantly different with those in the control group. Serum FAS levels of

  10. Chemical pathology of homocysteine. V. Thioretinamide, thioretinaco, and cystathionine synthase function in degenerative diseases.

    Science.gov (United States)

    McCully, Kilmer S

    2011-01-01

    Hyperhomocysteinemia was first associated with degenerative disease by observation of accelerated arteriosclerosis in children with inherited disorders of cystathionine synthase, methionine synthase, and methylene tetrohydrofolate reductase. The metabolic blockade of sulfate synthesis from homocysteine thiolactone in malignant cells is ascribed to a deficiency of a chemopreventive derivative of homocysteine thiolactone that occurs in normal cells. Its chemical structure was elucidated by the organic synthesis of thioretinamide from retinoic acid and homocysteine thiolactone. Oxidation of the sulfur atom of homocysteine is inhibited in scorbutic guinea pigs, demonstrating ascorbate function in sulfate synthesis from homocysteine. Studies of homocysteine metabolism in protein energy malnutrition led to the conclusion that the biosynthesis of thioretinamide from the retinol of transthyretin is catalyzed by dehydroascorbate and superoxide generated from the heme oxygenase group of cystathionine synthase. Newly synthesized thioretinamide is complexed with cobalamin to form thioretinaco, which is activated by ozone and oxygen to function as the active site of oxidative phosphorylation. In accordance with the trophoblastic theory of cancer, pancreatic enzymes are believed to be oncolytic because they hydrolyze the homocysteinylated proteins, nucleic acids and glycosaminoglycans of malignant tissues. The clonal selection of malignant cells that are deficient in the heme oxygenase function of cystathionine synthase produces cells dependent upon glycolysis for ATP synthesis, since they are deficient in synthesis of thioretinamide, thioretinaco and thioretinaco ozonide. The vulnerable plaque of arteriosclerosis originates from complexes of microbes with homocysteinylated lipoproteins, obstructing vasa vasorum narrowed by endothelial dysfunction, causing arterial ischemia, and intimal micro-abscesses. Degenerative diseases may be ameliorated by a proposed therapeutic protocol

  11. Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocycl opropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

    Institute of Scientific and Technical Information of China (English)

    Jian-Feng LI; Robert QI; Liang-Hu QU; Autar K Mattoo; Ning LI

    2005-01-01

    l-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv.Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the Nterminus of LeACS2. Four C-terminus-deleted (26-50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser460 for this metalloprotease.Furhermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.

  12. Antinociceptive effect of clavulanic acid and its preventive activity against development of morphine tolerance and dependence in animal models.

    Science.gov (United States)

    Hajhashemi, V; Dehdashti, Kh

    2014-01-01

    Glutamate has a key role in pain perception and also development of tolerance and dependence to morphine. It has been reported that clavulanic acid affects glutamatergic transmission via activation of glutamate transporter. Therefore the present study was aimed to evaluate the possible antinociceptive effect of clavulanic acid and its preventive activity against development of morphine tolerance and dependence in animal models. Male Swiss mice (25-30 g) were used in this study. Acetic acid-induced writhing, formalin test and hot plate method were used to assess the antinociceptive effect of clavulanic acid. Morphine (30 mg/kg, s.c.) was administered to the mice two times a day (8 AM and 4 PM) for 3 days in order to produce tolerance. To develop morphine dependence, morphine sulfate (50, 50 and 75 mg/kg) was injected at 8 and 12 AM and 16 PM respectively and for 3 consecutive days. Naloxone (5 mg/kg, i.p) was used to induce morphine withdrawal syndrome and the number of jumps and presence of ptosis, piloerection, tremor, sniffing and diarrhea were recorded and compared with control group. Clavulanic acid at doses of 10, 20 and 40 mg/kg inhibited abdominal constriction and licking behavior of acetic acid and formalin-induced pain respectively. Clavulanic acid was not able to show any antinociception in hot plate model and could not prevent development of tolerance and dependence to morphine. Clavulanic acid has considerable antinociceptive activity and further studies are needed to clarify its exact mechanism.

  13. Opioid Tolerance and Physical Dependence: Role of Spinal Neuropeptides, Excitatory Amino Acids and Their Messengers

    Directory of Open Access Journals (Sweden)

    Khem Jhamandas

    2000-01-01

    Full Text Available Chronic opioid treatment results in the development of tolerance and physical dependence. The mechanisms underlying opioid tolerance and/or physical dependence are unclear. Recent studies suggest that opioid receptor or nociceptive, neural network-based adaptations contribute to this phenomenon. At the spinal level, the genesis of tolerance and physical dependence is associated with increased excitatory amino acid activity expressed through N-methyl-D-aspartate receptors in the dorsal horn. However, recent evidence also implicates spinal neuropeptide transmitters such as calcitonin gene-related peptide (CGRP and  substance P in the development of opioid tolerance. Long term spinal morphine treatment increases CGRP-like immunostaining in the dorsal horn, and coadministration of morphine with CGRP8-37, a competitive CGRP1 receptor antagonist, prevents this response as well as loss of the analgesic potency. CGRP8-37, like N-methyl-D-aspartate receptor antagonists, has the potential to restore morphine potency in experimental animals who are already tolerant to the opioid agonist. Recent evidence suggests that the effects of excitatory amino acid and neuropeptide receptor activity may be expressed through the generation of messengers such as nitric oxide and prostanoids. Agents that inhibit the synthesis of nitric oxide and prostanoids have the potential to inhibit and reverse spinal opioid tolerance, suggesting that this phenomenon may be expressed through the activity of these mediators. Nociceptive transmission in the dorsal horn of the spinal cord also involves activity of a number of other mediators including morphine modulatory neuropeptides, neuropeptide FF  and neuropeptide SF. The role of these mediators and their relationship with other factors implicated in tolerance remain to be determined.

  14. Ursolic acid and luteolin-7-glucoside improves rat plasma lipid profile and increases liver glycogen content through glycogen synthase kinase-3

    OpenAIRE

    Azevedo, Marisa; Camsari, Çagri; Sá, Carla M.; Lima, Cristóvão F.; Ferreira, Manuel Fernandes; Wilson, Cristina Pereira

    2010-01-01

    Documento submetido para revisão pelos pares. A publicar em Phytotherapy Research. ISSN 0951-418X In the present study, two phytochemicals – ursolic acid (UA) and luteolin-7-glucoside (L7G) – were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profi le (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucos...

  15. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  16. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    Science.gov (United States)

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  17. Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea.

    Science.gov (United States)

    Kravchuk, Zhana; Vicedo, Begonya; Flors, Víctor; Camañes, Gemma; González-Bosch, Carmen; García-Agustín, Pilar

    2011-03-01

    Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses. Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment. We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.

  18. Ca2+- and PKC-dependent stimulation of PGE2 synthesis by deoxycholic acid in human colonic fibroblasts.

    Science.gov (United States)

    Zhu, Yingting; Hua, Ping; Rafiq, Shazia; Waffner, Eric J; Duffey, Michael E; Lance, Peter

    2002-09-01

    We investigated prostanoid biogenesis by human colonic fibroblasts (CCD-18Co cells and nine primary fibroblast cultures) exposed to a primary (cholic, CA) or a secondary (deoxycholic, DCA) bile acid. Basal PGE2 levels in CCD-18Co cultures and fibroblast strains initiated from normal and adenocarcinomatous colon, respectively, were 1.7 +/- 0.3, 4.0 +/- 2.0, and 15.0 +/- 4.8 ng/mg protein. Peak levels 24 h after exposure to DCA (300 microM) rose, respectively, seven-, six- and sevenfold, but CA elicited no such responses. Increases in PGE2 synthesis were preceded by sequential increases in PGH synthase-2 mRNA and protein expression and were fully prevented by a nonselective (indomethacin) or a selective (celecoxib) nonsteroidal anti-inflammatory drug. DCA, but not CA, caused abrupt, transient increases in fibroblast intracellular Ca2+ concentration ([Ca2+]i) approximately 1 min after exposure. Increased [Ca2+]i was required for DCA-mediated induction of PGE2 synthesis, and protein kinase C was a further essential component of this signaling pathway. Colonic fibroblasts may be a major target for prostanoid biogenesis induced by fecal bile acids and, potentially, other noxious actions of these agents. PMID:12181161

  19. [Gamma-hydroxybutyric acid (GHB) dependence and the GHB withdrawal syndrome: diagnosis and treatment].

    Science.gov (United States)

    van Noorden, Martijn S; Kamal, Rama; de Jong, Cor A J; Vergouwen, A C M Ton; Zitman, Frans G

    2010-01-01

    Gamma-hydroxybutyric acid (GHB) is a neurotransmitter that occurs naturally in the brain and is increasingly being used as a 'party drug' because of its relaxing and euphoria-inducing effects. GHB has a limited medical use in the treatment of narcolepsy. GHB-intoxications occur often in non-medical use, and generally result in a coma of short duration. GHB use several times a day can lead to tolerance and dependence. After sudden cessation or reduction of intensive GHB use, a severe withdrawal syndrome may occur with symptoms varying from tremor, anxiety and agitation to autonomic instability, hallucinations and delirium. Treatment of the GHB withdrawal syndrome consists of supportive care and benzodiazepines, often in high doses. The controlled detoxification of GHB using pharmaceutical GHB in an adjusted dose is currently being investigated in the Netherlands. There is no literature concerning the treatment of patients following GHB intoxication or after detoxification.

  20. Observations of nitrous acid and its relative humidity dependence in Shanghai

    Institute of Scientific and Technical Information of China (English)

    HAO Nan; ZHOU Bin; CHEN Dan; CHEN Li-min

    2006-01-01

    Nitrous acid, HNO2, is an important precursor of OH radicals in the troposphere. Measurements of HNO2 and NO2, using differential optical absorption spectroscopy (DOAS), were performed in Shanghai, China for a period from October 22, 2004 to January 4, 2005. The mean (and median) hourly concentrations of HNO2 and NO2 during this period were 1.1 (0.7) ppb and 24 (21.4)ppb respectively. A correlation between HNO2/NO2 and PM10 mass concentrations was obtained. This correlation suggests that significant heterogeneous chemical production of HNO2 may occur through NO2 reactions on aerosol surfaces. This hypothesis was further supported by detailed analysis of selected pollution episodes in this study. At the same time, the water dependence of HNO2 formation was studied by analysis of relative humidity (RH). It showed that the maximum HNO2/NO2 ratio was increased along with RH below 70% and inhibited at RH>70%.

  1. Morphology-dependent capacitive properties of three nanostructured polyanilines through interfacial polymerization in various acidic media

    International Nuclear Information System (INIS)

    Highlights: • Preparing three PANI nanostructures in HCl, H2SO4, and HNO3 reaction media during a slow interfacial polymerization. • Obtaining nanofibrous, nanogranular and hollow ball–like morphologies in the order of oxidizability of acids. • Demonstrating high specific capacitance, good rate performance and long cycle life of PANI nanofibers. - Abstract: Three nanostructured polyanilines (PANIs) were prepared by interfacial polymerization using different inorganic acids including HCl, H2SO4, and HNO3 as reaction media, respectively. The morphology-dependent structure and properties of as-prepared PANIs were characterized by means of scanning electron microscope, ultraviolet-visible spectra, Fourier transform infrared spectroscopy and X-ray diffraction patterns. Meanwhile, the electrochemical performance of the fabricated electrodes was evaluated by cyclic voltammetry, galvanostatic charge/discharge measurement, and electrochemical impedance spectroscopy. It is found that the reaction medium plays a vital role in deciding the final morphology and structure of product when a slow reaction rate occurred in interfacial polymerization. The as-prepared PANIs exhibited nanofibrous, nanogranular and hollow ball-like morphologies according to the order of the relative oxidizing ability of HCl < H2SO4 < HNO3, respectively. Furthermore, the capacitive properties of these composites as electrode materials highly depended not only on their morphologies but also on the conductivity, crystalline property and the inhibitory role of the lattice during the redox process, as well as the interparticle contact resistance. It is demonstrated that PANI nanofibers prepared in HCl medium exhibited high specific capacitance, good rate performance and long cycle life for the supercapacitor application

  2. Suppression of jasmonic acid-dependent defense in cotton plant by the mealybug Phenacoccus solenopsis.

    Directory of Open Access Journals (Sweden)

    Pengjun Zhang

    Full Text Available The solenopsis mealybug, Phenacoccus solenopsis, has been recently recognized as an aggressively invasive pest in China, and is now becoming a serious threat to the cotton industry in the country. Thus, it is necessary to investigate the molecular mechanisms employed by cotton for defending against P. solenopsis before the pest populations reach epidemic levels. Here, we examined the effects of exogenous jasmonic acid (JA, salicylic acid (SA, and herbivory treatments on feeding behavior and on development of female P. solenopsis. Further, we compared the volatile emissions of cotton plants upon JA, SA, and herbivory treatments, as well as the time-related changes in gossypol production and defense-related genes. Female adult P. solenopsis were repelled by leaves from JA-treated plant, but were not repelled by leaves from SA-treated plants. In contrast, females were attracted by leaves from plants pre-infested by P. solenopsis. The diverse feeding responses by P. solenopsis were due to the difference in volatile emission of plants from different treatments. Furthermore, we show that JA-treated plants slowed P. solenopsis development, but plants pre-infested by P. solenopsis accelerated its development. We also show that P. solenopsis feeding inhibited the JA-regulated gossypol production, and prevented the induction of JA-related genes. We conclude that P. solenopsis is able to prevent the activation of JA-dependent defenses associated with basal resistance to mealybugs.

  3. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    C.C.N. van Schie; M.A. Haring; R.C. Schuurink

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  4. Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid.

    Science.gov (United States)

    Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Stec-Martyna, Emilia; Pawlowska, Zofia; Watala, Cezary

    2016-06-25

    Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1

  5. Use of linalool synthase in genetic engineering of scent production

    Energy Technology Data Exchange (ETDEWEB)

    Pichersky, Eran (Chelsea, MI)

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  6. Use of linalool synthase in genetic engineering of scent production

    Energy Technology Data Exchange (ETDEWEB)

    Pichersky, E.

    1998-12-15

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

  7. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  8. Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

    KAUST Repository

    Bastian, René

    2010-03-01

    An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

  9. A "Whirly" transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis.

    Science.gov (United States)

    Desveaux, Darrell; Subramaniam, Rajagopal; Després, Charles; Mess, Jean-Nicholas; Lévesque, Caroline; Fobert, Pierre R; Dangl, Jeffery L; Brisson, Normand

    2004-02-01

    Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response. PMID:14960277

  10. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.

    Science.gov (United States)

    Gibert, Yann; Bernard, Laure; Debiais-Thibaud, Melanie; Bourrat, Franck; Joly, Jean-Stephane; Pottin, Karen; Meyer, Axel; Retaux, Sylvie; Stock, David W; Jackman, William R; Seritrakul, Pawat; Begemann, Gerrit; Laudet, Vincent

    2010-09-01

    One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity. PMID:20445074

  11. Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle

    OpenAIRE

    1992-01-01

    The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)- 2-methylp...

  12. Computational studies of acid sites in ZSM-5: Dependence on cluster size

    International Nuclear Information System (INIS)

    An ab initio molecular orbital study of the proton affinities, OH stretching frequencies, and ammonia interactions of SixTOyHz (T = Si, Al) clusters representing the ZSM-5 zeolite framework has been carried out. The dependence of these properties on cluster size was examined. The proton affinity of the central bridging Si-O-Al site was found to be slowly convergent with cluster size. In calculations on clusters containing up to two shells of silicons and two shells of oxygens beyond the bridge, the proton affinity varies by as much as 30 kcal/mol with each additional shell. Addition of a silicon shell reduces the proton affinity varies by as much as 30 kcal/mol with each additional shell. Addition of a silicon shell reduces the proton affinity and addition of an oxygen shell increases the proton affinity. This is due to the long range electrostatic interaction of the added shells on the bridging hydroxyl site. The clusters containing a central Si-O-Si bridge exhibit a similar slow convergence of the proton affinity. The OH stretching frequencies are much less dependent on cluster size than the proton affinities. The theoretical proton affinities and OH stretching frequencies are much less dependent on cluster size than the proton affinities. The theoretical proton affinities and OH stretching frequencies from the T = Al clusters are consistent with experiment. The interaction energy of the 'ionic' structure that results from interaction of the ammonia with the acid site, Z-...HNH3+, has a slow convergence with cluster size similar to the proton affinity and the 'ionic' structure increases instability relative to the 'covalent' structure, ZH...NH3, with increasing cluster size. The NH3 desorption energy is inversely proportional to the proton affinities of the clusters of the same size representing the ZSM-5 zeolite. 54 refs., 8 figs., 7 tabs

  13. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass.

    Science.gov (United States)

    Solloway, Mark J; Madjidi, Azadeh; Gu, Chunyan; Eastham-Anderson, Jeff; Clarke, Holly J; Kljavin, Noelyn; Zavala-Solorio, Jose; Kates, Lance; Friedman, Brad; Brauer, Matt; Wang, Jianyong; Fiehn, Oliver; Kolumam, Ganesh; Stern, Howard; Lowe, John B; Peterson, Andrew S; Allan, Bernard B

    2015-07-21

    Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation. PMID:26166562

  14. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

    Directory of Open Access Journals (Sweden)

    Mark J. Solloway

    2015-07-01

    Full Text Available Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

  15. Correction of Hypertension by Normalization of Endothelial Levels of Fibroblast Growth Factor and Nitric Oxide Synthase in Spontaneously Hypertensive Rats

    Science.gov (United States)

    Cuevas, Pedro; Garcia-Calvo, Margarita; Carceller, Fernando; Reimers, Diana; Zazo, Mercedes; Cuevas, Begona; Munoz-Willery, Isabel; Martinez-Coso, Victoria; Lamas, Santiago; Gimenez-Gallego, Guillermo

    1996-10-01

    Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

  16. CD36 binds oxidized low density lipoprotein (LDL) in a mechanism dependent upon fatty acid binding.

    Science.gov (United States)

    Jay, Anthony G; Chen, Alexander N; Paz, Miguel A; Hung, Justin P; Hamilton, James A

    2015-02-20

    The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

  17. The kinetics of process dependent ammonia inhibition of methanogenesis from acetic acid.

    Science.gov (United States)

    Wilson, Christopher Allen; Novak, John; Takacs, Imre; Wett, Bernhard; Murthy, Sudhir

    2012-12-01

    Advanced anaerobic digestion processes aimed at improving the methanization of sewage sludge may be potentially impaired by the production of inhibitory compounds (e.g. free ammonia). The result of methanogenic inhibition is relatively high effluent concentrations of acetic acid and other soluble organics, as well as reduced methane yields. An extreme example of such an advanced process is the thermal hydrolytic pretreatment of sludge prior to high solids digestion (THD). Compared to a conventional mesophilic anaerobic digestion process (MAD), THD operates in a state of constant inhibition driven by high free ammonia concentrations, and elevated pH values. As such, previous investigations of the kinetics of methanogenesis from acetic acid under uninhibited conditions do not necessarily apply well to the modeling of extreme processes such as THD. By conducting batch ammonia toxicity assays using biomass from THD and MAD reactors, we compared the response of these communities over a broad range of ammonia inhibition. For both processes, increased inhibitor concentrations resulted in a reduction of biomass growth rate (r(max) = μ(max)∙X) and a resulting decrease in the substrate half saturation coefficient (K(S)). These two parameters exhibited a high degree of correlation, suggesting that for a constant transport limited system, the K(S) was mostly a linear function of the growth rate. After correcting for reactor pH and temperature, we found that the THD and MAD biomass were both able to perform methanogenesis from acetate at high free ammonia concentrations (equivalent to 3-5 g/L total ammonia nitrogen), albeit at less than 30% of their respective maximum rates. The reduction in methane production was slightly less pronounced for the THD biomass than for MAD, suggesting that the long term exposure to ammonia had selected for a methanogenic pathway less dependent on those organisms most sensitive to ammonia inhibition (i.e. aceticlastic methanogens). PMID

  18. Neighbor-dependent Ramachandran probability distributions of amino acids developed from a hierarchical Dirichlet process model.

