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Sample records for carboxylase

  1. Molecular evolution of urea amidolyase and urea carboxylase in fungi

    Directory of Open Access Journals (Sweden)

    Harris Steven D

    2011-03-01

    Full Text Available Abstract Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms. Results Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota. It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina. Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina. Conclusions We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal

  2. Pyruvate carboxylase is expressed in human skeletal muscle

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable...

  3. Chemical Genetics of Acetyl-CoA Carboxylases

    Directory of Open Access Journals (Sweden)

    Xuyu Zu

    2013-01-01

    Full Text Available Chemical genetic studies on acetyl-CoA carboxylases (ACCs, rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC and carboxyltransferase (CT activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

  4. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

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    Terekhova, I.V.; Chernyad' ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  5. Experiments on the formation of carboxylase and thiamine pyrophosphate in living bakers' yeast

    NARCIS (Netherlands)

    Leijnse, B.; Terpstra, W.

    1951-01-01

    The formation of carboxylase by living bakers' yeast was demonstrated upon incubation of the yeast with either thiamine or 2-methyl-4-amino-5-ethoxymethylpyrimidine, in the presence and in the absence of glucose. Carboxylase is also formed upon incubation of the yeast with NH4 sulfate and glucose. I

  6. The dynamic organization of fungal acetyl-CoA carboxylase

    Science.gov (United States)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  7. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Gokarn, R.R.; Eiteman, M.A.; Altman, E.

    2000-05-01

    Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99a-pyc than by cells which overproduced PPC(JCL1242/pPC201, ppc{sup +}), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc{sup +}) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc{sup +} strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc{sup +} strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.

  8. Decreased renal vitamin K-dependent γ-glutamyl carboxylase activity in calcium oxalate calculi patients

    Institute of Scientific and Technical Information of China (English)

    陈俊汇; 刘继红; 章咏裳; 叶章群; 王少刚

    2003-01-01

    Objective To study the activity of vitamin K-dependent γ-glutamyl carboxylase in patients with calcium oxalate (CaOx) urolithiasis compared with healthy individuals and to assess its relationship to the renal calcium oxalate urolithiasis. Methods Renal parenchymas were harvested from urolithic patients and renal tumor patients undergoing nephrectomy. The renal carboxylase activity was evaluated as the radioactivity of [14C] labeled sodium bicarbonate in carboxylic reactions in vitro using β-liquid scintillation counting. Results Significantly reduced activity of renal vitamin K-dependent γ-glutamyl carboxylase was observed in the urolithic group as compared with normal controls (P<0.01). Conclusion It suggests that the reduced carboxylase activity observed in the urolithic patients may play an important role in the course of renal calcium oxalate urolithiasis.

  9. Crystallization and structure of a recombinant ribulose-1,5-bisphosphate carboxylase

    Science.gov (United States)

    Schneider, Gunter; Lindqvist, Ylva; Brändén, Carl-Ivar; Lorimer, George

    1988-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

  10. Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

    Science.gov (United States)

    Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

    1983-08-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

  11. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

    Science.gov (United States)

    Broussard, Tyler C; Pakhomova, Svetlana; Neau, David B; Bonnot, Ross; Waldrop, Grover L

    2015-06-23

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.

  12. Pyruvate carboxylase deficiency: An underestimated cause of lactic acidosis

    Directory of Open Access Journals (Sweden)

    F. Habarou

    2015-03-01

    Full Text Available Pyruvate carboxylase (PC is a biotin-containing mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, thereby being involved in gluconeogenesis and in energy production through replenishment of the tricarboxylic acid (TCA cycle with oxaloacetate. PC deficiency is a very rare metabolic disorder. We report on a new patient affected by the moderate form (the American type A. Diagnosis was nearly fortuitous, resulting from the revision of an initial diagnosis of mitochondrial complex IV (C IV defect. The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Intellectual disability was noted at this time. Amino acids in plasma and organic acids in urine did not show patterns of interest for the diagnostic work-up. In skin fibroblasts PC showed no detectable activity whereas biotinidase activity was normal. We had previously reported another patient with the severe form of PC deficiency and we show that she also had secondary C IV deficiency in fibroblasts. Different anaplerotic treatments in vivo and in vitro were tested using fibroblasts of both patients with 2 different types of PC deficiency, type A (patient 1 and type B (patient 2. Neither clinical nor biological effects in vivo and in vitro were observed using citrate, aspartate, oxoglutarate and bezafibrate. In conclusion, this case report suggests that the moderate form of PC deficiency may be underdiagnosed and illustrates the challenges raised by energetic disorders in terms of diagnostic work-up and therapeutical strategy even in a moderate form.