    Directory of Open Access Journals (Sweden)

    Daniel Ting

    2010-04-01

    Full Text Available Distributions of the backbone dihedral angles of proteins have been studied for over 40 years. While many statistical analyses have been presented, only a handful of probability densities are publicly available for use in structure validation and structure prediction methods. The available distributions differ in a number of important ways, which determine their usefulness for various purposes. These include: 1 input data size and criteria for structure inclusion (resolution, R-factor, etc.; 2 filtering of suspect conformations and outliers using B-factors or other features; 3 secondary structure of input data (e.g., whether helix and sheet are included; whether beta turns are included; 4 the method used for determining probability densities ranging from simple histograms to modern nonparametric density estimation; and 5 whether they include nearest neighbor effects on the distribution of conformations in different regions of the Ramachandran map. In this work, Ramachandran probability distributions are presented for residues in protein loops from a high-resolution data set with filtering based on calculated electron densities. Distributions for all 20 amino acids (with cis and trans proline treated separately have been determined, as well as 420 left-neighbor and 420 right-neighbor dependent distributions. The neighbor-independent and neighbor-dependent probability densities have been accurately estimated using Bayesian nonparametric statistical analysis based on the Dirichlet process. In particular, we used hierarchical Dirichlet process priors, which allow sharing of information between densities for a particular residue type and different neighbor residue types. The resulting distributions are tested in a loop modeling benchmark with the program Rosetta, and are shown to improve protein loop conformation prediction significantly. The distributions are available at http://dunbrack.fccc.edu/hdp.

  19. Cellulose synthase complexes: structure and regulation

    Directory of Open Access Journals (Sweden)

    Lei eLei

    2012-04-01

    Full Text Available This review is to update the most recent progress on characterization of the composition, regulation, and trafficking of cellulose synthase complexes. We will highlight proteins that interact with cellulose synthases, e.g. cellulose synthase-interactive protein 1 (CSI1. The potential regulation mechanisms by which cellulose synthase interact with cortical microtubules in primary cell walls will be discussed.

  20. Temperature dependent emission of formic and acetic acid from paper, and its consequences for the air quality in archives

    DEFF Research Database (Denmark)

    Hjerrild Smedemark, Signe; Ryhl-Svendsen, Morten; Vinther Hansen, Birgit;

    2014-01-01

    ) the reaction rate is therefore, among other things, depending on temperature. Some of the emission products from paper, e.g., acetic and formic acid, are problematic from a conservation point of view, because they will re-react with the paper and other archival materials and cause further deterioration......Paper records affect the indoor environment in archives, because the paper may give off acid compounds to the air. These emission products origins from the decay of cellulose and lignin in the paper. As the off-gassing origins from chemical processes within the material (e.g., acid hydrolysis...

  1. Mitochondria-dependent apoptosis of con A-activated T lymphocytes induced by asiatic acid for preventing murine fulminant hepatitis.

    Science.gov (United States)

    Guo, Wenjie; Liu, Wen; Hong, Shaocheng; Liu, Hailiang; Qian, Cheng; Shen, Yan; Wu, Xuefeng; Sun, Yang; Xu, Qiang

    2012-01-01

    Selectively facilitating apoptosis of activated T cells is essential for the clearance of pathogenic injurious cells and subsequent efficient resolution of inflammation. However, few chemicals have been reported to trigger apoptosis of activated T cells for the treatment of hepatitis without affecting quiescent T cells. In the present study, we found that asiatic acid, a natural triterpenoid, selectively triggered apoptosis of concanavalin A (Con A)-activated T cells in a mitochondria-dependent manner indicated by the disruption of the mitochondrial transmembrane potential, release of cytochrome c from mitochondria to cytosol, caspases activation, and cleavage of PARP. In addition, asiatic acid also induced the cleavage of caspase 8 and Bid and augmented Fas expression in Con A-activated T cells. However, following activation of T cells from MRL(lpr/lpr) mice with mutation of Fas demonstrated a similar susceptibility to asiatic acid-induced apoptosis compared with normal T cells, suggesting that Fas-mediated death-receptor apoptotic pathway does not mainly contribute to asiatic acid-induced cell death. Furthermore, asiatic acid significantly alleviated Con A-induced T cell-dependent fulminant hepatitis in mice, as assessed by reduced serum transaminases, pro-inflammatory cytokines, and pathologic parameters. Consistent with the in vitro results, asiatic acid also induced apoptosis of activated CD4(+) T cells in vivo. Taken together, our results demonstrated that the ability of asiatic acid to induce apoptosis of activated T cells and its potential use in the treatment of T-cell-mediated inflammatory diseases.

  2. Structure-dependent activities of polybrominated diphenyl ethers and hydroxylated metabolites on zebrafish retinoic acid receptor.

    Science.gov (United States)

    Zhao, Jing; Zhu, Xiangwei; Xu, Ting; Yin, Daqiang

    2015-02-01

    Polybrominated diphenyl ethers (PBDEs), a group of potential endocrine-disrupting chemicals (EDCs) have been shown to disrupt retinoid homeostasis in different species in both laboratory and field studies. However, the molecular mechanisms of interactions with the retinoic acid receptor (RAR) are not fully understood. Zebrafish have proven useful for investigating mechanisms of chemical toxicity. In the present study, a reporter gene assay was used to investigate the activities of 11 PBDEs and six OH-PBDEs with different degrees of bromination on zebrafish RAR. All tested OH-PBDEs induced RAR transcriptional activity; however, of the 11 PBDEs examined, only BDE28 and BDE154 affected the RAR transcriptional activity. Homology modeling and molecular docking were employed to simulate the interactions of PBDEs/OH-PBDEs with zebrafish RARs and to identify binding affinities to analyze the specialization of the interaction between RARs and PBDEs/OH-PBDEs. The results showed that although these compounds could bind with RARs, the effects of PBDEs/OH-PBDEs on RAR transcriptional activity did not depend on their RAR-binding abilities. The present study is the first attempt to demonstrate that OH-PBDEs could induce RAR transcriptional activity by binding directly with RAR; these effects are possibly related to the structure of the compounds, especially their hydroxylation and bromination. Most of the PBDEs could not directly interact with the RAR. PMID:25077655

  3. Observations of nitrous acid and its relative humidity dependence in Shanghai.

    Science.gov (United States)

    Hao, Nan; Zhou, Bin; Chen, Dan; Chen, Li-min

    2006-01-01

    Nitrous acid, HNO2, is an important precursor of OH radicals in the troposphere. Measurements of HNO2 and NO2, using differential optical absorption spectroscopy (DOAS), were performed in Shanghai, China for a period from October 22, 2004 to January 4, 2005. The mean (and median) hourly concentrations of HNO2 and NO2 during this period were 1.1 (0.7) ppb and 24 (21.4) ppb respectively. A correlation between HNO2/NO2 and PMI,( mass concentrations was obtained. This correlation suggests that significant heterogeneous chemical production of HNO2 may occur through NO2 reactions on aerosol surfaces. This hypothesis was further supported by detailed analysis of selected pollution episodes in this study. At the same time, the water dependence of HNO2 formation was studied by analysis of relative humidity (RH). It showed that the maximum HNO2/NO2 ratio was increased along with RH below 70% and inhibited at RH>70%. PMID:17278746

  4. Chlorogenic acid differentially affects postprandial glucose and glucose-dependent insulinotropic polypeptide response in rats.

    Science.gov (United States)

    Tunnicliffe, Jasmine M; Eller, Lindsay K; Reimer, Raylene A; Hittel, Dustin S; Shearer, Jane

    2011-10-01

    Regular coffee consumption significantly lowers the risk of type 2 diabetes (T2D). Coffee contains thousands of compounds; however, the specific component(s) responsible for this reduced risk is unknown. Chlorogenic acids (CGA) found in brewed coffee inhibit intestinal glucose uptake in vitro. The objective of this study was to elucidate the mechanisms by which CGA acts to mediate blood glucose response in vivo. Conscious, unrestrained, male Sprague-Dawley rats were chronically catheterized and gavage-fed a standardized meal (59% carbohydrate, 25% fat, 12% protein), administered with or without CGA (120 mg·kg(-1)), in a randomized crossover design separated by a 3-day washout period. Acetaminophen was co-administered to assess the effects of CGA on gastric emptying. The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) were measured. GLP-1 response in the presence of glucose and CGA was further examined, using the human colon cell line NCI-H716. Total area under the curve (AUC) for blood glucose was significantly attenuated in rats fed CGA (p gastric emptying was not altered. Plasma GIP response was blunted in rats fed CGA, with a lower peak concentration and AUC up to 180 min postprandially (p alterations seen in GIP concentrations. Given the widespread consumption and availability of coffee, CGA may be a viable prevention tool for T2D. PMID:21977912

  5. Targeting sialic acid dependent and independent pathways of invasion in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rosalynn Louise Ord

    Full Text Available The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH family. Ligands from the EBL family largely govern the sialic acid (SA dependent pathways of invasion and the RH family ligands (except for RH1 mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion.

  6. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    Science.gov (United States)

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  7. Membrane sialic acid influences basophil histamine release by interfering with calcium dependence

    DEFF Research Database (Denmark)

    Jensen, C; Norn, S; Skov, P S;

    1987-01-01

    The influence of the cell membrane content of sialic acid on basophil histamine release was examined in vitro in allergic patients and normal controls. Enzymatical removal of sialic acid enhanced histamine release induced by allergen and anti-IgE, whereas an increase in membrane sialic acid content...

  8. Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

    Directory of Open Access Journals (Sweden)

    Nils Widderich

    Full Text Available Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa, we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (SaEctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of

  9. Interleukin-10 modifies the effects of interleukin-1beta and tumor necrosis factor-alpha on the activity and expression of prostaglandin H synthase-2 and the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase in cultured term human villous trophoblast and chorion trophoblast cells.

    Science.gov (United States)

    Pomini, F; Caruso, A; Challis, J R

    1999-12-01

    The concentrations of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), two inflammatory cytokines in amniotic fluid, have been shown to rise during chorioamnionitis. This is probably related to activation of the immune system in order to intensify the inflammatory process and to protect the maternal and fetal organism from infectious agents. These cytokines activate the PG biosynthetic pathway in several tissues, but few studies have examined effects on PG-metabolizing enzymes. When PGs are produced by increased synthesis and/or decreased metabolism at the chorio-decidual interface, labor can be induced. Interleukin-10 (IL-10) is known to act as an antiinflammatory cytokine. The goals of this study were to evaluate the interaction of IL-10 with IL-1beta and TNFalpha on PG synthesis and to determine the effects of IL-10, IL-1beta, and TNFalpha on PG metabolism using purified cultures of villous trophoblast and chorion trophoblast cells prepared from placentas of patients at term. Cells were treated with IL-1beta and TNFalpha with or without IL-10 for various times up to 24 h. Levels of messenger ribonucleic acid (mRNA) encoding PGH synthase-2 (PGHS-2) and NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) were quantified by Northern blotting, and PGE2 and 13,14-dihydro-15-keto-PGF2alpha (PGFM) output in the medium was measured by RIA. IL-1beta increased PGHS-2 mRNA and PGE2 output from villous and chorion trophoblasts and decreased PGDH mRNA in villous trophoblasts (all P < 0.05). These effects were reversed by IL-10. We found no change in PGHS-2 mRNA or PGE2 output in either trophoblast type treated with TNFalpha, but TNFalpha reduced PGDH mRNA in villous trophoblast, and this effect was reversed by IL-10 (both P < 0.05). We conclude that proinflammatory cytokines can influence PG output through effects on PG synthesis and metabolism and that these effects may be opposed by an antiinflammatory cytokine. These interactions may be

  10. Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yang [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100080 (China); Wang Jianshe; Wei Yanhong; Zhang Hongxia; Xu Muqi [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China); Dai Jiayin [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Beijing 100101 (China)], E-mail: daijy@ioz.ac.cn

    2008-09-29

    The effects of acute perfluorododecanoic acid (PFDoA) exposure on the induction of oxidative stress and alteration of mitochondrial gene expression were studied in the livers of female zebrafish (Danio rerio). Female zebrafish were exposed to PFDoA via a single intraperitoneal injection (0, 20, 40, or 80 {mu}g PFDoA/g body weight) and were then sacrificed 48 h, 96 h, or seven days post-PFDoA administration. PFDoA-treated fish exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. Glutathione (GSH) content and catalase (CAT) activity decreased significantly at 48 h post-injection while superoxide dismutase (SOD) activity was initially decreased at 48 h post-injection but was then elevated by seven days post-injection. The activity of glutathione peroxidase (GPx) increased at 48 h and seven days compared to control fish, although the increased level at seven days post-injection was decreased compared to the level at 48 h post-injection. Lipid peroxidation levels were increased at seven days post-injection, while no apparent induction was observed at 48 h or 96 h post-injection. The mRNA expression of medium-chain fatty acid dehydrogenase (MCAD) was induced, while the transcriptional expression of liver fatty acid binding protein (L-FABP), peroxisome proliferating activating receptor {alpha} (PPAR{alpha}), carnitine palmitoyl-transferase I (CPT-I), uncoupling protein 2 (UCP-2), and Bcl-2 were significantly inhibited. Furthermore, the transcriptional expression of peroxisomal fatty acyl-CoA oxidase (ACOX), very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD) did not exhibit significant changes following PFDoA treatment. No significant changes were noted in the transcriptional expression of genes involved in mitochondrial respiratory chain and ATP synthesis, including cytochrome c oxidase subunit I (COXI), NADH dehydrogenase subunit I (NDI), and ATP synthase F0 subunit 6

  11. Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver

    International Nuclear Information System (INIS)

    The effects of acute perfluorododecanoic acid (PFDoA) exposure on the induction of oxidative stress and alteration of mitochondrial gene expression were studied in the livers of female zebrafish (Danio rerio). Female zebrafish were exposed to PFDoA via a single intraperitoneal injection (0, 20, 40, or 80 μg PFDoA/g body weight) and were then sacrificed 48 h, 96 h, or seven days post-PFDoA administration. PFDoA-treated fish exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. Glutathione (GSH) content and catalase (CAT) activity decreased significantly at 48 h post-injection while superoxide dismutase (SOD) activity was initially decreased at 48 h post-injection but was then elevated by seven days post-injection. The activity of glutathione peroxidase (GPx) increased at 48 h and seven days compared to control fish, although the increased level at seven days post-injection was decreased compared to the level at 48 h post-injection. Lipid peroxidation levels were increased at seven days post-injection, while no apparent induction was observed at 48 h or 96 h post-injection. The mRNA expression of medium-chain fatty acid dehydrogenase (MCAD) was induced, while the transcriptional expression of liver fatty acid binding protein (L-FABP), peroxisome proliferating activating receptor α (PPARα), carnitine palmitoyl-transferase I (CPT-I), uncoupling protein 2 (UCP-2), and Bcl-2 were significantly inhibited. Furthermore, the transcriptional expression of peroxisomal fatty acyl-CoA oxidase (ACOX), very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain acyl-CoA dehydrogenase (LCAD) did not exhibit significant changes following PFDoA treatment. No significant changes were noted in the transcriptional expression of genes involved in mitochondrial respiratory chain and ATP synthesis, including cytochrome c oxidase subunit I (COXI), NADH dehydrogenase subunit I (NDI), and ATP synthase F0 subunit 6 (ATPo6). These

  12. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar t...... into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein....

  13. Alpha-picolinic Acid Activates Diverse Defense Responses of Salicylic Acid-, Jasmonic Acid/Ethylene- and Ca2 -dependent Pathways in Arabidopsis and Rice Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGHai-Kuo; ZHANGXin; LIQun; HEZu-Hua

    2004-01-01

    Alpha-picolinic acid (PA) is an apoptosis inducer in animal cells, and could elicit hypersensitiv eresponse (HR) in rice, a monocotyledonous model plant. Here we report that PA is an HR inducer in plants. It induced HR in Arabidopsis, a dicotyledonous model plant, including the oxidative burst and cell death. We investigated defense signal transduction activated by PA through marker genes of particular defense pathways in Arabidopsis. The result indicated that both the salicylic acid-dependent and jasmonic acid/ethylene-dependent pathways were activated by PA, in which the marker defense genes PR-1, PR-2 and PDF 1.2 were all induced in dose-dependent and time-course manners. We also observed that the PAinduced reactive oxygen species (ROS) production in rice suspension cells was Ca2+-dependent. Together with our previous studies of PA-induced defense activation in rice, we conclude that PA acts as a nonspecific elicitor in plant defense and has a potential utilization in cellular model establishment of systemicac quired resistance (SAR) activation.

  14. Comparison of various models to describe the charge-pH dependence of poly(acrylic acid)

    NARCIS (Netherlands)

    Lützenkirchen, J.; Male, van J.; Leermakers, F.A.M.; Sjöberg, S.

    2011-01-01

    The charge of poly(acrylic acid) (PAA) in dilute aqueous solutions depends on pH and ionic strength. We report new experimental data and test various models to describe the deprotonation of PAA in three different NaCl concentrations. A simple surface complexation approach is found to be very success

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

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

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

  16. 3-Nitropropionic acid neurotoxicity in organotypic striatal and corticostriatal slice cultures is dependent on glucose and glutamate

    DEFF Research Database (Denmark)

    Storgaard, J; Kornblit, B T; Zimmer, J;

    2000-01-01

    Mitochondrial inhibition by 3-nitropropionic acid (3-NPA) causes striatal degeneration reminiscent of Huntington's disease. We studied 3-NPA neurotoxicity and possible indirect excitotoxicity in organotypic striatal and corticostriatal slice cultures. Neurotoxicity was quantified by assay...... of lactate dehydrogenase in the medium and glutamic acid decarboxylase in tissue homogenates. 3-NPA toxicity (25-100 microM in 5 mM glucose, 24-48 h) appeared to be highly dependent on culture medium glucose levels. 3-NPA treatment caused also a dose-dependent lactate increase, reaching a maximum...... striatum without cortex and tetrodotoxin, MK-801, and d-2-amino-5-phosphonopentanoic acid prevented or attenuated 3-NPA neurotoxicity, suggesting that membrane depolarization and/or neuronal activity of the glutamatergic corticostriatal pathway contributes to striatal pathology. The results indicate...

  17. Fatty acid content of honeycombs depending on the level of technogenic loading on the environment

    Directory of Open Access Journals (Sweden)

    V. Y. Vishchur

    2016-02-01

    Full Text Available The content of heavy metals, non-etherified and anionic forms of fatty acids, and also fatty acids of common lipids in honeycomb cells, obtained from hives placed in areas with high, medium and low levels of technogenic loading is investigated. The hives were located at the educational apiary of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj (area with very heavy traffic and industrial activity and in private beekeeping farms in the town Vynnyky and the village Chyzhyky, Pustomyty district, Lviv region (areas with lower intensity of traffic and industrial activity. Our research has shown that the newly-built honeycomb cells obtained from hives placed in the territory with medium and low levels of technogenic loading contain a lower quantity of such heavy metals as iron, zinc, cuprum, chromium, nickel, lead and cadmium. The total amount of non-etherified fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids of n-7 and n-9 families and polyunsaturated fatty acids of n-3 and n-6 families was higher in these honeycombs. However, the total concentration of anionic forms of fatty acids with even and odd number of carbon atoms in the chain, monounsaturated fatty acids of n-7 and n-9 families and polyunsaturated fatty acids of n-3 and n-6 families was lower in these honeycombs. Due to the above-mentioned monounsaturated and polyunsaturated fatty acids the amount of fatty acids of common lipids rises. It leads to increase in the antibacterial and antifungal properties of honeycomb cells and reduction of fragility of their walls. The greatest changes in concentration of heavy metals, non-etherified and anionic form of fatty acids, and also fatty acids of common lipids occur in the newly built honeycomb cells in the territory with low technogenic loading on the environment.

  18. A new member of the chalcone synthase (CHS family in sugarcane

    Directory of Open Access Journals (Sweden)

    Contessotto Miriam G.G.

    2001-01-01

    Full Text Available Sequences from the sugarcane expressed sequence tag (SUCEST database were analyzed based on their identities to genes encoding chalcone-synthase-like enzymes. The sorghum (Sorghum bicolor chalcone-synthase (CHS, EC 2.3.1.74 protein sequence (gi|12229613 was used to search the SUCEST database for clusters of sequencing reads that were most similar to chalcone synthase. We found 121 reads with homology to sorghum chalcone synthase, which we were then able to sort into 14 clusters which themselves were divided into two groups (group 1 and group 2 based on the similarity of their deduced amino acid sequences. Clusters in group 1 were more similar to the sorghum enzyme than those in group 2, having the consensus sequence of the active site of chalcone and stilbene synthase. Analysis of gene expression (based on the number of reads from a specific library present in each group indicated that most of the group 1 reads were from sugarcane flower and root libraries. Group 2 clusters were more similar to the amino acid sequence of an uncharacterized pathogen-induced protein (PI1, gi|9855801 from the S. bicolor expressed sequence tag (EST database. The group 2 clusters sequences and PI1 proteins are 90% identical, having two amino acid changes at the chalcone and stilbene synthase consensi but conserving the cysteine residue at the active site. The PI1 EST has not been previously associated with chalcone synthase and has a different consensus sequence from the previously described chalcone synthase of sorghum. Most of the group 2 reads were from libraries prepared from sugarcane roots and plants infected with Herbaspirillum rubrisubalbicans and Gluconacetobacter diazotroficans. Our results indicate that we have identified a sugarcane chalcone synthase similar to the pathogen-induced PI1 protein found in the sorghum cDNA libraries, and it appears that both proteins represent new members of the chalcone and stilbene synthase super-family.

  19. Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes.

    Science.gov (United States)

    Susukida, Takeshi; Sekine, Shuichi; Ogimura, Eiichiro; Aoki, Shigeki; Oizumi, Kumiko; Horie, Toshiharu; Ito, Kousei

    2015-10-01

    The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs. PMID:26055650

  20. How Imaging Glutamate, γ-Aminobutyric Acid, and Dopamine Can Inform the Clinical Treatment of Alcohol Dependence and Withdrawal.

    Science.gov (United States)

    Hillmer, Ansel T; Mason, Graeme F; Fucito, Lisa M; O'Malley, Stephanie S; Cosgrove, Kelly P

    2015-12-01

    Neuroimaging studies have dramatically advanced our understanding of the neurochemical basis of alcohol dependence, a major public health issue. In this paper, we review the research generated from neurochemical specific imaging modalities including magnetic resonance spectroscopy, positron emission tomography, and single-photon emission computed tomography in studies of alcohol dependence and withdrawal. We focus on studies interrogating γ-aminobutyric acid (GABA), glutamate, and dopamine, as these are prominent neurotransmitter systems implicated in alcohol dependence. Highlighted findings include diminished dopaminergic functioning and modulation of the GABA system by tobacco smoking during alcohol withdrawal. Then, we consider how these findings impact the clinical treatment of alcohol dependence and discuss directions for future experiments to address existing gaps in the literature, for example, sex differences and smoking comorbidity. These and other considerations provide opportunities to build upon the current neurochemistry imaging literature of alcohol dependence and withdrawal, which may usher in improved therapeutic and relapse prevention strategies. PMID:26510169

  1. Rosmarinic Acid Methyl Ester Inhibits LPS-Induced NO Production via Suppression of MyD88- Dependent and -Independent Pathways and Induction of HO-1 in RAW 264.7 Cells.

    Science.gov (United States)

    So, Yangkang; Lee, Seung Young; Han, Ah-Reum; Kim, Jin-Baek; Jeong, Hye Gwang; Jin, Chang Hyun

    2016-01-01

    In this study, we investigated the anti-inflammatory effect of rosmarinic acid methyl ester (RAME) isolated from a mutant cultivar of Perilla frutescens (L.) Britton. We found that RAME inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with an IC50 of 14.25 µM, in RAW 264.7 cells. RAME inhibited the LPS-induced expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, monocyte chemoattractant protein-1, interferon-β, and inducible nitric oxide synthase (iNOS). Moreover, RAME suppressed the activation of nuclear factor kappa B. These results suggest that the downregulation of iNOS expression by RAME was due to myeloid differentiation primary response gene 88 (MyD88)-dependent and -independent pathways. Furthermore, RAME induced the expression of heme oxygenase-1 (HO-1) through activation of nuclear factor-erythroid 2-related factor 2. Treatment with tin protoporphyrin, an inhibitor of HO-1, reversed the RAME-induced suppression of NO production. Taken together, RAME isolated from P. frutescens inhibited NO production in LPS-treated RAW 264.7 cells through simultaneous induction of HO-1 and inhibition of MyD88-dependent and -independent pathways. PMID:27548124

  2. Redox-dependent anti-inflammatory signaling actions of unsaturated fatty acids.

    Science.gov (United States)

    Delmastro-Greenwood, Meghan; Freeman, Bruce A; Wendell, Stacy Gelhaus

    2014-01-01

    Unsaturated fatty acids are metabolized to reactive products that can act as pro- or anti-inflammatory signaling mediators. Electrophilic fatty acid species, including nitro- and oxo-containing fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,β-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases. PMID:24161076

  3. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  4. Properties of phosphorylated thymidylate synthase.

    Science.gov (United States)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  5. Morphology-dependent Electrochemical Enhancements of Porous Carbon as Sensitive Determination Platform for Ascorbic Acid, Dopamine and Uric Acid

    Science.gov (United States)

    Cheng, Qin; Ji, Liudi; Wu, Kangbing; Zhang, Weikang

    2016-02-01

    Using starch as the carbon precursor and different-sized ZnO naoparticles as the hard template, a series of porous carbon materials for electrochemical sensing were prepared. Experiments of scanning electron microscopy, transmission electron microscopy and Nitrogen adsorption-desorption isotherms reveal that the particle size of ZnO has big impacts on the porous morphology and surface area of the resulting carbon materials. Through ultrasonic dispersion of porous carbon and subsequent solvent evaporation, different sensing interfaces were constructed on the surface of glassy carbon electrode (GCE). The electrochemical behaviors of ascorbic acid (AA), dopamine (DA) and uric acid (UA) were studied. On the surface of porous carbon materials, the accumulation efficiency and electron transfer ability of AA, DA and UA are improved, and consequently their oxidation signals enhance greatly. Moreover, the interface enhancement effects of porous carbon are also controlled by the particle size of hard template. The constructed porous carbon interface displays strong signal amplification ability and holds great promise in constructing a sensitive platform for the simultaneous determination of AA, DA and UA.

  6. pH-, Lactic Acid-, and Non-Lactic Acid-Dependent Activities of Probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium

    Science.gov (United States)

    Fayol-Messaoudi, Domitille; Berger, Cédric N.; Coconnier-Polter, Marie-Hélène; Liévin-Le Moal, Vanessa; Servin, Alain L.

    2005-01-01

    The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect. PMID:16204515

  7. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway1[OPEN

    Science.gov (United States)

    Légeret, Bertrand; Mirabella, Boris; Guédeney, Geneviève; Jetter, Reinhard; Peltier, Gilles

    2016-01-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

  8. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    DEFF Research Database (Denmark)

    Skov, Søren; Pedersen, Marianne Terndrup; Andresen, Lars;

    2005-01-01

    We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...... apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3...... by small interfering RNA or by different inhibitors showed that GSK-3 activity is essential for the induced MICA/B expression. We thus present evidence that cancer cells which survive the direct induction of cell death by HDAC inhibitors become targets for NKG2D-expressing cells like NK cells, gammadelta T...

  9. The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

    Science.gov (United States)

    Singh, Dhirendra; Sielaff, Hendrik; Sundararaman, Lavanya; Bhushan, Shashi; Grüber, Gerhard

    2016-02-01

    A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.

  10. Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum

    DEFF Research Database (Denmark)

    Egebo, L A; Nielsen, S V; Jochimsen, B U

    1991-01-01

    Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires...... an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation...

  11. Gamma Hydroxybutyric Acid (GHB for the Treatment of Alcohol Dependence: A Review

    Directory of Open Access Journals (Sweden)

    Mauro Bernardi

    2009-06-01

    Full Text Available Gamma-hydroxybutyric acid (GHB is a short-chain fatty acid structurally similar to the inhibitory neurotransmitter γ-aminobutyric acid. Clinical trials have demonstrated that 50-100 mg/kg of GHB fractioned into three or six daily doses is able to suppress alcohol withdrawal symptoms and facilitates the maintenance of abstinence from alcohol. These studies have also shown that GHB craving episodes are a very limited phenomenon (about 10-15%. Thus, physicians with access should consider the clinical efficacy of GHB as a valid pharmacological tool for the treatment of alcohol addiction.

  12. Retinoic acid stimulation of human dermal fibroblast proliferation is dependent on suboptimal extracellular Ca2+ concentration.

    OpenAIRE

    Varani, J.; Shayevitz, J.; Perry, D; Mitra, R. S.; Nickoloff, B J; Voorhees, J. J.

    1990-01-01

    Human dermal fibroblasts failed to proliferate when cultured in medium containing 0.15 mmol/l (millimolar) Ca2+ (keratinocyte growth medium [KGM]) but did when the external Ca2+ concentration was raised to 1.4 mmol/l. All-trans retinoic acid (retinoic acid) stimulated proliferation in KGM but did not further stimulate growth in Ca2(+)-supplemented KGM. The ability of retinoic acid to stimulate proliferation was inhibited in KGM prepared without Ca2+ or prepared with 0.03 mmol/l Ca2+ and in KG...

  13. Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study.

    Science.gov (United States)

    Jacques, Paul F; Bostom, Andrew G; Selhub, Jacob; Rich, Sharron; Ellison, R Curtis; Eckfeldt, John H; Gravel, Roy A; Rozen, Rima

    2003-01-01

    The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, methionine synthase and methionine synthase reductase, have also been identified. Methionine synthase catalyzes the remethylation of homocysteine to form methionine and methionine synthase reductase is required for the reductive activation of the cobalamin-dependent methionine synthase. The methionine synthase gene (MTR) mutation is an A to G substitution, 2756A-->G, which converts an aspartate to a glycine codon. The methionine synthase reductase gene (MTRR) mutation is an A to G substitution, 66A-->G, that converts an isoleucine to a methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a methionine load, a weak positive association was observed between change in homocysteine after a methionine load and the number of mutant MTR alleles (P-trend=0.04), but this association was not statistically significant according to the overall F-statistic (P=0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine. PMID:12482550

  14. The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant-negative effect on cellulose synthesis and plant growth.

    Science.gov (United States)

    Daras, Gerasimos; Rigas, Stamatis; Penning, Bryan; Milioni, Dimitra; McCann, Maureen C; Carpita, Nicholas C; Fasseas, Constantinos; Hatzopoulos, Polydefkis

    2009-01-01

    Genetic functional analyses of mutants in plant genes encoding cellulose synthases (CesAs) have suggested that cellulose deposition requires the activity of multiple CesA proteins. Here, a genetic screen has led to the identification of thanatos (than), a semi-dominant mutant of Arabidopsis thaliana with impaired growth of seedlings. Homozygous seedlings of than germinate and grow but do not survive. In contrast to other CesA mutants, heterozygous plants are dwarfed and display a radially swollen root phenotype. Cellulose content is reduced by approximately one-fifth in heterozygous and by two-fifths in homozygous plants, showing gene-dosage dependence. Map-based cloning revealed an amino acid substitution (P578S) in the catalytic domain of the AtCesA3 gene, indicating a critical role for this residue in the structure and function of the cellulose synthase complex. Ab initio analysis of the AtCesA3 subdomain flanking the conserved proline residue predicted that the amino acid substitution to serine alters protein secondary structure in the catalytic domain. Gene dosage-dependent expression of the AtCesA3 mutant gene in wild-type A. thaliana plants resulted in a than dominant-negative phenotype. We propose that the incorporation of a mis-folded CesA3 subunit into the cellulose synthase complex may stall or prevent the formation of functional rosette complexes. PMID:19645738

  15. Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

    Directory of Open Access Journals (Sweden)

    Saito Koji

    2005-08-01

    Full Text Available Abstract Background In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1 is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS, in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. Results Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. Conclusion These results suggest that ETO1

  16. Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

    Science.gov (United States)

    Horváth, Edit; Csiszár, Jolán; Gallé, Ágnes; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2015-07-01

    The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

  17. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Jie; Krausz, Kristopher W. [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Li, Feng; Ma, Xiaochao [Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, 4089 KLSIC, MS 1018, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Gonzalez, Frank J., E-mail: fjgonz@helix.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-15

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  18. Dose-dependent improvement of myoclonic hyperkinesia due to Valproic acid in eight Huntington's Disease patients: a case series

    Directory of Open Access Journals (Sweden)

    Przuntek Horst

    2006-02-01

    Full Text Available Abstract Background Chorea in Huntington's Disease (HD is usually treated with antidopaminergic neuroleptics like haloperidol, olanzapine and tiaprid or dopamine depleting drugs like tetrabenazine. Some patients with hyperkinesia, however, react to treatment with antidopaminergic drugs by developing extrapyramidal side effects. In earlier studies valproic acid showed no beneficial effect on involuntary choreatic movements. Myoclonus is rare in HD and is often overseen or misdiagnosed as chorea. Methods In this report, we present eight patients whose main symptom is myoclonic hyperkinesia. All patients were treated with valproic acid and scored by using the Unified Huntington's Disease Rating Scale (UHDRS motor score before and after treatment. In addition to this, two patients agreed to be videotaped. Results In seven patients myoclonus and, therefore the UHDRS motor score improved in a dose dependent manner. In three of these patients antidopaminergic medication could be reduced. Conclusion In the rare subgroup of HD patients suffering from myoclonic hyperkinesia, valproic acid is a possible alternative treatment.

  19. Identification of a transcription factor controlling pH-dependent organic acid response in Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Lars Poulsen

    Full Text Available Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0. Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis.

  20. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

    OpenAIRE

    Mark J. Solloway; Azadeh Madjidi; Chunyan Gu; Jeff Eastham-Anderson; Holly J. Clarke; Noelyn Kljavin; Jose Zavala-Solorio; Lance Kates; Brad Friedman; Matt Brauer; Jianyong Wang; Oliver Fiehn; Ganesh Kolumam; Howard Stern; John B. Lowe

    2015-01-01

    Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor i...

  1. In Vitro Gender-Dependent Inhibition of Porcine Cytochrome P450 Activity by Selected Flavonoids and Phenolic Acids

    Directory of Open Access Journals (Sweden)

    Bo Ekstrand

    2015-01-01

    Full Text Available We investigated gender-related differences in the ability of selected flavonoids and phenolic compounds to modify porcine hepatic CYP450-dependent activity. Using pools of microsomes from male and female pigs, the inhibition of the CYP families 1A, 2A, 2E1, and 3A was determined. The specific CYP activities were measured in the presence of the following selected compounds: rutin, myricetin, quercetin, isorhamnetin, p-coumaric acid, gallic acid, and caffeic acid. We determined that myricetin and isorhamnetin competitively inhibited porcine CYP1A activity in the microsomes from both male and female pigs but did not affect the CYP2A and CYP2E1. Additionally, isorhamnetin competitively inhibited CYP3A in both genders. Noncompetitive inhibition of CYP3A activity by myricetin was observed only in the microsomes from male pigs, whereas CYP3A in female pigs was not affected. Quercetin competitively inhibited CYP2E1 and CYP1A activity in the microsomes from male pigs and irreversibly CY3A in female pigs. No effect of quercetin on CYP2E1 was observed in the microsomes from female pigs. Neither phenolic acids nor rutin affected CYP450 activities. Taken together, our results suggest that the flavonoids myricetin, isorhamnetin, and quercetin may affect the activities of porcine CYP1A, CYP3A, and CYP2E1 in a gender-dependent manner.

  2. Role of acid sphingomyelinase in the age-dependent dysregulation of sphingolipids turnover in the tissues of rats.

    Science.gov (United States)

    Babenko, Nataliya A; Garkavenko, Vladimir V; Storozhenko, Galina V; Timofiychuk, Olga A

    2016-04-01

    Old age-associated pathologies usually coincide with altered sphingolipid metabolism. In the present article, the role of acid sphingomyelinase (ASMase) in the age-dependent changes of sphingomyelin (SM) and ceramide contents in the tissues has been investigated by means of ASMase inhibitors, imipramine and zoledronic acid. It has been determined that ceramide content and ceramide/SM ratio increased, while SM level decreased in the heart, liver, blood serum and skeletal muscles of 24-month old rats in contrast to 3-month old animals. Injections of imipramine or zoledronic acid to 24-month old rats resulted in significant downregulation of ASMase in the liver and skeletal and heart muscles. The both inhibitors decreased the ceramide content and ceramide/SM ratio and increased the SM content in all tissues studied, except the heart, of old rats to the levels close to those observed in the young animals. Long-term treatment of rats by inhibitors, which have different mechanisms of action on ASMase, exerts the similar, but not equal effects on enzyme activity and SM turnover. In summary, the data above strongly suggest that the age-dependent up-regulation of ASMase plays an important role in the modulation of ceramide and SM contents in rat tissues and that imipramine and zoledronic acid are useful tools for SM turnover manipulation at old age. PMID:26830134

  3. Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

    International Nuclear Information System (INIS)

    The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated

  4. Characterizing Time-Dependent Contact Angles for Sands Hydrophobized with Oleic and Stearic Acids

    DEFF Research Database (Denmark)

    Subedi, Shaphal; Kawamoto, Ken; Jayarathna, Lakmal;

    2012-01-01

    -frequency precipitation. A potential solution is to alter soil grain surfaces to become water repellent by mixing or coating the soil cover material with hydrophobic agents (HAs). In this study, hydrophobic CBs comprised of sands mixed with environmentally friendly HAs (oleic acid [OA] and stearic acid [SA]) were studied....... Water repellency (WR) characteristics for hydrophobized sand samples with different HA contents and representing different coating methods (mixing in and solvent aided) were measured. Initial contact angles (αi) for OA-coated samples sharply increased with increasing HA content and reached peak values...

  5. Liver Fatty Acid Binding Protein Gene Ablation Enhances Age-Dependent Weight Gain in Male Mice

    OpenAIRE

    Martin, Gregory G.; Atshaves, Barbara P.; McIntosh, Avery L.; Payne, H. Ross; Mackie, John T.; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Although studies performed in vitro and with transfected cells in culture suggest a role for liver fatty acid binding protein (L-FABP) in regulating fatty acid oxidation and fat deposition, the physiological significance of this possibility is not completely clear. To begin to address this question, the effect of L-FABP gene ablation on phenotype of standard rodent chow-fed male mice was examined with increasing age up to 18 mo. While young (2-3 mo) L-FABP null mice displayed no visually obvi...

  6. Higher diet-dependent renal acid load associates with higher glucocorticoid secretion and potentially bioactive free glucocorticoids in healthy children.

    Science.gov (United States)

    Esche, Jonas; Shi, Lijie; Sánchez-Guijo, Alberto; Hartmann, Michaela F; Wudy, Stefan A; Remer, Thomas

    2016-08-01

    Metabolic acidosis induces elevated glucocorticoid (GC) levels. However, the influence of less strong daily acid loads on GCs is largely unexplored. To investigate this, we studied whether higher acid loads in children, fully within the normal range of habitual diets, associate with endogenous GCs. In a specific quasi-experimental design, we examined 200 6- to 10-year-old healthy participants of the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study equally divided to either high or low 24-hour renal net acid excretion. Major urinary GC metabolites were analyzed by gas chromatography-mass spectrometry to assess daily adrenal GC secretion and metabolites of tissue cortisol catabolism (6β-hydroxycortisol and 20α-dihydrocortisol). Liquid chromatography-mass spectrometry was used to quantify urinary free cortisol and cortisone. After confounder adjustment, significant positive associations were unmasked for urinary potential renal acid load and net acid excretion with adrenal GC secretion, free cortisone, free cortisone plus cortisol, 6β-hydroxycortisol, and 20α-dihydrocortisol. An inverse association emerged for an enzymatic marker (5β-reductase) of irreversible GC inactivation. Our data suggest that existing moderate elevations in diet-dependent acid loads suffice to raise GCs and affect cortisol metabolism. Thus, potential detrimental effects of high acid loading appear to be mediated, in part, by increased GC activity via increased GC secretion and/or reduced GC inactivation. Higher cortisone levels, directly available for intracrine activation to cortisol may play a special role. PMID:27165611

  7. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

    Science.gov (United States)

    Kaya, Yusuf; Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-04-30

    TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

  8. Salt dependent stability of stearic acid Langmuir-Blodgett films exposed to aqueous electrolytes

    NARCIS (Netherlands)

    Kumar, Naveen; Wang, Lei; Siretanu, Igor; Duits, Michel; Mugele, Frieder

    2013-01-01

    We use contact angle goniometry, imaging ellipsometry, and atomic force microscopy to study the stability and wettability of Langmuir–Blodgett (LB) monolayers of stearic acid on silica substrates, upon drying and exposure to aqueous solutions of varying salinity. The influences of Ca2+ and Na+ ions

  9. Coral cavity sponges depend on reef-derived food resources: stable isotope and fatty acid constraints

    NARCIS (Netherlands)

    van Duyl, F.C.; Moodley, L.; Nieuwland, G.; van Ijzerloo, L.; van Soest, R.W.M.; Houtekamer, M.; Meesters, E.H.; Middelburg, J.J.