  13. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Science.gov (United States)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  14. Resistance to acetyl-CoA carboxylase-inhibiting herbicides.

    Science.gov (United States)

    Kaundun, Shiv S

    2014-09-01

    Resistance to acetyl-CoA carboxylase herbicides is documented in at least 43 grass weeds and is particularly problematic in Lolium, Alopecurus and Avena species. Genetic studies have shown that resistance generally evolves independently and can be conferred by target-site mutations at ACCase codon positions 1781, 1999, 2027, 2041, 2078, 2088 and 2096. The level of resistance depends on the herbicides, recommended field rates, weed species, plant growth stages, specific amino acid changes and the number of gene copies and mutant ACCase alleles. Non-target-site resistance, or in essence metabolic resistance, is prevalent, multigenic and favoured under low-dose selection. Metabolic resistance can be specific but also broad, affecting other modes of action. Some target-site and metabolic-resistant biotypes are characterised by a fitness penalty. However, the significance for resistance regression in the absence of ACCase herbicides is yet to be determined over a practical timeframe. More recently, a fitness benefit has been reported in some populations containing the I1781L mutation in terms of vegetative and reproductive outputs and delayed germination. Several DNA-based methods have been developed to detect known ACCase resistance mutations, unlike metabolic resistance, as the genes remain elusive to date. Therefore, confirmation of resistance is still carried out via whole-plant herbicide bioassays. A growing number of monocotyledonous crops have been engineered to resist ACCase herbicides, thus increasing the options for grass weed control. While the science of ACCase herbicide resistance has progressed significantly over the past 10 years, several avenues provided in the present review remain to be explored for a better understanding of resistance to this important mode of action.

  15. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    Science.gov (United States)

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  16. Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Fontaine, V.; Pelloux, J.; Afif, D.; Gerant, D.; Dizengremel, P. [Univ. Henri Poincare-Nancy 1, Lab. de Biologie Forestiere, Vandauvre les Nancy cedex (France); Podor, M.; Grieu, P. [ENSAIA-INRA, Lab. Agronomie Environnement, Vandauvre les Nancy cedex (France)

    1999-06-01

    The effects of ozone exposure on carbon-fixation-related processes in Pinus halepensis Mill. needles were assessed over 3 months under controlled conditions. Ozone fumigation (200 ppb) did not induce a modification of either net CO{sub 2} assimilation or stomatal conductance in 1-year-old needles, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity was shown to be reduced by a half. Moreover, this ozone-induced reduction in Rubisco activity was associated with a decrease in the quantity of Rubisco, as determined by the decrease in the large subunit (LSU). On the other hand, 200-ppb ozone fumigation induced a strong increase in both activity and quantity of another carboxylating enzyme, phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), generally considered in C{sub 3} plants to participate in carbon catabolism processes. Ozone induced a significant decrease in the Rubisco/PEPC activity ratio which promotes the role of PEPC in trees under ozone stress. The role of this carboxylase will be discussed. (au) 42 refs.

  17. A Patient With Pyruvate Carboxylase Deficiency and Nemaline Rods on Muscle Biopsy

    DEFF Research Database (Denmark)

    Unal, Ozlem; Orhan, Diclehan; Ostergaard, Elsebet;

    2013-01-01

    and nemaline rods detected on muscle biopsy. The nemaline rods may be due to cellular energy shortage and altered energy metabolism in pyruvate carboxylase deficiency, similar to that in the previously reported patients. The mechanism of nemaline rod formation may be associated with the role of pyruvate...

  18. Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii.