    2011-01-01

    The diet of cavity sponges on the narrow fringing reefs of Cura double dagger ao, Caribbean was studied. The origin and resources of the bulk food of these sponges, i.e., dissolved organic matter (DOM), were identified using stable carbon and nitrogen isotopes and fatty acid biomarkers. We found tha

  10. Coral cavity sponges depend on reef-derived food resources: stable isotope and fatty acid constraints

    NARCIS (Netherlands)

    F.C. van Duyl; L. Moodley; G. Nieuwland; L. van IJzerloo; R.W.M. van Soest; M. Houtekamer; E.H. Meesters; J.J. Middelburg

    2011-01-01

    The diet of cavity sponges on the narrow fringing reefs of Curaçao, Caribbean was studied. The origin and resources of the bulk food of these sponges, i.e., dissolved organic matter (DOM), were identified using stable carbon and nitrogen isotopes and fatty acid biomarkers. We found that phytoplankto

  11. Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger

    DEFF Research Database (Denmark)

    Poulsen, Lars; Andersen, Mikael Rørdam; Lantz, Anna Eliasson;

    2012-01-01

    overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms...

  12. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    NARCIS (Netherlands)

    Madsen, L.; Pedersen, L.M.; Liaset, B.; Ma, T.; Petersen, R.K.; Berg, S. van den; Pan, J.; Müller-Decker, K.M.; Dülsner, E.D.; Kleemann, R.; Kooistra, T.; Døskeland, S.O.; Kristiansen, K.

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-ad

  13. Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.

    Science.gov (United States)

    Mills, Jeremy H; Khare, Sagar D; Bolduc, Jill M; Forouhar, Farhad; Mulligan, Vikram Khipple; Lew, Scott; Seetharaman, Jayaraman; Tong, Liang; Stoddard, Barry L; Baker, David

    2013-09-11

    Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2'-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ∼40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 Å respectively over all atoms in the binding site.

  14. Coral cavity sponges depend on reef-derived food resources: stable isotope and fatty acid constraints

    NARCIS (Netherlands)

    Van Duyl, F.C.; Moodley, L.; Nieuwland, G.; Van IJzerloo, L.P.; Van Soest, R.W.M.; Houtekamer, M.J.; Meesters, E.H.W.G.; Middelburg, J.J.

    2011-01-01

    The diet of cavity sponges on the narrow fringing reefs of Curac¸ao, Caribbean was studied. The origin and resources of the bulk food of these sponges, i.e., dissolved organic matter (DOM), were identified using stable carbon and nitrogen isotopes and fatty acid biomarkers. We found that phytoplankt

  15. Polymorph-dependent titanium dioxide nanoparticle dissolution in acidic and alkali digestions

    Science.gov (United States)

    Multiple polymorphs (anatase, brookite and rutile) of titanium dioxide nanoparticles (TiO2-NPs) with variable structures were quantified in environmental matrices via microwave-based hydrofluoric (HF) and nitric (HNO3) mixed acid digestion and muffle furnace (MF)-based potassium ...

  16. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Romagnoli, G.; Luttik, M.A.H.; Kötter, P.; Pronk, J.T.; Daran, J.M.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share seque

  17. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function.

    Science.gov (United States)

    Kampranis, Sotirios C; Ioannidis, Daphne; Purvis, Alan; Mahrez, Walid; Ninga, Ederina; Katerelos, Nikolaos A; Anssour, Samir; Dunwell, Jim M; Degenhardt, Jörg; Makris, Antonios M; Goodenough, Peter W; Johnson, Christopher B

    2007-06-01

    Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases. PMID:17557809

  18. Fatty Acid Composition of Tropical Fish Depends on Reservoir Trophic Status and Fish Feeding Habit.

    Science.gov (United States)

    Gomes, Aline D; Tolussi, Carlos E; Boëchat, Iola G; Pompêo, Marcelo L M; Cortez, Maíra P T; Honji, Renato M; Moreira, Renata G

    2016-10-01

    Eutrophication results in a deficiency of n-3 LC-PUFA (long-chain polyunsaturated fatty acids) in aquatic food chains, affecting fish nutrition and physiology. The trophic transfer of FA (fatty acids) to fish species of different feeding habits was investigated in two reservoirs in southeast Brazil-the mesotrophic Ponte Nova Reservoir (PN) and the hypereutrophic Billings Reservoir (Bil). Total FA profile of stomach contents and adipose tissue, triacylglycerols (TAG), and phospholipids (PL) from liver and muscle of the omnivorous Astyanax fasciatus and the carnivorous Hoplias malabaricus were analyzed by gas chromatography. A prevalence of n-6PUFA, as 18:2n-6 (linoleic acid) and 20:4n-6 (arachidonic acid, ARA) was observed in the stomach contents and in the tissues of A. fasciatus from the PN reservoir. In contrast, n-3 LC-PUFA, as 20:5n-3 (eicosapentaenoic acid, EPA) was accumulated in fish tissues from Bil, resulting in higher n3/n6 and EPA/ARA ratios, compared to fish from PN. This differential FA accumulation was also observed for H. malabaricus, but differences were slightly minor, and no changes were observed in the EPA/ARA ratios between fish from both reservoirs. Regardless reservoir, FA profiles of TAG resembled that of their diet, whereas FA profiles of PL were more conservative and mainly comprised by LC-PUFA. We conclude that reservoir trophic status affected the FA composition of food resources available to these fish species, resulting in differential allocation of n-3 and n-6 FA. As expected, FA profile of the investigated fish species also reflected their feeding habit and physiological demands. PMID:27631676

  19. A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

    Science.gov (United States)

    Sato-Masumoto, Naoko; Ito, Michiho

    2014-06-01

    Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group.

  20. Structure and Mechanism of Human UDP-xylose Synthase

    OpenAIRE

    Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

    2012-01-01

    UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with ...

  1. The Domain Responsible for Sphingomyelin Synthase (SMS) Activity

    OpenAIRE

    Yeang, Calvin; Varsheny, Shweta; Wang, Renxiao; ZHANG, YA; Ye, Deyong; Jiang, Xian-Cheng

    2008-01-01

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this s...

  2. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  3. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E;

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...... have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA...

  4. Rom2-dependent phosphorylation of Elo2 controls the abundance of very long chain fatty acids

    DEFF Research Database (Denmark)

    Olson, Daniel K; Fröhlich, Florian; Christiano, Romain;

    2015-01-01

    Sphingolipids are essential components of eukaryotic membranes, where they serve to maintain membrane integrity. They are important components of membrane trafficking and function in signaling as messenger molecules. Sphingolipids are synthesized de novo from very long chain fatty acids (VLCFA) a...... with sphingolipid metabolism and link signal transduction pathways from the plasma membrane to the regulation of lipids for membrane homeostasis....... that Elo2, a key enzyme of VLCFA synthesis, is controlled by signaling of the guanine nucleotide exchange factor Rom2, initiating at the plasma membrane. This pathway controls Elo2 phosphorylation state and VLCFA synthesis. Our data identify a regulatory mechanism for coordinating VLCFA synthesis......Sphingolipids are essential components of eukaryotic membranes, where they serve to maintain membrane integrity. They are important components of membrane trafficking and function in signaling as messenger molecules. Sphingolipids are synthesized de novo from very long chain fatty acids (VLCFA...

  5. OXIDATIVE STRESS TRIGGERS CA2+-DEPENDENT LYSOSOME TRAFFICKING AND ACTIVATION OF ACID SPHINGOMYELINASE

    OpenAIRE

    Li, Xiang; Gulbins, Erich; Zhang, Yang

    2012-01-01

    Recent studies demonstrate that rapid translocation of the acid sphingomyelinase (ASM), a lysosomal hydrolase, to the outer leaflet of the cell membrane and concomitant release of ceramide constitute a common cellular signaling cascade to various stimuli including CD95 ligation, UV-irradiation, bacterial and viral infections. Reactive oxygen species (ROS) were shown to play a crucial role in regulating this signaling cascade at least for some bacterial infections and UV-irradiation. However, ...

  6. Coral cavity sponges depend on reef-derived food resources: stable isotope and fatty acid constraints

    OpenAIRE

    van Duyl, F.C.; Moodley, L; Nieuwland, G.; IJzerloo, L. van; van Soest, R.W.M.; Houtekamer, M.; Meesters, E.H.; Middelburg, J. J.

    2011-01-01

    The diet of cavity sponges on the narrow fringing reefs of Cura double dagger ao, Caribbean was studied. The origin and resources of the bulk food of these sponges, i.e., dissolved organic matter (DOM), were identified using stable carbon and nitrogen isotopes and fatty acid biomarkers. We found that phytoplankton and its derived DOM from the adjacent open sea and from reef overlying water is not the main source of food for most of the sponges examined nor is bacterioplankton. Interestingly, ...

  7. Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

    Science.gov (United States)

    Cuadra, Giancarlo A; Frantellizzi, Ashley J; Gaesser, Kimberly M; Tammariello, Steven P; Ahmed, Anika

    2016-07-01

    Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation. PMID:27350615

  8. The temperature dependence of the rate constant for the reaction of hydroxyl radicals with nitric acid

    Science.gov (United States)

    Kurylo, M. J.; Cornett, K. D.; Murphy, J. L.

    1982-01-01

    The rate constant for the reaction of hydroxyl radicals with nitric acid in the 225-443 K temperature range has been measured by means of the flash photolysis resonance fluorescence technique. Above 300 K, the rate constant levels off in a way that can only be explained by the occurrence of two reaction channels, of which one, operative at low temperatures, proceeds through the formation of an adduct intermediate. The implications of these rate constant values for stratospheric reaction constants is discussed.

  9. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling

    OpenAIRE

    Gibert, Yann; Bernard, Laure; Debiais-Thibaud, Melanie; Bourrat, Franck; Joly, Jean-Stephane; Pottin, Karen; Meyer, Axel; Retaux, Sylvie; Stock, David W.; Jackman, William R.; Seritrakul, Pawat; Begemann, Gerrit; Laudet, Vincent

    2010-01-01

    One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth develo...

  10. Study on the Ascorbic Acid Content of rose Hip fruit Depending on Stationary Conditions

    Directory of Open Access Journals (Sweden)

    Sorina Ropciuc

    2011-10-01

    Full Text Available The dog rose area includes southern and central Europe, reaching southern Scandinavia; it is also found in Asia Minor, Central Asia and North Africa. In our country, Rosa canina is the most widespread of the many species of Rosa, common in all areas. The components of the station, known also as the „stationary factors” affect differently the plant life and their chemical composition. Cynosbati fructus is the false fruit of Rosa canina L. (Rosaceae, known in Romanian as rose hip. The rose hip contain as active ingredients 500-1000 % vitamin C, 600-10000 mg% carotenoids, pectin, dextrin, vitamins B2, E, PP, flavone, sugars, organic acids, tannins, volatile oil, vanillin, triterpenoid saponosides, beta-sitosterol, fat (lecithin, glycerides of fatty acids in seeds, minerals (potassium, calcium, magnesium, iron. Vitamin C (ascorbic acid plays an important role in human body. The greatest amount is found in plants which reached maturity. It is concentrated in the rose hip flesh. Solutions easily destroyit in the presence of UV, of copper, silver, iron and oxidative enzymes traces. Vitamin C participates actively in all processes of oxidoreductions of the living cell. Its lack in food causes the disease called scurvy which manifests itself by inflammated and bleeding gums, tooth loss. Rose hip fruits are known as medicines since prehistoric times.

  11. Molecular cloning and expression profile of ß-ketoacyl-acp synthase gene from tung tree (Vernicia fordii Hemsl.)

    Science.gov (United States)

    Tung tree (Vernicia fordii) is an important woody oil tree. Tung tree seeds contain 50-60% oil with approximately 80 mole a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Fatty acid synthesis is catalyzed by the concerted action of acetyl-CoA carboxylase and fatty acid synthase, a ...

  12. Cross-Linked Dependency of Boronic Acid-Conjugated Chitosan Nanoparticles by Diols for Sustained Insulin Release

    Directory of Open Access Journals (Sweden)

    Nabil A. Siddiqui

    2016-10-01

    Full Text Available Boronic acids have been widely investigated for their potential use as glucose sensors in glucose responsive polymeric insulin delivery systems. Interactions between cyclic diols and boronic acids, anchored to polymeric delivery systems, may result in swelling of the delivery system, releasing the drug. In this study, 4-formylphenylboronic acid conjugated chitosan was formulated into insulin containing nanoparticles via polyelectrolyte complexation. The nanoparticles had an average diameter of 140 ± 12.8 nm, polydispersity index of 0.17 ± 0.1, zeta potential of +19.1 ± 0.69 mV, encapsulation efficiency of 81% ± 1.2%, and an insulin loading capacity of 46% ± 1.8% w/w. Changes in size of the nanoparticles and release of insulin were type of sugar- and concentration-dependent. High concentration of diols resulted in a sustained release of insulin due to crosslink formation with boronic acid moieties within the nanoparticles. The formulation has potential to be developed into a self-regulated insulin delivery system for the treatment of diabetes.

  13. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    Science.gov (United States)

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

  14. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice

    NARCIS (Netherlands)

    Bodewes, Frank A. J. A.; Bijvelds, Marcel J.; de Vries, Willemien; Baller, Juul F. W.; Gouw, Annette S. H.; de Jonge, Hugo R.; Verkade, Henkjan J.

    2015-01-01

    The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts

  15. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice

    NARCIS (Netherlands)

    F.A.J.A. Bodewes (Frank); M.J.C. Bijvelds (Marcel); De Vries, W. (Willemien); Baller, J.F.W. (Juul F. W.); A.S.H. Gouw (Annette); H.R. de Jonge (Hugo); H.J. Verkade (Henkjan)

    2015-01-01

    textabstractThe cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobi

  16. Burdock fructooligosaccharide induces fungal resistance in postharvest Kyoho grapes by activating the salicylic acid-dependent pathway and inhibiting browning.

    Science.gov (United States)

    Sun, Fei; Zhang, Pengying; Guo, Moran; Yu, Wenqian; Chen, Kaoshan

    2013-05-01

    Burdock fructooligosaccharide (BFO) is a natural elicitor from Arcitum lappa. The effects of BFO in controlling postharvest disease in grape, apple, banana, kiwi, citrus, strawberry, and pear were investigated. The disease index, decay percentage, and area under the disease progress curve indicated that BFO has general control effects on postharvest disease of fruits. Kyoho grapes were studied to elucidate the mechanism of BFO in boosting the resistance of grapes to Botrytis cinerea infection. BFO treatment induced upregulation of the npr1, pr1, pal, and sts genes, and inhibited the total phenol content decrease, which activated chitinase and β-1,3-glucanase. These results indicated that the salicylic acid-dependent signalling pathway was induced. The delayed colour change and peroxidase and polyphenoloxidase activity suggested that BFO delayed grape browning. The reduced respiration rate, weight loss, and titratable acidity prolonged the shelf life of postharvest grapes. BFO is a promising elicitor in postharvest disease control.

  17. Natural oils affect the human skin integrity and the percutaneous penetration of benzoic acid dose-dependently

    DEFF Research Database (Denmark)

    Nielsen, Jesper Bo

    2006-01-01

    Abstract: Natural oils are extensively used in cosmetics and as treatment for a growing number of more or less specific ailments. Skin irritation and cases of allergy have repeatedly been described in the literature following exposure to these oils. The present study evaluated the extent to which...... three natural oils (eucalyptus oil, tea tree oil, peppermint oil) would affect the skin integrity and the percutaneous penetration of benzoic acid when applied topically in relevant concentrations. An experimental in vitro model using static diffusion cells mounted with human breast or abdominal skin...... was applied. The three natural oils decreased the skin integrity dose-dependently. Concomitant dermal exposure to low concentrations of peppermint oil reduced the percutaneous penetration of benzoic acid. The present study lends support to the notion that low concentrations of peppermint oil may act...

  18. Lipopolysaccharide inhibits or accelerates biomedical titanium corrosion depending on environmental acidity.

    Science.gov (United States)

    Yu, Fei; Addison, Owen; Baker, Stephen J; Davenport, Alison J

    2015-09-14

    Titanium and its alloys are routinely used as biomedical implants and are usually considered to be corrosion resistant under physiological conditions. However, during inflammation, chemical modifications of the peri-implant environment including acidification occur. In addition certain biomolecules including lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls and driver of inflammation have been shown to interact strongly with Ti and modify its corrosion resistance. Gram-negative microbes are abundant in biofilms which form on dental implants. The objective was to investigate the influence of LPS on the corrosion properties of relevant biomedical Ti substrates as a function of environmental acidity. Inductively coupled plasma mass spectrometry was used to quantify Ti dissolution following immersion testing in physiological saline for three common biomedical grades of Ti (ASTM Grade 2, Grade 4 and Grade 5). Complementary electrochemical tests including anodic and cathodic polarisation experiments and potentiostatic measurements were also conducted. All three Ti alloys were observed to behave similarly and ion release was sensitive to pH of the immersion solution. However, LPS significantly inhibited Ti release under the most acidic conditions (pH 2), which may develop in localized corrosion sites, but promoted dissolution at pH 4-7, which would be more commonly encountered physiologically. The observed pattern of sensitivity to environmental acidity of the effect of LPS on Ti corrosion has not previously been reported. LPS is found extensively on the surfaces of skin and mucosal penetrating Ti implants and the findings are therefore relevant when considering the chemical stability of Ti implant surfaces in vivo.

  19. Lipopolysaccharide inhibits or accelerates biomedical titanium corrosion depending on environmental acidity

    Institute of Scientific and Technical Information of China (English)

    Fei Yu; Owen Addison; Stephen J Baker; Alison J Davenport

    2015-01-01

    Titanium and its alloys are routinely used as biomedical implants and are usually considered to be corrosion resistant under physiological conditions. However, during inflammation, chemical modifications of the peri-implant environment including acidification occur. In addition certain biomolecules including lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls and driver of inflammation have been shown to interact strongly with Ti and modify its corrosion resistance. Gram-negative microbes are abundant in biofilms which form on dental implants. The objective was to investigate the influence of LPS on the corrosion properties of relevant biomedical Ti substrates as a function of environmental acidity. Inductively coupled plasma mass spectrometry was used to quantify Ti dissolution following immersion testing in physiological saline for three common biomedical grades of Ti (ASTM Grade 2, Grade 4 and Grade 5). Complementary electrochemical tests including anodic and cathodic polarisation experiments and potentiostatic measurements were also conducted. All three Ti alloys were observed to behave similarly and ion release was sensitive to pH of the immersion solution. However, LPS significantly inhibited Ti release under the most acidic conditions (pH 2), which may develop in localized corrosion sites, but promoted dissolution at pH 4–7, which would be more commonly encountered physiologically. The observed pattern of sensitivity to environmental acidity of the effect of LPS on Ti corrosion has not previously been reported. LPS is found extensively on the surfaces of skin and mucosal penetrating Ti implants and the findings are therefore relevant when considering the chemical stability of Ti implant surfaces in vivo.

  20. Oxidation of intramyocellular lipids is dependent on mitochondrial function and the availability of extracellular fatty acids

    DEFF Research Database (Denmark)

    Corpeleijn, Eva; Hessvik, Nina P; Bakke, Siril S;

    2010-01-01

    Obesity and insulin resistance are related to both enlarged intramyocellular triacylglycerol stores and accumulation of lipid intermediates. We investigated how lipid overflow can change the oxidation of intramyocellular lipids (ICL(OX)) and intramyocellular lipid storage (ICL). These experiments...... were extended by comparing these processes in primary cultured myotubes established from healthy lean and obese type 2 diabetic (T2D) individuals, two extremes in a range of metabolic phenotypes. ICLs were prelabeled for 2 days with 100 microM [(14)C]oleic acid (OA). ICL(OX) was studied using a (14)CO...

  1. Length Scale Dependence of the Dynamic Properties of Hyaluronic Acid Solutions in the Presence of Salt

    Energy Technology Data Exchange (ETDEWEB)

    Horkay, Ferenc; Falus, Peter; Hecht, Anne-Marie; Geissler, Erik (CNRS-UMR); (NIH); (ILL)

    2010-12-07

    In solutions of the charged semirigid biopolymer hyaluronic acid in salt-free conditions, the diffusion coefficient D{sub NSE} measured at high transfer momentum q by neutron spin echo is more than an order of magnitude smaller than that determined by dynamic light scattering, D{sub DLS}. This behavior contrasts with neutral polymer solutions. With increasing salt content, D{sub DLS} approaches D{sub NSE}, which is independent of ionic strength. Contrary to theoretical expectation, the ion-polymer coupling, which dominates the low q dynamics of polyelectrolyte solutions, already breaks down at distance scales greater than the Debye-Hueckel length.

  2. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    DEFF Research Database (Denmark)

    Madsen, Lise; Pedersen, Lone Møller; Liaset, Bjørn;

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti...... pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity....

  3. Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid.

    Science.gov (United States)

    Buchenau, Bärbel; Thauer, Rudolf K

    2004-10-01

    Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 microM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.

  4. Purification, crystallization and preliminary crystallographic analysis of human cystathionine β-synthase

    International Nuclear Information System (INIS)

    This article describes the crystallization and preliminary crystallographic analysis of a protein construct (hCBS516–525) that contains the full-length cystathionine β-synthase from Homo sapiens (hCBS) and just lacks amino-acid residues 516–525. Human cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent hemeprotein, whose catalytic activity is regulated by S-adenosylmethionine. CBS catalyzes the β-replacement reaction of homocysteine (Hcy) with serine to yield cystathionine. CBS is a key regulator of plasma levels of the thrombogenic Hcy and deficiency in CBS is the single most common cause of homocystinuria, an inherited metabolic disorder of sulfur amino acids. The properties of CBS enzymes, such as domain organization, oligomerization degree or regulatory mechanisms, are not conserved across the eukaryotes. The current body of knowledge is insufficient to understand these differences and their impact on CBS function and physiology. To overcome this deficiency, we have addressed the crystallization and preliminary crystallographic analysis of a protein construct (hCBS516–525) that contains the full-length CBS from Homo sapiens (hCBS) and just lacks amino-acid residues 516–525, which are located in a disordered loop. The human enzyme yielded crystals belonging to space group I222, with unit-cell parameters a = 124.98, b = 136.33, c = 169.83 Å and diffracting X-rays to a resolution of 3.0 Å. The crystal structure appears to contain two molecules in the asymmetric unit which presumably correspond to a dimeric form of the enzyme

  5. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  6. An overlooked riddle of life's origins: energy-dependent nucleic acid unzipping.

    Science.gov (United States)

    Kovác, Ladislav; Nosek, Jozef; Tomáska, L'ubomír

    2003-01-01

    The imposing progress in understanding contemporary life forms on Earth and in manipulating them has not been matched by a comparable progress in understanding the origins of life. This paper argues that a crucial problem of unzipping of the double helix molecule of nucleic acid during its replication has been underrated, if not plainly overlooked, in the theories of life's origin and evolution. A model is presented of how evolution may have solved the problem in its early phase. Similar to several previous models, the model envisages the existence of a protocell, in which osmotic disbalance is being created by accumulation of synthetic products resulting in expansion and division of the protocell. Novel in the model is the presence in the protocell of a double-stranded nucleic acid, with each of its two strands being affixed by its 3'-terminus to the opposite sides of the membrane of a protocell. In the course of the protocell expansion, osmotic force is utilized to pull the two strands longitudinally in opposite directions, unzipping the helix and partitioning the strands between the two daughter protocells. The model is also being used as a background for arguments of why life need operate in cycles. Many formal models of life's origin and evolution have not taken into account the fact that logical possibility does not equal thermodynamic feasibility. A system of self-replication has to consist of both replicators and replicants. PMID:15008415

  7. Conformation of succinic acid: its pH dependence by Licry-NMR analysis

    Science.gov (United States)

    Chidichimo, G.; Formoso, P.; Golemme, A.; Imbardelli, D.

    The conformations of fully protonated (H2SA), fully deprotonated (SA=) and monoprotonated (HSA-) succinic acid have been investigated by means of nuclear magnetic resonance spectroscopy in liquid crystal mesophases (Licry-NMR). H-H and 13C-H direct dipolar couplings have been determined by measuring 1H-NMR and 13C-NMR spectra from quaternary nematic-lyotropic solutions of myristyltrimethylammonium bromide (MTAB), decanol, deuterated water and succinic acid (in each of its three different protonated forms). Direct dipolar couplings have been used to investigate the conformational equilibrium of the molecule in its three different protonation forms. Data could be interpreted in terms of a single conformation for each of the investigated forms. The dihedral angle between the H3-C5'-C5 and the C5'-C5-H1 planes gradually increases when going from the fully protonated H2SA species to the SA= ions. Our findings are different from those obtained by other authors by analysis of Jij couplings. In that case an equilibrium conformation between the gauche and trans conformers had been obtained.

  8. Salicylic acid modulates levels of phosphoinositide dependent-phospholipase C substrates and products to remodel the Arabidopsis suspension cell transcriptome

    Directory of Open Access Journals (Sweden)

    Eric eRuelland

    2014-11-01

    Full Text Available Basal phosphoinositide-dependent phospholipase C (PI-PLC activity controls gene expression in Arabidopsis suspension cells and seedlings. PI-PLC catalyzes the production of phosphorylated inositol and diacylglycerol (DAG from phosphoinositides. It is not known how PI-PLC regulates the transcriptome although the action of DAG-kinase (DGK on DAG immediately downstream from PI-PLC is responsible for some of the regulation. We previously established a list of genes whose expression is affected in the presence of PI-PLC inhibitors. Here this list of genes was used as a signature in similarity searches of curated plant hormone response transcriptome data. The strongest correlations obtained with the inhibited PI-PLC signature were with salicylic acid (SA treatments. We confirm here that in Arabidopsis suspension cells SA treatment leads to an increase in phosphoinositides, then demonstrate that SA leads to a significant 20% decrease in phosphatidic acid, indicative of a decrease in PI-PLC products. Previous sets of microarray data were re-assessed. The SA response of one set of genes was dependent on phosphoinositides. Alterations in the levels of a second set of genes, mostly SA-repressed genes, could be related to decreases in PI-PLC products that occur in response to SA action. Together, the two groups of genes comprise at least 40% of all SA-responsive genes. Overall these two groups of genes are distinct in the functional categories of the proteins they encode, their promoter cis-elements and their regulation by DGK or phospholipase D. SA-regulated genes dependent on phosphoinositides are typical SA response genes while those with an SA response that is possibly dependent on PI-PLC products are less SA-specific. We propose a model in which SA inhibits PI-PLC activity and alters levels of PI-PLC products and substrates, thereby regulating gene expression divergently.

  9. Knowledge and use of periconceptual folic acid supplements by British Forces Germany personnel and dependents.

    Science.gov (United States)

    Roberts, L J

    1996-10-01

    Periconceptual dietary folic acid supplementation reduces the incidence of neural tube defects (NTDs). Their use has been advocated by the Chief Medical Officer (CMO), the Consultant Advisor in Obstetrics & Gynaecology (O&G), the Senior Consultant in O&G British Forces in Germany (BFG) and by Medical Administration & Technical Instruction (MA&TI) in all NAAFI outlets. An audit of awareness of the recommendations and use of such supplements amongst pregnant women in BFG was performed over a 12 week period in BMH Rinteln. 16.6% (5/6) and 17.6% (34/193) of those with and without a history of a pregnancy affected by a NTD respectively had taken the correct dose before conception. Knowledge of and compliance with the recommendations is poor. An opportunity to positively influence health in a "captive" population is being lost.

  10. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

    Science.gov (United States)

    Grassian, Alexandra R; Parker, Seth J; Davidson, Shawn M; Divakaruni, Ajit S; Green, Courtney R; Zhang, Xiamei; Slocum, Kelly L; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D; Straub, Christopher; Growney, Joseph D; Vander Heiden, Matthew G; Murphy, Anne N; Pagliarini, Raymond; Metallo, Christian M

    2014-06-15

    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

  11. A Smoking- dependent Risk of Coronary Artery Disease Associated With A Polymorphism of The Endothelial Nitric Oxide Synthase Gene%内皮型NO合酶基因多态性与冠心病、吸烟的关系

    Institute of Scientific and Technical Information of China (English)

    齐霁; 代春华; 曾定尹; 黄涛; 马跃; 袁大军

    2003-01-01

    目的探讨内皮型一氧化氮合酶( endothelial nitric oxide synthase, eNOS)基因多态性与冠心病、吸烟的关系.方法依据 eNOS基因外显子 7G894T位点设计引物,通过巢式聚合酶链反应( PCR)扩增目的片段,限制性内切酶消化目的片段,琼脂糖凝胶电泳,紫外透射分析仪检测,计数 117例 CAD患者和有吸烟的 CAD患者及 100例健康者基因型及突变基因频率,通过χ 2检验有无统计学意义.结果 eNOS基因外显子 7的 894位点有 3种基因型: GG、 GT、 TT. CAD组 117例中 31例发生 G894T突变,纯合子 TT 3例,杂合子 GT 28例.对照组 100例中 15例发生 G894T突变,均为杂合子. CAD组基因突变频数明显高于对照组,两组 GT+ TT型χ 2检验结果:χ 2=4.265 P< 0.05,等位基因频数χ 2=5.321, P< 0.05.吸烟比例在 CAD组明显高于对照组χ 2=17.921,P< 0.01.另外,基因突变频数在 CAD吸烟组明显高于 CAD非吸烟组χ 2=4.966,P< 0.05.结论 eNOS基因 894位点 G→ T突变与吸烟和 CAD发病密切相关.

  12. Purification and activity evaluation of methionine synthase%蛋氨酸合酶活性筛选体系的建立

    Institute of Scientific and Technical Information of China (English)

    郭莹; 李超; 张志丽; 田超; 王孝伟; 刘俊义

    2012-01-01

    钴胺素依赖的蛋氨酸合酶催化N5-甲基四氢叶酸转移甲基至同型半胱氨酸生成蛋氨酸和四氢叶酸,直接参与蛋氨酸循环、叶酸循环及含硫氨基酸代谢,与DNA、蛋白质合成及生物甲基化有密切关系.本研究采用蛋白层析技术,将大鼠肝匀浆经超声破碎和高速离心处理后,依次经过DE-52批处理、Q Sepharose Fast Flow离子交换层析和CHT陶瓷羟基磷灰石吸附柱层析进行纯化,并对纯化产物进行了SDS-PAGE和Western blotting 鉴定.采用分光光度法测定蛋氨酸合酶的活性,对纯化酶的酶促反应动力学进行了研究,确定了最佳反应条件,动力学结果显示蛋氨酸合酶的双底物酶促反应的机制为乒乓机制.研究表明,采用层析技术纯化得到的蛋氨酸合酶适用于以其为靶点的化合物高通量筛选.%Methionine synthase (MS, EC2.1.1.13), a key enzyme in the folate metabolism area catalyzing methyl transfer from N5-methyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine, takes a core position in folate cycle, one-carbon-unit transfer and sculpture amino acid pathways. Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme was purified 609-fold to near homogeneity by batch chromatography on DE-52, anion-exchange chromatography on Q Sepharose Fast Flow and CHT-I hydroxyapatite column and was identified by SDS-PAGE and Western blotting. The enzyme activity was determined by spectrophotometric assay. In addition, the influencing factor and optimal reaction condition were performed. The steady state kinetic of rat liver methionine synthase was similar to that of other mammalian cobalamin-dependent methionine synthase which employed a Ping-Pong mechanism. The result indicated that cobalamin-dependent methionine synthase purified from rat liver is suitable for screening and studying methionine synthase specific inhibitors.

  13. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Abuzar Elnager

    2015-01-01

    Full Text Available Background. Caffeic acid phenethyl ester (CAPE has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM. After 3 hours, D-dimer (DD levels and WB clot weights were measured for each concentration. Thromboelastography (TEG parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM. The 50% effective dose (ED50 of CAPE (based on DD was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

  14. Amino acids and carbohydrates absorption by Na+-dependent transporters in the pyloric ceca of Hoplias malabaricus (Erythrinidae

    Directory of Open Access Journals (Sweden)

    Vieira Vania Lucia Pimentel

    2001-01-01

    Full Text Available Information about amino acids and carbohydrate absorption in fish is important to formulate an adequate diet to obtain optimal growth. Therefore, the objective of this study was to investigate if Na+-dependent transporters are involved on the absorption of glycine, L-glutamine, L-leucine, L-lysine, L-proline, L-alanine, and the carbohydrates fructose and glucose in the pyloric ceca of Hoplias malabaricus. The pyloric ceca were mounted in a system of continuous perfusion "in vitro". Amino acids and carbohydrates were placed on the mucosal side at concentrations of 10, 20, and 40mM. The serosal side of the pyloric ceca was positive in relation to the mucosal side. The addition of glycine, L-glutamine, L-leucine, L-lysine, L-proline (all tested concentrations, and glucose (at concentrations of 20 and 40mM increased the positivity of the serosal side, indicating the presence of Na+-dependent transporters in the absorption of these substances. L-alanine and fructose did not change the positivity of the serosal side. The pyloric ceca seem to be the main site of nutrient absorption in the digestive tract of H. malabaricus.

  15. Theoretical insight into the wavelength-dependent photodissociation mechanism of nitric acid.

    Science.gov (United States)

    Xiao, Hongyan; Maeda, Satoshi; Morokuma, Keiji

    2016-09-21

    Photodissociation pathways of HNO3 involving the four lowest electronic singlet states (S0, S1, S2 and S3) were studied by the MS-CAS(12e,8o)PT2/6-31+G* method. All critical points, i.e. minima, transition states and minimum energy conical intersections, were explored systematically by the global reaction route mapping (GRRM) strategy utilizing the anharmonic downward distortion following (ADDF) method. Some key structures were also optimized at the MS-CAS(16e,12o)PT2/aug-cc-pVDZ level. Based on structures and relative energies of these critical points, we discussed the wavelength-dependent photodissociation mechanism of HNO3 in detail. The OH(X(2)Π) dissociation was found to occur through four pathways on the S1, S2, and S3 surfaces depending on the excitation energy, and one of the four pathways was shown to undergo the excited state roaming mechanism. The experimental appearance energy of the O((1)D) dissociation channel at 538.8 kJ mol(-1) was attributed to a conical intersection between the S2 and S3 surfaces. It has been shown experimentally that the O((1)D) channel was dominant at the higher excitation energy. In this study, its mechanistic reason was explained by the shape of the S3 surface and locations of important critical points. PMID:27538805

  16. Measuring acetic and formic acid by proton transfer reaction-mass spectrometry: sensitivity, humidity dependence, and quantifying interferences

    Directory of Open Access Journals (Sweden)

    M. Baasandorj

    2014-10-01

    Full Text Available We present a detailed investigation of the factors governing the quantification of formic acid (FA, acetic acid (AA and their relevant mass analogues by proton transfer reaction-mass spectrometry (PTR-MS, assess the underlying fragmentation pathways and humidity dependencies, and present a new method for separating FA and AA from their main isobaric interferences. PTR-MS sensitivities towards glycolaldehyde, ethyl acetate and peroxyacetic acid at m/z 61 are comparable to that for AA; when present, these species will interfere with ambient AA measurements by PTR-MS. Likewise, when it is present, dimethyl ether can interfere with FA measurements. On the other hand, for E/N = 125 Townsend (Td, the PTR-MS sensitivity towards ethanol at m/z 47 is 5–20× lower than for FA; ethanol will then only be an important interference when present in much higher abundance than FA. Sensitivity towards 2-propanol is m/z 79, which is also commonly used to measure benzene. However, the resulting interference for benzene is only significant when E/N is low (E/N, but decreases with humidity at high E/N due to water-driven fragmentation. Sensitivity towards FA decreases with humidity throughout the full range of E/N. For glycoaldehyde and the alcohols, the sensitivity increases with humidity due to ligand switching reactions (at low E/N and reduced fragmentation in the presence of water (at high E/N. Their role as interferences will typically be greatest at high humidity. For compounds such as AA where the humidity effect depends strongly on the collisional energy in the drift tube, simple humidity correction factors (XR will only be relevant for a specific instrumental configuration. We recommend E/N∼125 Td as an effective condition for AA and FA measurements by PTR-MS, as it optimizes between the competing E/N-dependent mechanisms controlling their sensitivities and those of the interfering species. Finally, we present the design and evaluation of an online acid

  17. A pH dependent Raman and surface enhanced Raman spectroscopic studies of citrazinic acid aided by theoretical calculations.

    Science.gov (United States)

    Sarkar, Sougata; Chowdhury, Joydeep; Dutta, Soumen; Pal, Tarasankar

    2016-12-01

    A pH dependent normal Raman scattering (NRS) and surface enhanced Raman scattering (SERS) spectral patterns of citrazinic acid (CZA), a biologically important molecule, have been investigated. The acid, with different pKa values (~4 and ~11) for the two different functional groups (-COOH and -OH groups), shows interesting range of color changes (yellow at pH~14 and brown at pH~2) with the variation in solution pH. Thus, depending upon the pH of the medium, CZA molecule can exist in various protonated and/or deprotonated forms. Here we have prescribed the existence different possible forms of CZA at different pH (Forms "C", "H" and "Dprot" at pH~14 and Forms "A", "D", and "P" at pH~2 respectively). The NRS spectra of these solutions and their respective SERS spectra over gold nanoparticles were recorded. The spectra clearly differ in their spectral profiles. For example the SERS spectra recorded with the CZA solution at pH~2 shows blue shift for different bands compared to its NRS window e.g. 406 to 450cm(-1), 616 to 632cm(-1), 1332 to 1343cm(-1) etc. Again, the most enhanced peak at ~1548cm(-1) in NRS while in the SERS window this appears at ~1580cm(-1). Similar observation was also made for CZA at pH~14. For example, the 423cm(-1) band in the NRS profile experience a blue shift and appears at ~447cm(-1) in the SERS spectrum as well as other bands at ~850, ~1067 and ~1214cm(-1) in the SERS window are markedly enhanced. It is also worth noting that the SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Therefore based on the experimental findings we propose different possible molecular forms of CZA at different pH (acidic and alkaline) conditions. For example forms 'A', 'D' and 'P' existing in acidic pH (pH~2) and three other deprotonated forms 'C', 'H' and 'Dprot' in alkaline pH (pH~14). The DFT calculations for these

  18. Cell survival during complete nutrient deprivation depends on lipid droplet-fueled β-oxidation of fatty acids.

    Science.gov (United States)

    Cabodevilla, Ainara G; Sánchez-Caballero, Laura; Nintou, Eleni; Boiadjieva, Violeta G; Picatoste, Fernando; Gubern, Albert; Claro, Enrique

    2013-09-27

    Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.

  19. Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type

    Directory of Open Access Journals (Sweden)

    Fujiwara Kazuo

    2012-08-01

    Full Text Available Abstract Background A large number of studies have been carried out to obtain amino acid propensities for α-helices and β-sheets. The obtained propensities for α-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the β-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects β-sheet propensity. Results We calculated amino acid propensities for α-helices and β-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that α-helix propensities do not differ significantly by fold, but β-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for α-helix, but such is not the case for the β-sheet propensities. We also found some fold dependence on amino acid frequency in β-strands. Folds with a high Ser, Thr and Asn content at exposed sites in β-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = −0.90 and to have flat β-sheets. At buried sites in β-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = −0.93. "All-β" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas "α/β" proteins tend to have a higher content of Val, Ile and Leu. Conclusions The α-helix propensities are similar for all folds and for exposed and buried residues. However, β-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in β-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding

  20. Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice

    OpenAIRE

    Deng, Liyuan; Elmore, C. Lee; Lawrance, Andrea K.; Matthews, Rowena G.; Rozen, Rima

    2008-01-01

    Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B12-dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we u...