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    Lanaras, T; Codd, G A

    1981-11-01

    Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.

  19. Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

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    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

  20. Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley.

    Science.gov (United States)

    Rundle, S J; Zielinski, R E

    1991-03-15

    We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.

  1. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  2. Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

    OpenAIRE

    Stefan Christen; Doriane Lorendeau; Roberta Schmieder; Dorien Broekaert; Kristine Metzger; Koen Veys; Ilaria Elia; Joerg Martin Buescher; Martin Franz Orth; Shawn Michael Davidson; Thomas Georg Philipp Grünewald; Katrien De Bock; Sarah-Maria Fendt

    2016-01-01

    Cellular proliferation depends on refilling the tricarboxylic acid (TCA) cycle to support biomass production (anaplerosis). The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC) and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis ...

  3. Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.

    OpenAIRE

    Lopez, J.M.; Bennett, M K; Sanchez, H B; Rosenfeld, J M; Osborne, T E

    1996-01-01

    Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for th...

  4. (4-Piperidinyl)-piperazine: a new platform for acetyl-CoA carboxylase inhibitors.

    Science.gov (United States)

    Chonan, Tomomichi; Oi, Takahiro; Yamamoto, Daisuke; Yashiro, Miyoko; Wakasugi, Daisuke; Tanaka, Hiroaki; Ohoka-Sugita, Ayumi; Io, Fusayo; Koretsune, Hiroko; Hiratate, Akira

    2009-12-01

    Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the synthesis and structure-activity relationships of a series of disubstituted (4-piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors.

  5. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    Science.gov (United States)

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

  6. Attempts to apply affinity labeling techniques to ribulosebisphosphate carboxylase/oxygenase. [Comparison of spinach leaf and Rhodospirillum rubrum

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, F. C.; Norton, I. L.; Stringer, C. D.; Schloss, J. V.

    1978-01-01

    Studies on carboxylases/oxygenases from different species may be necessary to confirm that a residue implicated as essential is indeed an active-site component. To provide an especially stringent test case for the identification of species invariant structural features the enzymes from two phylogenetically distant species, spinach and Rhodospirillum rubrum, were compared. To date, the reactions of Br-butanone-P/sub 2/ and BrAcNHEtOP with the spinach enayme have been rather thoroughly characterized; only preliminary experiments have been completed with the R. rubrum enzyme. Both enzymes were isolated and assayed for carboxylase activity (spectrophotometrically or /sup 14/CO/sub 2/-fixation) and for oxygenase activity.

  7. Dark/Light Modulation of Ribulose Bisphosphate Carboxylase Activity in Plants from Different Photosynthetic Categories 1

    Science.gov (United States)

    Vu, J. Cu V.; Allen, Leon H.; Bowes, George

    1984-01-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO3− and Mg2+ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C3); P. maximum (C4 phosphoenolpyruvate carboxykinase); P. milioides (C3/C4); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C3); P. miliaceum (C4 NAD malic enzyme); Zea mays and Sorghum bicolor (C4 NADP malic enzyme); Moricandia arvensis (C3/C4); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C3 species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO2 and Mg2+ activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  8. Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories.

    Science.gov (United States)

    Vu, J C; Allen, L H; Bowes, G

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light.

  9. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

    Science.gov (United States)

    Lietzan, Adam D; Lin, Yi; St Maurice, Martin

    2014-11-15

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

  10. Biotin deficiency in the cat and the effect on hepatic propionyl CoA carboxylase.

    Science.gov (United States)

    Carey, C J; Morris, J G

    1977-02-01

    Biotin deficiency was produced in growing kittens by feeding a diet containing dried, raw egg white. After receiving either an 18.5% egg white diet for 25 weeks, or a 32% egg white diet for 12 weeks, they exhibited dermal lesions characterized by alopecia, scaly dermatitis and achromotrichia, which increased in severity with the deficiency. Females developed accumulations of dried salivary, nasal and lacrymal secretions in the facial region although a male did not. There was a loss of body weight in all cats as the deficiency progressed. Hepatic propionyl CoA carboxylase activities were measured on biopsy samples of liver during biotin deficiency and after biotin supplementation. In the deficient state, activities were 4% and 24% of that following biotin supplementation. Propionyl carboxylase activity in the liver of the cat was comparable to that reported in the rat and chick in the deficient and normal states. Subcutaneous injection of 0.25 mg biotin every other day while continuing to receive the egg white diet caused remission of clinical signs, a body weight gain and increased food intake.