  1. Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1.

    Science.gov (United States)

    Dilshara, Matharage Gayani; Lee, Kyoung-Tae; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Kang, Chang-Hee; Park, Sang Rul; Choi, Yung Hyun; Choi, Il-Whan; Hyun, Jin-Won; Chang, Weon-Young; Kim, Yeon-Su; Lee, Hak-Ju; Kim, Gi-Young

    2014-01-01

    Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE2 without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE2 production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE2, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression. PMID:24291391

  2. Maternal Nutritional Imbalance between Linoleic Acid and Alpha-Linolenic Acid Increases Offspring's Anxious Behavior with a Sex-Dependent Manner in Mice.

    Science.gov (United States)

    Sakayori, Nobuyuki; Tokuda, Hisanori; Yoshizaki, Kaichi; Kawashima, Hiroshi; Innis, Sheila M; Shibata, Hiroshi; Osumi, Noriko

    2016-01-01

    Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are essential nutrients for normal brain development. The principal dietary n-6 and n-3 PUFAs are linoleic acid (LA) and α-linolenic acid (ALA), respectively, We have previously shown that maternal dietary imbalance between these PUFAs, i.e., rich in LA and poor in ALA, affected brain development and increased anxiety-related behavior in the mouse offspring. Here we further addressed sex difference in anxiety-related behavior in the offspring exposed to maternal LA:ALA imbalance. We fed pregnant mice a LA excess/ALA deficient (LA(ex)/ALA(def)) diet, and raised their offspring on a well-balanced LA:ALA diet from an early lactation period. When the offspring were grown to adulthood, they were subjected to behavioral and biochemical analyses. We found that both male and female offspring exposed to the LA(ex)/ALA(def) diet showed increased anxiety-related behavior compared to those exposed to the control diet, which was differently observed between the sexes. The female offspring also exhibited hyperactivity by maternal intake of the LA(ex)/ALA(def) diet. On the other hand, abnormal depressive behavior was undetected in both sexes. We also found that the ratio of n-6 to n-3 PUFAs in the brain was unaffected regardless of maternal diet or offspring's sex. Since the n-6/n-3 ratio is known to influence emotional behavior, it is reasonable to assume that LA:ALA imbalance exposed during brain development is the key for causing enhanced anxiety in adulthood. The present study indicates that maternal dietary imbalance between LA and ALA increases offspring's anxiety-related behavior with a sex-dependent manner. PMID:27558477

  3. Hyaluronic acid as a rescue therapy for trinitrobenzene sulfonic acid-induced colitis through Cox-2 and PGE2 in a Toll-like receptor 4-dependent way

    Institute of Scientific and Technical Information of China (English)

    Huan CHEN; Mahesh MAHASETH; Yah ZHANG

    2011-01-01

    We hypothesized whether systemic administration of high-molecular-weight hyaluronic acid (HMW HA) could rescue trinitrobenzene sulfonic acid (TNBS)-induced colitis through Toll-like receptor 4 (TLR4) signal.C3H/HeN mice and C3H/HeJ mice were used.Mice were divided into four groups:control,50% ethanol treatment group,TNBS treatment group,and TNBS plus HA treatment group.The weight changes,clinical scores,macroscopic scores,and histological scores were recorded.Cyclooxygenase 2 (Cox-2) and prostaglandin E2 (PGE2) expressions were measured both in colons and peritoneal macrophages from these mice.HA was a rescue therapy for the colitis induced by TNBS only in C3H/HeN mice.The clinical score,macroscopic score,and histological score were much lower in C3H/HeN mice receiving TNBS plus HA treatment.Cox-2 and PGE2 expressions only increased in C3H/HeN mice.These Cox-2 expressing cells were macrophages.HA can also promote the production of Cox-2 and PGE2 in peritoneal macrophages from C3H/HeN mice.Our data demonstrated that HMW HA can rescue TNBS-induced colitis through inducing Cox-2 and PGE2 expressions in a TLR4-dependent way.Macrophages may be the effector cells of HMW HA.

  4. Structure and Function of Microsomal Prostaglandin E Synthase-1

    OpenAIRE

    Pawelzik, Sven-Christian

    2010-01-01

    The glutathione-dependent enzyme microsomal prostaglandin E synthase-1 (MPGES1) plays a pivotal role in inflammatory diseases. MPGES1 is up-regulated by pro-inflammatory cytokines in concert with cyclooxygenase (COX) -2, and the concerted action of both enzymes leads to the production of induced prostaglandin E2 (PGE2), a potent lipid mediator of inflammation, pain, and fever. Non-steroidal anti-inflammatory drugs (NSAIDs) as well as COX-2 specific inhibitors (COXIBs) are widely u...

  5. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    Energy Technology Data Exchange (ETDEWEB)

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  6. Ozone stress induces the expression of ACC synthase in potato plants

    Energy Technology Data Exchange (ETDEWEB)

    Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J. (Pennsylvania State Univ., University Park (United States))

    1993-05-01

    When potato plants (Solanum tuberosum L. cv Norland) are subjected to oxone stress ethylene is emitted. Increases in ethylene production are often the result of increased expression of the enzyme ACC synthase. We used the polymerase chain reaction (PCR) to clone a cDNA encoding an ozone-induced ACC synthase. After treating potato plants with 300 ppb ozone for 4 h, RNA was extracted using a guanidinium isothiocyanate method. Using degenerate oligonucleotides corresponding to several conserved regions of ACC synthase sequences reported from different plant tissues as primers, we were able to reverse transcribe the RNA and amplify a cDNA for ACC synthase. The clone is 1098 bp in length encoding for 386 amino acids comprising [approximately]80% of the protein. Computer analysis of the deduced amino acid sequence showed that our clone is 50-70% homologous with ACC synthase genes cloned from other plant tissues. Using the cDNA as a probe in northern analysis we found that there is little or no expression in control tissue: however there is a large increase in the expression of the ACC synthase message in response to ozone treatment.

  7. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    Science.gov (United States)

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  8. Suberoyl bis-hydroxamic acid induces p53-dependent apoptosis of MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-gang ZHUANG; Fei FEI; Ying CHEN; Wei JIN

    2008-01-01

    Aim: To study the effects of suberoyl bis-hydroxamic acid (SBHA), an inhibitor of histone deacetylases, on the apoptosis of MCF-7 breast cancer cells. Meth-ods: Apoptosis in MCF-7 cells induced by SBHA was demonstrated by flow cytometric analysis, morphological observation, and DNA ladder. Mitochondrial membrane potential (△ψm) was measured using the fluorescent probe JC-1. The expressions of p53, p21, Bax, and PUMA were determined using RT-PCR or Western blotting analysis after the MCF-7 cells were treated with SBHA or p53 siRNA. Results: SBHA induced apoptosis in MCF-7 cells. The expressions of p53, p21, Bax, and PUMA were induced, and △ψm collapsed after treatment with SBHA. p53 siRNA abrogated the SBHA-induced apoptosis and the expressions of p53, p21, Bax, and PUMA. Conclusion: The activation of the p53 pathway is involved in SBHA-induced apoptosis in MCF-7 cells.

  9. Nutritional balance of essential amino acids and carbohydrates of the adult worker honeybee depends on age.

    Science.gov (United States)

    Paoli, Pier P; Donley, Dion; Stabler, Daniel; Saseendranath, Anumodh; Nicolson, Susan W; Simpson, Stephen J; Wright, Geraldine A

    2014-06-01

    Dietary sources of essential amino acids (EAAs) are used for growth, somatic maintenance and reproduction. Eusocial insect workers such as honeybees are sterile, and unlike other animals, their nutritional needs should be largely dictated by somatic demands that arise from their role within the colony. Here, we investigated the extent to which the dietary requirements of adult worker honeybees for EAAs and carbohydrates are affected by behavioural caste using the Geometric Framework for nutrition. The nutritional optimum, or intake target (IT), was determined by confining cohorts of 20 young bees or foragers to liquid diets composed of specific proportions of EAAs and sucrose. The IT of young, queenless bees shifted from a proportion of EAAs-to-carbohydrates (EAA:C) of 1:50 towards 1:75 over a 2-week period, accompanied by a reduced lifespan on diets high in EAAs. Foragers required a diet high in carbohydrates (1:250) and also had low survival on diets high in EAA. Workers exposed to queen mandibular pheromone lived longer on diets high in EAA, even when those diets contained 5× their dietary requirements. Our data show that worker honeybees prioritize their intake of carbohydrates over dietary EAAs, even when overeating EAAs to obtain sufficient carbohydrates results in a shorter lifespan. Thus, our data demonstrate that even when young bees are not nursing brood and foragers are not flying, their nutritional needs shift towards a diet largely composed of carbohydrates when they make the transition from within-hive duties to foraging.

  10. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  11. Nutritional balance of essential amino acids and carbohydrates of the adult worker honeybee depends on age.

    Science.gov (United States)

    Paoli, Pier P; Donley, Dion; Stabler, Daniel; Saseendranath, Anumodh; Nicolson, Susan W; Simpson, Stephen J; Wright, Geraldine A

    2014-06-01

    Dietary sources of essential amino acids (EAAs) are used for growth, somatic maintenance and reproduction. Eusocial insect workers such as honeybees are sterile, and unlike other animals, their nutritional needs should be largely dictated by somatic demands that arise from their role within the colony. Here, we investigated the extent to which the dietary requirements of adult worker honeybees for EAAs and carbohydrates are affected by behavioural caste using the Geometric Framework for nutrition. The nutritional optimum, or intake target (IT), was determined by confining cohorts of 20 young bees or foragers to liquid diets composed of specific proportions of EAAs and sucrose. The IT of young, queenless bees shifted from a proportion of EAAs-to-carbohydrates (EAA:C) of 1:50 towards 1:75 over a 2-week period, accompanied by a reduced lifespan on diets high in EAAs. Foragers required a diet high in carbohydrates (1:250) and also had low survival on diets high in EAA. Workers exposed to queen mandibular pheromone lived longer on diets high in EAA, even when those diets contained 5× their dietary requirements. Our data show that worker honeybees prioritize their intake of carbohydrates over dietary EAAs, even when overeating EAAs to obtain sufficient carbohydrates results in a shorter lifespan. Thus, our data demonstrate that even when young bees are not nursing brood and foragers are not flying, their nutritional needs shift towards a diet largely composed of carbohydrates when they make the transition from within-hive duties to foraging. PMID:24623119

  12. 2-Dimensional Fluorescence Spectroscopy: Determining the Temperature-Dependent Conformations of Porphyrin Dimers and Nucleic Acids

    Science.gov (United States)

    Widom, Julia; Perdomo-Ortiz, Alejandro; Aspuru-Guzik, Alan; Marcus, Andrew

    2012-02-01

    I will describe spectroscopic studies on a covalently-linked zinc tetraphenylporphyrin dimer embedded in a phospholipid bilayer membrane. Using phase-modulation 2-Dimensional Fluorescence Spectroscopy (2D FS, a fluorescence-detected version of 2D electronic spectroscopy) along with linear absorption and fluorescence spectroscopy, it was found that the dimer adopts two predominant conformations in the membrane, and that the relative populations of these two states change as a function of temperature. Simultaneously fitting the linear absorption spectrum and the 2D FS spectra at four different excitation wavelengths revealed a wealth of information about these two states, including their relative populations, relative fluorescence quantum yields, the strength of the exciton coupling present in each state, and the approximate angles between the electronic transition dipole moments of the two porphyrins. Ongoing analysis focuses on elucidating the relaxation and energy transfer dynamics of this system through the population time dependence of the 2D spectra. Finally, I will present preliminary results from experiments in which 2D FS was performed with ultraviolet excitation to study the conformations of DNA constructs labeled with a fluorescent analogue of guanine.

  13. Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice.

    Directory of Open Access Journals (Sweden)

    Frank A J A Bodewes

    Full Text Available The cause of Cystic fibrosis liver disease (CFLD, is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous or chronic (three weeks via the diet. In Cftr-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.

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

    Institute of Scientific and Technical Information of China (English)

    XU; Maojun; DONG; Jufang; ZHU; Muyuan

    2006-01-01

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

  15. Mitochondrial ATP synthase is a target for TNBS-induced protein carbonylation in XS-106 dendritic cells.

    Science.gov (United States)

    Je, Jeong Hwan; Lee, Tae Hyung; Kim, Dong Hyun; Cho, Young Hun; Lee, Ju Hee; Kim, Soo Chan; Lee, Sang-Kyou; Lee, Jaewon; Lee, Min-Geol

    2008-06-01

    ROS are produced in dendritic cells (DCs) during antigen presentation in contact hypersensitivity (CHS). As a result, ROS cause a number of nonenzymatic protein modifications, including carbonylation, which is the most widely used marker of oxidative stress. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) is a well-characterized contact allergen that results in the formation of ROS. However, proteins that are carbonylated in DCs in response to TNBS have not been identified. To study ROS-dependent protein carbonylation in response to TNBS, we used the well-established mouse DC line, XS-106. We focused on the effects of TNBS on oxidation by examining selected oxidative markers. We identified TNBS-induced ROS and myeloperoxidase (MPO) proteins and demonstrated that the increase in ROS resulted in IL-12 production. The increase in oxidation was further confirmed by an oxidation-dependent increase in protein modifications, such as carbonylation. In fact, TNBS strongly induced carbonylation of mitochondrial adenosine triphosphate (ATP) synthase in XS-106 DCs, as determined by MALDI-TOF analysis and 2-D Western blotting. ROS production and protein carbonylation were confirmed in human monocyte-derived DCs (Mo-DCs). Furthermore, glutathione (GSH) decreased ROS and protein carbonylation in Mo-DCs. Carbonylation of ATP synthase in DCs may contribute to the pathophysiology of CHS.

  16. Amino acid modifications on tRNA

    Institute of Scientific and Technical Information of China (English)

    Jing Yuan; Kelly Sheppard; Dieter S(o)ll

    2008-01-01

    The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation.Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa.tRNA synthetases.However,in the case of four amino acids (Gin,Asn,Cys and Sec),aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life.The process begins with the charging ofnoncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNAcys formation or by synthetases with relaxed specificity,such as the non-discriminating glutamyl-tRNA,non-discriminating aspartyl-tRNA and seryl-tRNA synthetases.The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA,which is catalyzed by a group of tRNA-dependent modifying enzymes,such as tRNA-dependent amidotransferases,Sep-tRNA:Cys-tRNA synthase,O-phosphoseryi-tRNA kinase and Sep-tRNA:Sec-tRNA synthase.The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.

  17. Arabidopsis Indole Synthase,a Homolog of Tryptophan Synthase Alpha,is an Enzyme Involved in the Trp-independent Indole-containing Metabolite Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Rui Zhang; Bing Wang; Jian Ouyang; Jiayang Li; Yonghong Wang

    2008-01-01

    The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.

  18. Similar peptides from two beta cell autoantigens, proinsulin and glutamic acid decarboxylase, stimulate T cells of individuals at risk for insulin-dependent diabetes.

    OpenAIRE

    Rudy, G; N. Stone; Harrison, L C; Colman, P. G.; McNair, P; Brusic, V.; French, M. B.; Honeyman, M. C.; Tait, B.; Lew, A M

    1995-01-01

    BACKGROUND: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T c...

  19. pH dependence of iron photoreduction in a rocky mountain stream affected by acid mine drainage

    Science.gov (United States)

    McKnight, Diane M.; Kimball, B.A.; Runkel, R.L.

    2001-01-01

    The redox speciation of dissolved iron and the transport of iron in acidic, metal-enriched streams is controlled by precipitation and dissolution of iron hydroxides, by photoreduction of dissolved ferric iron and hydrous iron oxides, and by oxidation of the resulting dissolved ferrous iron. We examined the pH dependence of these processes in an acidic mine-drainage stream, St Kevin Gulch, Colorado, by experimentally increasing the pH of the stream from about 4.0 to 6.5 and following the downstream changes in iron species. We used a solute transport model with variable flow to evaluate biogeochemical processes controlling downstream transport. We found that at pH 6.4 there was a rapid and large initial loss of ferrous iron concurrent with the precipitation of aluminium hydroxide. Below this reach, ferrous iron was conservative during the morning but there was a net downstream loss of ferrous iron around noon and in the afternoon. Calculation of net oxidation rates shows that the noontime loss rate was generally much faster than rates for the ferrous iron oxidation at pH 6 predicted by Singer and Stumm (1970. Science 167: 1121). The maintenance of ferrous iron concentrations in the morning is explained by the photoreduction of photoreactive ferric species, which are then depleted by noon. Copyright ?? 2001 John Wiley & Sons, Ltd.

  20. Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

    NARCIS (Netherlands)

    Cai, G.; Faleri, C.; Casino, C.; Emons, A.M.C.; Cresti, M.

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tub

  1. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael (MSU); (NWU)

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  2. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung [Hanyang Univ., Seoul (Korea, Republic of); Koo, Bonsung [Rural Development Administration, Suwon (Korea, Republic of)

    2013-02-15

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS.

  3. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  4. Demonstration of disturbed free fatty acid metabolism of myocardium in patients with non-insulin-dependent diabetes mellitus as measured with iodine-123-heptadecanoic acid

    Energy Technology Data Exchange (ETDEWEB)

    Kuikka, J.T. (Kuopio Central Hospital (Finland). Dept. of Clinical Physiology); Mustonen, J.N.; Uusitupa, M.I.J.; Rautio, P.; Vanninen, E.; Laakso, M.; Laensimies, E. (Kuopio Central Hospital (Finland). Dept. of Medicine Kuopio Central Hospital (Finland). Dept. of Clinical Nutrition)

    1991-07-01

    Myocardial free fatty acid metabolism and left ventricular function were evaluated in 15 middle-aged patients with non-insulin dependent diabetes mellitus (NIDDM) and in 8 healthy control subjects. The study subjects had no evidence of coronary heart disease on the basis of clinical history, exercise ECG or myocardial perfusion scintigraphy. During peak exercise, iodine-123 hepatadecanoic acid (HDA) was intravenously injected. Myocardial activity distribution of {sup 123}I-HDA was measured 10, 30, and 50 min after exercise using single-photon emission tomography (SPET); and then further corrected by free {sup 123}I-iodine. Venous blood samples were drawn for detecting the plasma activity of {sup 123}I. The net extraction of {sup 123}I-HDA into the myocardium was obtained by dividing the corrected tissue {sup 123}I concentration by the integral of the plasma time activity curve. The net extraction was 0.40+-0.06 min{sup {minus}1} (mean+-SD) patients with NIDDM and 0.38+-0.006 min{sup {minus}1} in control subjects (P>0.1), respectively. The faster elimination rate of {sup 123}I-HDA was found in patients with NIDDM (0.029+-0.008 min{sup {minus}1}) than in control subjects (0.022+-0.004 min{sup {minus}1}); (P<0.01). There no statistically significant difference in left ventricular ejection fraction (LVEF) at rest between patients with NIDDM (53+-9%) and control subjects (56+-2%), whereas the increase of LVEF during exercise remained lower in patients with NIDDM (3.4+-8.2) than in control subjects (11.8+-5.8%; P<0.025). A significant correlation (r=0.64; P<0.01) was found between the net extraction of {sup 123}I-HDA and the change of LVEF, as well as with exercise load (r=0.68; P<0.01). In conclusion, evidence of an increased fatty acid utilization and triglyceride synthesis rate was observed in the diabetic myocardium. (orig.).

  5. Effect of gamma-aminobutyric acid B receptor on nitric oxide/nitric oxide synthase system during recurrent febrile seizures%反复热性惊厥过程中γ-氨基丁酸B受体对一氧化氮/一氧化氮合酶体系的调节作用

    Institute of Scientific and Technical Information of China (English)

    韩颖; 秦炯; 卜定方; 杨志仙; 常杏芝; 杜军保

    2006-01-01

    目的:探讨γ-氨基丁酸B受体(γ-aminobutyric acid B receptor,GABABR)对热性惊厥(febrile seizure,FS)大鼠一氧化氮(nitric oxide,NO)/一氧化氮合酶(nitric oxide synthase,NOS)体系表达的影响.方法:将21 d龄SD大鼠随机分为对照组、FS组、FS+巴氯芬(baclofen)组和FS+法克罗芬(phaclofen)组.采用热水浴诱导大鼠FS,隔日诱导1次,共10次.采用分光光度计法测定大鼠血浆中NO含量;用原位杂交方法观察神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)mRNA表达情况;用免疫组化方法观察nNOS蛋白表达情况.结果:FS+baclofen组NO含量低于FS组[(19.02±9.31)μmol/L比(40.03±9.12)μmol/L],同时nNOS蛋白和mRNA表达也较FS组减弱;而FS+phaclofen组NO含量高于FS组[(66.46±8.15)μmol/L比(40.03±9.12)μmol/L],同时nNOS蛋白和mRNA表达也较FS组增强.结论:反复热性惊厥过程中,GABABR的改变可影响NO/NOS体系的表达.

  6. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    Science.gov (United States)

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies.

  7. RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice

    OpenAIRE

    Ali, Nusrat; Paul, Soumitra; Gayen, Dipak; Sarkar, Sailendra Nath; Datta, Swapan K.; Datta, Karabi

    2013-01-01

    Background Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also stu...

  8. Maternal folic acid supplementation modulates DNA methylation and gene expression in the rat offspring in a gestation period-dependent and organ-specific manner.

    Science.gov (United States)

    Ly, Anna; Ishiguro, Lisa; Kim, Denise; Im, David; Kim, Sung-Eun; Sohn, Kyoung-Jin; Croxford, Ruth; Kim, Young-In

    2016-07-01

    Maternal folic acid supplementation can alter DNA methylation and gene expression in the developing fetus, which may confer disease susceptibility later in life. We determined which gestation period and organ were most sensitive to the modifying effect of folic acid supplementation during pregnancy on DNA methylation and gene expression in the offspring. Pregnant rats were randomized to a control diet throughout pregnancy; folic acid supplementation at 2.5× the control during the 1st, 2nd or 3rd week of gestation only; or folic acid supplementation throughout pregnancy. The brain, liver, kidney and colon from newborn pups were analyzed for folate concentrations, global DNA methylation and gene expression of the Igf2, Er-α, Gr, Ppar-α and Ppar-γ genes. Folic acid supplementation during the 2nd or 3rd week gestation or throughout pregnancy significantly increased brain folate concentrations (Pfolic acid supplementation throughout pregnancy significantly increased liver folate concentrations (P=.005), in newborn pups. Brain global DNA methylation incrementally decreased from early to late gestational folic acid supplementation and was the lowest with folic acid supplementation throughout pregnancy (P=.026). Folic acid supplementation in late gestation or throughout pregnancy significantly decreased Er-α, Gr and Ppar-α gene expression in the liver (Pfolic acid supplementation. Maternal folic acid supplementation affects tissue folate concentrations, DNA methylation and gene expression in the offspring in a gestation-period-dependent and organ-specific manner. PMID:27152636

  9. The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

    Directory of Open Access Journals (Sweden)

    Santos Diógenes S

    2008-04-01

    Full Text Available Abstract Background The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB. The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS, molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and

  10. Translocation of the potato 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase into isolated spinach chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Jianmin; Weaver, L.M.; Herrmann, K.M. (Purdue Univ., West Lafayette, IN (USA))

    1990-05-01

    A cDNA for potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, encodes a 56 KD polypeptide whose amino terminus resembles a chloroplast transit sequence. The cDNA was placed downstream of the phage T7 polymerase recognition sequence in plasmid pGEM-3Z. DNA of the resulting plasmid pGEM-DWZ directed T7 polymerase to synthesize potato DAHP synthase mRNA in vitro. The mRNA was used in wheat germ and rabbit reticulocyte lysates for the synthesis of {sup 35}S-labeled pro-DAHP synthase. The predominant translation product is a 59 KD polypeptide that can be immunoprecipitated by rabbit polyclonal antibodies raised against the 53 KD DAHP synthase purified from potato tubers. Isolated spinach chloroplasts process the 59 KD pro-DAHP synthase to a 50 KD polypeptide. The processed polypeptide is protected from protease degradation, suggesting uptake of the enzyme into the cell organelle. Fractionation of reisolated chloroplasts after import of pro-DAHP synthase showed mature enzyme in the stroma. The uptake and processing of DAHP synthase is inhibited by antibodies raised against the mature enzyme. Our results are consistent with the assumption that potato contains a nuclear DNA encoded DAHP synthase that is synthesized as a proenzyme and whose mature form resides in the chloroplasts. Our data provide further evidence that green plants synthesize aromatic amino acids in plastids.

  11. Fatty Acids Composition of Vegetable Oils and Its Contribution to Dietary Energy Intake and Dependence of Cardiovascular Mortality on Dietary Intake of Fatty Acids

    OpenAIRE

    Jana Orsavova; Ladislava Misurcova; Jarmila Vavra Ambrozova; Robert Vicha; Jiri Mlcek

    2015-01-01

    Characterizations of fatty acids composition in % of total methylester of fatty acids (FAMEs) of fourteen vegetable oils—safflower, grape, silybum marianum, hemp, sunflower, wheat germ, pumpkin seed, sesame, rice bran, almond, rapeseed, peanut, olive, and coconut oil—were obtained by using gas chromatography (GC). Saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA), palmitic acid (C16:0; 4.6%–20.0%), oleic acid (C18:1; 6.2%–71.1%) and linoleic acid (C18:2; 1.6%–79%)...

  12. Amino acid behavior in aqueous amide solutions: Temperature dependence of the L-phenylalanine–N,N-dimethylformamide interaction

    Energy Technology Data Exchange (ETDEWEB)

    Kustov, Andrey V., E-mail: kustov@isuct.ru

    2013-08-20

    Highlights: • Thermodynamics of amino acid solvation in aqueous DMF solutions was studied at 288–318 K. • The pair interaction parameters were compared with those for urea solutions. • For hydrophobic solutes enthalpies and entropies of interaction reveal strong temperature changes. • The relationship between the temperature dependence of solvation and solute–solute interactions was found. - Abstract: We have studied thermodynamics of the L-phenylalanine (Phe) pair interaction with denaturing agents – urea (U) and dimethylformamide (DMF) at 288–318 K. Our study does indicate that enthalpies and entropies of the Phe–U interaction reveal the anomalous temperature dependence which does not occur for DMF solutions. The anomalous Phe behavior in U solutions appears to be closely related to peculiarities of U hydration. One more result is in the fact that for hydrophobic solutes such as L-phenylalanine and substituted amides it is not justified to use the results obtained at 298 K for predicting the solute behavior at physiological temperatures.

  13. Pseudolaric Acid B Induces Caspase-Dependent and Caspase-Independent Apoptosis in U87 Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Muhammad Khan

    2012-01-01

    Full Text Available Pseudolaric acid B (PLAB is one of the major bioactive components of Pseudolarix kaempferi. It has been reported to exhibit inhibitory effect on cell proliferation in several types of cancer cells. However, there is no report elucidating its effect on glioma cells and organ toxicity in vivo. In the present study, we found that PLAB inhibited growth of U87 glioblastoma cells in a dose-dependent manner with IC50~10 μM. Flow cytometry analysis showed that apoptotic cell death mediated by PLAB was accompanied with cell cycle arrest at G2/M phase. Using Western blot, we found that PLAB induced G2/M phase arrest by inhibiting tubulin polymerization in U87 cells. Apoptotic cell death was only partially inhibited by pancaspase inhibitor, z-VAD-fmk, which suggested that PLAB-induced apoptosis in U87 cells is partially caspase-independent. Further mechanistic study demonstrated that PLAB induced caspase-dependent apoptosis via upregulation of p53, increased level of proapoptotic protein Bax, decreased level of antiapoptotic protein Bcl-2, release of cytochrome c from mitochondria, activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose polymerase (PARP and caspase-independent apoptosis through apoptosis inducing factor (AIF. Furthermore, in vivo toxicity study demonstrated that PLAB did not induce significant structural and biochemical changes in mouse liver and kidneys at a dose of 25 mg/kg. Therefore, PLAB may become a potential lead compound for future development of antiglioma therapy.

  14. A TDG/CBP/RARα Ternary Complex Mediates the Retinoic Acid-dependent Expression of DNA Methylation-sensitive Genes

    Directory of Open Access Journals (Sweden)

    Hélène Léger

    2014-02-01

    Full Text Available The thymine DNA glycosylase (TDG is a multifunctional enzyme, which is essential for embryonic development. It mediates the base excision repair (BER of G:T and G:U DNA mismatches arising from the deamination of 5-methyl cytosine (5-MeC and cytosine, respectively. Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands. TDG interacts with the histone acetylase CREB-binding protein (CBP to activate CBP-dependent transcription. In addition, TDG also interacts with the retinoic acid receptor α (RARα, resulting in the activation of RARα target genes. Here we provide evidence for the existence of a functional ternary complex containing TDG, CBP and activated RARα. Using global transcriptome profiling, we uncover a coupling of de novo methylation-sensitive and RA-dependent transcription, which coincides with a significant subset of CBP target genes. The introduction of a point mutation in TDG, which neither affects overall protein structure nor BER activity, leads to a significant loss in ternary complex stability, resulting in the deregulation of RA targets involved in cellular networks associated with DNA replication, recombination and repair. We thus demonstrate for the first time a direct coupling of TDG’s epigenomic and transcription regulatory function through ternary complexes with CBP and RARα.

  15. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  16. HLA class II DR-DQ amino acids and insulin-dependent diabetes mellitus: Application of the haplotype method

    Energy Technology Data Exchange (ETDEWEB)

    Valdes, A.M.; McWeeney, S.; Thomson, G. [Univ. of California, Berkeley, CA (United States)

    1997-03-01

    Insulin-dependent diabetes mellitus (IDDM) HLA class III DRB1-DQA1-DQB1 data from four populations (Norwegian, Sardinian, Mexican American, and Taiwanese) have been analyzed to detect the amino acids involved in the disease process. The combination of sites DRB1 No. 67 and 86; DQA1 No. 47; and DQB1 No. 9, 26, 57, and 70 predicts the IDDM component in these four populations, when the results and criteria of the haplotype method for amino acids, developed in the companion paper in this issue of the journal, are used. The following sites, either individually, or in various combinations, previously have been suggested as IDDM components: DRB1 No. 57, 70, 71, and 86; DQA1 No. 52; and DQB1 No. 13, 45, and 57 (DQB1 No. 13 and 45 correlates 100% with DQB1 No. 9 and 26). We propose that DQA1 No. 47 is a better predictor of IDDM than is the previously suggested DQA1 No. 52, and we add DRB1 No. 67 and DQB1 No. 70 to the HLA DR-DQ IDDM amino acids. We do not claim to have identified all HLA DR-DQ amino acids - or highly correlated sites - involved in IDDM. The frequencies and predisposing/protective effects of the haplotypes defined by these seven sites have been compared, and the effects on IDDM are consistent across the populations. The strongest susceptible effects came from haplotypes DRB1*0301/DQA1*0501/ DQB1*0201 and DRB1*0401-5-7-8/DQA1*0301/DQB1*0302. The number of strong protective haplotypes observed was larger than the number of susceptible ones; some of the predisposing haplotypes were present in only one or two populations. Although the sites under consideration do not necessarily have a functional involvement in IDDM, they should be highly associated with such sites and should prove to be useful in risk assessment. 48 refs., 2 figs., 4 tabs.

  17. Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits

    Energy Technology Data Exchange (ETDEWEB)

    Olson, D.C.; White, J.A.; Edelman, L.; Kende, H. (Michigan State Univ., East Lansing (United States)); Harkins, R.N. (Berlex Biosciences, Alameda, CA (United States))

    1991-06-15

    1-Aminocyclopropane-1-carboxylate synthase is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits. The authors report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5{prime} and 3{prime} ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.

  18. Product Variability of the ‘Cineole Cassette'Monoterpene Synthases of Related Nicotiana Species

    Institute of Scientific and Technical Information of China (English)

    Anke F(a)hnrich; Katrin Krause; Birgit Piechulla

    2011-01-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassere' comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,and α-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoterpenes of this ‘cineole cassette' with α-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99% identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the ‘cineole cassette′ monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of N.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night (diurnal rhythm).

  19. Differences in the effect of ionizing radiation on NA+-dependent amino acid transport in human T (Molt-4) and human B (RPMI 1788) lymphoid cells

    International Nuclear Information System (INIS)

    We have investigated the effects of ionizing radiation (0.10 to 10 krad) on the Na+-dependent transport of 2-aminoisobutyric acid and the involvement of a plasma membrane sulfhydryl-protein component which modulates this transport system in a human T (Molt-4) and a B (RPMI 1788) lymphoid cell line. The radiosensitivity of this transport system and a putative sulfhydryl membrane regulatory component correlated with the known radiosensitivity of these two cell lines, i.e., DNA synthesis and survival, were much more sensitive to the effects of γ irradiation in Molt-4 than in RPMI 1788 cells. Ionizing radiation affected Na+-dependent amino acid transport in Molt-4 cells by a process which affects the maximal rate of uptake of the amino acid (i.e., V/sub max/) into the cell. No change in Na+-independent amino acid transport was observed when Molt-4 cells were subjected to radiation levels as high as 5 krad. These data coupled with the observations from other laboratories have led us to hypothesize that the radiation-induced interphase death of Molt-4 cells may be due to two factors: (i) damage to a plasma membrane sulfhydryl-protein component associated with the modulation of Na+-dependent transport of amino acids which are involved in DNA synthesis and (ii) damage to nuclear processes, which is enhanced when Na+-dependent amino acid transport is inhibited

  20. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    Science.gov (United States)

    Somerville, Chris R.; Scheible, Wolf

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  1. Molecular Cloning and Bacterial Expression of Germacrene A Synthase cDNA from Crepidiastrum sonchifolium

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Germacrene A synthase(GAS) catalyzes the biosynthesis of germacrene A, which is a key precursor for sesquiterpene lactones. Cloning of a novel full-length cDNA encoding GAS from the medicinal plant Crepidiastrum sonchifolium(designated CsGAS) is reported in this study. The cDNA is 1837 bp long and contains a 1680-bp open reading frame encoding a 559 amino-acid protein. The functional expression of the cDNA in Escherichia coli, as an N-terminal thioredoxin fusion protein, with the pET32a vector yielding a recombinant enzyme. Sequence analysis was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco, and the effect of possible involvement of a number of amino acids in sesquiterpene synthase on product specificity was also discussed.

  2. Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes.

    Science.gov (United States)

    Mott, David M; Stone, Karen; Gessel, Mary C; Bunt, Joy C; Bogardus, Clifton

    2008-02-01

    Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

  3. Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.

    Science.gov (United States)

    Parry, R J; Hoyt, J C

    1997-02-01

    Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase. PMID:9023226

  4. Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens.

    Science.gov (United States)

    Yotsui, Izumi; Serada, Satoshi; Naka, Tetsuji; Saruhashi, Masashi; Taji, Teruaki; Hayashi, Takahisa; Quatrano, Ralph S; Sakata, Yoichi

    2016-03-18

    Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation

  5. Co-occurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in subtropical acidic forest soils.

    Science.gov (United States)

    Meng, Han; Wang, Yong-Feng; Chan, Ho-Wang; Wu, Ruo-Nan; Gu, Ji-Dong

    2016-09-01

    Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two new processes of recent discoveries linking the microbial nitrogen and carbon cycles. In this study, 16S ribosomal RNA (rRNA) gene of anammox bacteria and pmoA gene of n-damo bacteria were used to investigate their distribution and diversity in natural acidic and re-vegetated forest soils. The 16S rRNA gene sequences retrieved featured at least three species in two genera known anammox bacteria, namely Candidatus Brocadia anammoxidans, Candidatus Brocadia fulgida, and Candidatus Kuenenia stuttgartiensis while the pmoA gene amplified was affiliated with two species of known n-damo bacteria Candidatus Methylomirabilis oxyfera and a newly established Candidatus Methylomirabilis sp. According to the results, the diversity of anammox bacteria in natural forests was lower than in re-vegetated forests, but no significant difference was observed in n-damo community between them. Quantitative real-time PCR showed that both anammox and n-damo bacteria were more abundant in the lower layer (10-20 cm) than the surface layer (0-5 cm). The abundance of anammox bacteria varied from 2.21 × 10(5) to 3.90 × 10(6) gene copies per gram dry soil, and n-damo bacteria quantities were between 1.69 × 10(5) and 5.07 × 10(6) gene copies per gram dry soil in the two different layers. Both anammox and n-damo bacteria are reported for the first time to co-occur in acidic forest soil in this study, providing a more comprehensive information on more defined microbial processes contributing to C and N cycles in the ecosystems. PMID:27178181

  6. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence.

    Science.gov (United States)

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

    Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (kcat/Km) in a wide range of temperatures, mainly due to an increase of k1 (substrate diffusion) and k2 (acylation) for V149I and of k2 and k3 (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔGa°) of activation for thermal denaturation may govern the thermostability. The value of ΔGa° is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases. PMID:25534779

  7. Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

    Directory of Open Access Journals (Sweden)

    Li HF

    2015-06-01

    Full Text Available Huai-Feng Li,1–3,* Xu-An Wang,1–3,* Shan-Shan Xiang,1–3,* Yun-Ping Hu,1–3 Lin Jiang,1–3 Yi-Jun Shu,1–3 Mao-Lan Li,1–3 Xiang-Song Wu,1–3 Fei Zhang,1–3 Yuan-Yuan Ye,1–3 Hao Weng,1–3 Run-Fa Bao,1–3 Yang Cao,1–3 Wei Lu,1–3 Qian Dong,1–3 Ying-Bin Liu1–3 1Department of General Surgery, 2Laboratory of General Surgery, 3Institute of Biliary Tract Disease, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Oleanolic acid (OA, a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma. Keywords: oleanolic acid, gallbladder carcinoma, apoptosis, cell cycle arrest, mitochondrial pathway

  8. Effects of leflunomide on hyaluronan synthases (HAS): NF-kappa B-independent suppression of IL-1-induced HAS1 transcription by leflunomide.

    Science.gov (United States)

    Stuhlmeier, Karl M

    2005-06-01

    Despite evidence that points to unfettered hyaluronic acid (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan synthase (HAS) genes. Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by (14)C-glucuronic acid incorporation assays. Additional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-kappaB inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1beta- nor with PMA-induced NF-kappaB translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1beta-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1beta-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the possible cause for unfettered HA production in rheumatoid arthritis and might explain, at least in part, the beneficial effects of leflunomide treatment. These findings also support the concept that IL-1beta-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-kappaB translocation.

  9. Use-dependent loss of active sympathetic neurogenic vasodilation after nitric oxide synthase inhibition in conscious rats. Evidence for the presence of preformed stores of nitric oxide-containing factors

    Science.gov (United States)

    Davisson, R. L.; Shaffer, R. A.; Johnson, A. K.; Lewis, S. J.

    1996-01-01

    In this study, we examined whether air-jet stress-induced active sympathetic hindlimb vasodilation in conscious rats involves the release of preformed stores of nitric oxide-containing factors. We determined the effects of repeated episodes of air-jet stress (six episodes given 5 minutes apart) on mean arterial pressure and vascular resistances in the mesenteric bed and intact and sympathetically denervated hindlimb beds of conscious rats treated with saline or the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 25 mumol/kg IV). In saline-treated rats, air-jet stress produced alerting behavior, minor changes in blood pressure, pronounced mesenteric vaso-constriction, and immediate and marked vasodilation in the sympathetically intact hindlimb but a minor vasodilation in the sympathetically denervated hindlimb. Each air-jet stress produced virtually identical responses. In L-NAME-treated rats, the first air-jet stress produced vasodilator responses in the sympathetically intact and sympathetically denervated hindlimbs that were similar to those in the saline-treated rats. However, each subsequent air-jet stress produced progressively smaller vasodilator responses in the sympathetically intact but not the sympathetically denervated hindlimb. There was no loss of air-jet stress-induced alerting behavior or mesenteric vasoconstriction, suggesting that L-NAME did not interfere with the central processing of the air-jet or the resultant changes in autonomic nerve activity. The progressive diminution of air-jet stress-induced vasodilation in the intact hindlimb of L-NAME-treated rats may be due to the use-dependent depletion of preformed stores of nitric oxide-containing factors that cannot be replenished in the absence of nitric oxide synthesis.

  10. Altering small and medium alcohol selectivity in the wax ester synthase.

    Science.gov (United States)

    Barney, Brett M; Ohlert, Janet M; Timler, Jacobe G; Lijewski, Amelia M

    2015-11-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.

  11. Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

    Full Text Available Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

  12. Effect of urine pH changed by dietary intervention on uric acid clearance mechanism of pH-dependent excretion of urinary uric acid

    Directory of Open Access Journals (Sweden)

    Kanbara Aya

    2012-06-01

    Full Text Available Abstract Background The finding reported in a previous paper - alkalization of urine facilitates uric acid excretion - is contradictory to what one might expect to occur: because food materials for the alkalization of urine contain fewer purine bodies than those for acidification, less uric acid in alkaline urine should have been excreted than in acid urine. To make clear what component of uric acid excretion mechanisms is responsible for this unexpected finding, we simultaneously collected data for the concentration of both creatinine and uric acid in serum as well as in urine, in order to calculate both uric acid and creatinine clearances. Methods Within the framework of the Japanese government’s health promotion program, we made recipes which consisted of protein-rich and less vegetable-fruit food materials for H + -load (acidic diet and others composed of less protein and more vegetable-fruit rich food materials (alkaline diet. This is a crossover study within some limitations. Healthy female students, who had no medical problems at the regular physical examination provided by the university, were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid, titratable acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+ and anions (Cl−,SO42−,PO4− necessary for the estimation of acid–base balance were measured. In the early morning before breakfast of the 1st, 3rd and 5th experimental day, we sampled 5 mL of blood to estimate the creatinine and uric acid concentration in serum. Results and discussion Urine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42−] + organic acid − gut alkaliwas linearly related with the excretion of acid (titratable acid + [NH4+] − [HCO3−], indicating that H + in urine is generated by the metabolic degradation of

  13. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis.

    Science.gov (United States)

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  14. Nutrient balancing of the adult worker bumblebee (Bombus terrestris) depends on the dietary source of essential amino acids.

    Science.gov (United States)

    Stabler, Daniel; Paoli, Pier P; Nicolson, Susan W; Wright, Geraldine A

    2015-03-01

    Animals carefully regulate the amount of protein that they consume. The quantity of individual essential amino acids (EAAs) obtained from dietary protein depends on the protein source, but how the proportion of EAAs in the diet affects nutrient balancing has rarely been studied. Recent research using the Geometric Framework for Nutrition has revealed that forager honeybees who receive much of their dietary EAAs from floral nectar and not from solid protein have relatively low requirements for dietary EAAs. Here, we examined the nutritional requirements for protein and carbohydrates of foragers of the buff-tailed bumblebee Bombus terrestris. By using protein (sodium caseinate) or an equimolar mixture of the 10 EAAs, we found that the intake target (nutritional optimum) of adult workers depended on the source and proportion of dietary EAAs. When bees consumed caseinate-containing diets in a range of ratios between 1:250 and 1:25 (protein to carbohydrate), they achieved an intake target (IT) of 1:149 (w/w). In contrast to those fed protein, bees fed the EAA diets had an IT more biased towards carbohydrates (1:560 w/w) but also had a greater risk of death than those fed caseinate. We also tested how the dietary source of EAAs affected free AAs in bee haemolymph. Bees fed diets near their IT had similar haemolymph AA profiles, whereas bees fed diets high in caseinate had elevated levels of leucine, threonine, valine and alanine in the haemolymph. We found that like honeybees, bumblebee workers prioritize carbohydrate intake and have a relatively low requirement for protein. The dietary source of EAAs influenced both the ratio of protein/EAA to carbohydrate and the overall amount of carbohydrate eaten. Our data support the idea that EAAs and carbohydrates in haemolymph are important determinants of nutritional state in insects. PMID:25617453

  15. Nutrient balancing of the adult worker bumblebee (Bombus terrestris) depends on the dietary source of essential amino acids.

    Science.gov (United States)

    Stabler, Daniel; Paoli, Pier P; Nicolson, Susan W; Wright, Geraldine A

    2015-03-01

    Animals carefully regulate the amount of protein that they consume. The quantity of individual essential amino acids (EAAs) obtained from dietary protein depends on the protein source, but how the proportion of EAAs in the diet affects nutrient balancing has rarely been studied. Recent research using the Geometric Framework for Nutrition has revealed that forager honeybees who receive much of their dietary EAAs from floral nectar and not from solid protein have relatively low requirements for dietary EAAs. Here, we examined the nutritional requirements for protein and carbohydrates of foragers of the buff-tailed bumblebee Bombus terrestris. By using protein (sodium caseinate) or an equimolar mixture of the 10 EAAs, we found that the intake target (nutritional optimum) of adult workers depended on the source and proportion of dietary EAAs. When bees consumed caseinate-containing diets in a range of ratios between 1:250 and 1:25 (protein to carbohydrate), they achieved an intake target (IT) of 1:149 (w/w). In contrast to those fed protein, bees fed the EAA diets had an IT more biased towards carbohydrates (1:560 w/w) but also had a greater risk of death than those fed caseinate. We also tested how the dietary source of EAAs affected free AAs in bee haemolymph. Bees fed diets near their IT had similar haemolymph AA profiles, whereas bees fed diets high in caseinate had elevated levels of leucine, threonine, valine and alanine in the haemolymph. We found that like honeybees, bumblebee workers prioritize carbohydrate intake and have a relatively low requirement for protein. The dietary source of EAAs influenced both the ratio of protein/EAA to carbohydrate and the overall amount of carbohydrate eaten. Our data support the idea that EAAs and carbohydrates in haemolymph are important determinants of nutritional state in insects.

  16. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    International Nuclear Information System (INIS)

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio [(activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)]. Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of 125I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms

  17. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  18. Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

    Science.gov (United States)

    Nong, Lidan; Ma, Jue; Zhang, Guangyan; Deng, Chunyu; Mao, Songsong; Li, Haifeng

    2016-01-01

    Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10–8~10–6 mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10–9 mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α2-adrenoceptor and nitric oxide synthase. PMID:27610030

  19. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  20. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  1. Enantioselective microbial synthesis of the indigenous natural product (-)-α-bisabolol by a sesquiterpene synthase from chamomile (Matricaria recutita).

    Science.gov (United States)

    Son, Young-Jin; Kwon, Moonhyuk; Ro, Dae-Kyun; Kim, Soo-Un

    2014-10-15

    (-)-α-Bisabolol, a sesquiterpene alcohol, is a major ingredient in the essential oil of chamomile (Matricaria recutita) and is used in many health products. The current supply of (-)-α-bisabolol is mainly dependent on the Brazilian candeia tree (Eremanthus erythropappus) by distillation or by chemical synthesis. However, the distillation method using the candeia tree is not sustainable, and chemical synthesis suffers from impurities arising from undesirable α-bisabolol isomers. Therefore enzymatic synthesis of (-)-α-bisabolol is a viable alternative. In the present study, a cDNA encoding (-)-α-bisabolol synthase (MrBBS) was identified from chamomile and used for enantioselective (-)-α-bisabolol synthesis in yeast. Chamomile MrBBS was identified by Illumina and 454 sequencing, followed by activity screening in yeast. When MrBBS was expressed in yeast, 8 mg of α-bisabolol was synthesized de novo per litre of culture. The structure of purified α-bisabolol was elucidated as (S,S)-α-bisabolol [or (-)-α-bisabolol]. Although MrBBS possesses a putative chloroplast-targeting peptide, it was localized in the cytosol, and a deletion of its N-terminal 23 amino acids significantly reduced its stability and activity. Recombinant MrBBS showed kinetic properties comparable with those of other sesquiterpene synthases. These data provide compelling evidence that chamomile MrBBS synthesizes enantiopure (-)-α-bisabolol as a single sesquiterpene product, opening a biotechnological opportunity to produce (-)-α-bisabolol. PMID:25048207

  2. Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

    Science.gov (United States)

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-01

    Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.

  3. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  4. 二甲双胍对人肝癌细胞 HepG2增殖及脂肪酸合酶的影响%Effects of metformin on cell proliferation and fatty acid synthase in human hepatocellular carcinoma cell line HepG2

    Institute of Scientific and Technical Information of China (English)

    彭晓韧; 刘燕; 邹大进; 李娟

    2015-01-01

    Objective The cancer risk of patients with diabetes mellitus who are treated by metformin declines remarkably in comparison to patients receiving other drug therapies.The article was to investigate the relationship between antineopastic activity and fatty acid synthase (FASN) of metformin in human hepatocellular carcinoma cell(HCC) line HepG2. Methods HepG2 cells were treated with various concentrations of metformin( 0, 1, 5, 10, 15 mmol/L) for 24, 48 and 72 h respectively and cell growth was assessed by CCK-8 assay.Positive control(paclitaxel 10μg/mL) and negative control(metformin 0mmol/L) were set up simultaneously.After being treated with doses of metformin(0, 5, 10,15mmol/L) for 72h, protein expression levels of AMPKα、P-AMPKα、FASN、P-mTOR and P-Akt were measured by western blotting analysis and FASN mRNA expression levels were measured by RT-PCR. Results Being treated with vari-ous doses of metformin(1, 5, 10, 15 mmol/L) for 24, 48 and 72 h, the growth of HepG2 cells were inhibited by metformin in dose-dependent and time-dependent manner( P0.05) .FASN mRNA expression levels decreased significantly in all metformin-treated groups( P<0.05) . Conclusion Met-formin actitiviates AMPK, inhibits mTOR and downregulates FASN, which are implicated in its antineopastic activity on HCC.Although metformin inhibits mTOR activation, it is not involved in Akt upregulation through a negative loop.%目的:二甲双胍治疗的糖尿病患者癌症发生风险较其他药物治疗者显著降低。探讨二甲双胍在人肝癌细胞HepG2中的抗肿瘤活性与脂肪酸合酶的关系。方法选取不同浓度(1、5、10、15 mmol/L)二甲双胍处理HepG2细胞24、48、72 h,用CCK-8法检测其对细胞增殖的影响。同时设阳性对照(紫杉醇10μg/mL),阴性对照(二甲双胍0 mmol/L)。设0、5、10、15 mmol/L二甲双胍处理72 h,用Western blot检测腺苷酸活化蛋白激酶( adenosine monophosphate activated protein

  5. Aromatic polyketide synthases from 127 Fusarium: pas de deux for chemical diversity

    Science.gov (United States)

    Fusarium species collectively cause disease on almost all crop plants and produce numerous natural products (NPs), including mycotoxins, of great concern. Many Fusarium NPs are derived from polyketide synthases (PKSs), large enzymes that catalyze the condensation of simple carboxylic acids. To gain ...

  6. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne;

    2015-01-01

    PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  7. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    OpenAIRE

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Edwin R Lampugnani; Persson, Staffan

    2016-01-01

    ABSTRACT Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated ...

  8. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

    Directory of Open Access Journals (Sweden)

    Justin W Leung

    2014-03-01

    Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

  9. Tapentadol and nitric oxide synthase systems.

    Science.gov (United States)

    Bujalska-Zadrożny, Magdalena; Wolińska, Renata; Gąsińska, Emilia; Nagraba, Łukasz

    2015-04-01

    Tapentadol, a new analgesic drug with a dual mechanism of action (μ-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10 mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10 mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2 mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia. PMID:25485639

  10. Ursodeoxycholic acid suppresses mitochondria-dependent programmed cell death induced by sodium nitroprusside in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Although ursodeoxycholic acid (UDCA) and its highly water-soluble formula (Yoo's solution; YS) have been shown to prevent neuronal damage, the effects of UDCA or YS against Parkinson's disease (PD)-related dopaminergic cell death has not been studied. This study investigated the protective effects of UDCA and YS on sodium nitroprusside (SNP)-induced cytotoxicity in human dopaminergic SH-SY5Y cells. Both UDCA (50–200 μM) and YS (100–200 μM) dose-dependently prevented SNP (1 mM)-induced cell death. Results showed that both UDCA and YS effectively attenuated the production of total reactive oxygen species (ROS), peroxynitrite (ONOO−) and nitric oxide (NO), and markedly inhibited the mitochondrial membrane potential (MMP) loss and intracellular reduced glutathione (GSH) depletion. SNP-induced programmed cell death events, such as nuclear fragmentation, caspase-3/7 and -9 activation, Bcl-2/Bax ratio decrease, and cytochrome c release, were significantly attenuated by both UDCA and YS. Furthermore, selective inhibitor of phosphatidylinositiol-3-kinase (PI3K), LY294002, and Akt/PKB inhibitor, triciribine, reversed the preventive effects of UDCA on the SNP-induced cytotoxicity and Bax translocation. These results suggest that UDCA can protect SH-SY5Y cells under programmed cell death process by regulating PI3K-Akt/PKB pathways.

  11. Cell nanomechanics and focal adhesions are regulated by retinol and conjugated linoleic acid in a dose-dependent manner

    International Nuclear Information System (INIS)

    Retinol and conjugated linoleic acid (CLA) have previously been shown to have an important role in gene expression and various cellular processes, including differentiation, proliferation and cell death. In this study we have investigated the effect of retinol and CLA, both individually and in combination, on the intracellular cytoskeleton, focal adhesions (FAs) and the nanomechanical properties of 3T3 fibroblasts. We observed a dose-dependent decrease in the formation of FAs following treatment with either compound, which was directly correlated to an increase in cell height (>30%) and a decrease in the measured Young's modulus (∼28%). Furthermore, treatments with both compounds demonstrated an increased effect and led to a reduction of>70% in the average number of FAs per cell and a decrease of >50% in average cell stiffness. These data reveal that retinol and CLA disrupt FA formation, leading to an increase in cell height and a significant decrease in stiffness. These results may broaden our understanding of the interplay between cell nanomechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease.

  12. Genetic analysis of central carbon metabolism unveils an amino acid substitution that alters maize NAD-dependent isocitrate dehydrogenase activity.

    Directory of Open Access Journals (Sweden)

    Nengyi Zhang

    Full Text Available BACKGROUND: Central carbon metabolism (CCM is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays, the most diverse model crop species, to study the genetics of CCM is a particularly attractive system. METHODOLOGY/PRINCIPAL FINDINGS: We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41, in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis, the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.

  13. NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Ahammed, Golam Jalal; Li, Xin; Yu, Jingquan; Shi, Kai

    2015-01-01

    Elevated CO2 can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO2-induced HS tolerance. Under ambient CO2 (380 µmol mol(-1)) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO2 (800 µmol mol(-1)) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO2-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO2-induced HS mitigation.

  14. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

    OpenAIRE

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-01-01

    Background Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Methods Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Results Each of the respective gene...

  15. The ent-15α-Acetoxykaur-16-en-19-oic Acid Relaxes Rat Artery Mesenteric Superior via Endothelium-Dependent and Endothelium-Independent Mechanisms

    OpenAIRE

    Êurica Adélia Nogueira Ribeiro; Edla de Azevedo Herculano; Cintia Danieli Ferreira da Costa; Fabiola Fialho Furtado; Emídio Vasconcelos Leitão da-Cunha; José Maria Barbosa-Filho; Marcelo Sobral da Silva; Isac Almeida de Medeiros

    2012-01-01

    The objective of the study was to investigate the mechanism of the relaxant activity of the ent-15 α -acetoxykaur-16-en-19-oic acid (KA-acetoxy). In rat mesenteric artery rings, KA-acetoxy induced a concentration-dependent relaxation in vessels precontracted with phenylephrine. In the absence of endothelium, the vasorelaxation was significantly shifted to the right without reduction of the maximum effect. Endothelium-dependent relaxation was significantly attenuated by pretreatment with L-NAM...

  16. Light- and metabolism-related regulation of the chloroplast ATP synthase has distinct mechanisms and functions.

    Science.gov (United States)

    Kohzuma, Kaori; Dal Bosco, Cristina; Meurer, Jörg; Kramer, David M

    2013-05-01

    The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

  17. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  18. Synthèse bibliographique: la divinyl éther synthase de plantes

    Directory of Open Access Journals (Sweden)

    Fauconnier M.L.

    2001-01-01

    Full Text Available Divinyl ether synthase in plants: a review. Divinyl ether synthase, an enzyme of the lipoxygenase pathway transforms, in potato tubers, 9-hydroperoxides of fatty acids into colneleic and colnelenic acid, two divinyl ethers of fatty acids. The enzyme has been described in a limited number of quite different plants. The enzyme has also been detected in tomato roots, garlic bulbs, tobacco plants and in marine algae. The enzyme is bound to membranes and is located in the microsomal fraction. The molecular weight of the enzyme exceeds 100,000 Da, its optimal pH is around 9 and its high specificity for 9-hydroperoxides as substrate is described. The reactional mechanism has been elucidated using radio-labelled molecules. Colneleic and colnelenic acid can be degraded enzymatically or not into aldehydes and oxo-acids. Those last compounds are also formed by the action of hydroperoxide lyase on 9-hydroperoxides of fatty acids. As other enzymes of the lipoxygenase pathway, reaction products of divinyl ether synthase are involved in pathogenic resistance. Colneleic and colnelenic acid content in potato plants has been corelated with resistance to Phytophthora infestans.

  19. Transport of Amino Acids in Lactobacillus casei by Proton-Motive-Force-Dependent and Non-Proton-Motive-Force-Dependent Mechanisms

    NARCIS (Netherlands)

    Strobel, Herbert J.; Russell, James B.; Driessen, Arnold J.M.; Konings, Wilhelmus

    1989-01-01

    Lactobacilus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition st

  20. Inducible nitric oxide synthase and inflammation.

    Science.gov (United States)

    Salvemini, D; Marino, M H

    1998-01-01

    Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications. PMID:15991919

  1. Nitric Oxide Synthases and Atrial Fibrillation

    OpenAIRE

    CynthiaAnnCarnes; ArunSridhar; SandorGyorke

    2012-01-01

    Oxidative stress has been implicated in the pathogenesis of atrial fibrillation. There are multiple systems in the myocardium which contribute to redox homeostasis, and loss of homeostasis can result in oxidative stress. Potential sources of oxidants include nitric oxide synthases, which normally produce nitric oxide in the heart. Two nitric oxide synthase isoforms (1 and 3) are normally expressed in the heart. During pathologies such as heart failure, there is induction of nitric oxide syn...

  2. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

  3. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.

  4. Unique animal prenyltransferase with monoterpene synthase activity

    Science.gov (United States)

    Gilg, Anna B.; Tittiger, Claus; Blomquist, Gary J.

    2009-06-01

    Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

  5. Characterization of α-humulene synthases responsible for the production of sesquiterpenes induced by methyl jasmonate in Aquilaria cell culture.

    Science.gov (United States)

    Kumeta, Yukie; Ito, Michiho

    2016-07-01

    The resinous portions of Aquilaria and Gyrinops plants are known as 'agarwood' and have a distinctive fragrance. To examine the biosynthesis of these fragrant compounds, we previously established cell cultures of Aquilaria crassna in which the production of three sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) could be induced by methyl jasmonate (MJ), and showed that cloned δ-guaiene synthase from MJ-treated cells is involved in the synthesis of these three compounds, although only very small amounts of α-humulene are produced. In the present study, cDNAs encoding α-humulene synthases were also isolated. Three putative sesquiterpene synthase clones (AcHS1-3) isolated from the MJ-treated cells had very similar amino acid sequences and shared 52 % identity with δ-guaiene synthases. The recombinant enzymes catalyzed the formation of α-humulene as a major product. Expression of transcripts of the α-humulene synthase and δ-guaiene synthase genes in cultured cells increased after treatment with MJ. These results revealed that these α-humulene and δ-guaiene synthases are involved in the synthesis of three sesquiterpenes induced by MJ treatment. PMID:27180085

  6. Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

    Science.gov (United States)

    Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

    2016-04-01

    Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. PMID:26880289

  7. Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Crock, John E. (Moscow, ID)

    1999-01-01

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha pi