  11. Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

    Science.gov (United States)

    Hagmann, Anna; Hunkeler, Moritz; Stuttfeld, Edward; Maier, Timm

    2016-08-01

    Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

  12. Dark/light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

    Energy Technology Data Exchange (ETDEWEB)

    Vu, J.C.V.; Allen, L.H. Jr.; Bowes, G.

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from light-exposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO/sub 3//sup -/ and Mg/sup 2 +/ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C/sub 3/); P. maximum (C/sub 4/ phosphoenolpyruvate carboxykinase); P. milioides (C/sub 3//C/sub 4/); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C/sub 3/); P. miliaceum (C/sub 4/ NAD malic enzyme); Zea mays and Sorghum bicolor (C/sub 4/ NADP malic enzyme); Moricandia arvensis (C/sub 3//C/sub 4/); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C/sub 3/ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO/sub 2/ and Mg/sup 2 +/ activation, but which can be converted to an activatable state upon exposure of the leaf to light. 16 references, 2 tables.

  13. A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.; Xiang, S; Lasso, G; Gil, D; Valle, M; Tong, L

    2009-01-01

    Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

  14. Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

    Science.gov (United States)

    Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

    2007-05-01

    The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

  15. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia : Mollusca)

    NARCIS (Netherlands)

    Cavanaugh, Colleen M.; Abbott, Marilyn S.; Veenhuis, Marten

    1988-01-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP2Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large

  16. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  17. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Jang Eun Lee

    Full Text Available Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2 regulates fatty acid oxidation (FAO by inhibiting carnitine palmitoyltransferase 1 (CPT1, a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

  18. In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase from Fully Denatured Subunits

    Institute of Scientific and Technical Information of China (English)

    Zhen-Hua YONG; Gen-Yun CHEN; Jiao-Nai SHI; Da-Quan XU

    2006-01-01

    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  19. Acetyl-CoA carboxylase inhibitors from avocado (Persea americana Mill) fruits.

    Science.gov (United States)

    Hashimura, H; Ueda, C; Kawabata, J; Kasai, T

    2001-07-01

    A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.

  20. CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

    Institute of Scientific and Technical Information of China (English)

    Li-Li Ling; Hong-Hui Lin; Ben-Hua Ji; De-Mao Jiao

    2006-01-01

    To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice(Oryza sativa L.) hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase (PEPC) transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and transferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield (AQY), photochemical efficiency of photosystem Ⅱ and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or maiate, or phosphoenolpyruvate.The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

  1. The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent.

    Science.gov (United States)

    Lin, Yi; Boese, Cody J; St Maurice, Martin

    2016-10-01

    Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO2 . UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.

  2. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    Science.gov (United States)

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  3. Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Singal, H R; Singh, R

    1986-02-01

    Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg(2+) ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K(m) of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO(3) (-) with S(0.5) value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, alpha-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca(2+) and Mn(2+). Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.

  4. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, R.B. [Georgia Univ., Athens, GA (United States). Dept. of Genetics

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  5. Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria

    Science.gov (United States)

    Takeya, Masahiro; Hirai, Masami Yokota; Osanai, Takashi

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2 fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders. PMID:28117365

  6. Cloning and characterization of cotton heteromeric acetyl-CoA carboxylase genes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Heteromeric acetyl-coanzyme A(CoA)carboxylese(ACCase)catalyzes the formation of malonyl-CoA from acetyl-CoA.It plays an essential role in fatty acid synthesis in prokaryotes and most of plants.The heteromeric ACCase is composed of four subunits:biotin carboxylase (BC),biotin carboxyl carrier protein (BCCP),and α-and β-subunits of carboxyltransferese(α-andβ-CT).In this study,we cloned five novel genes encoding the subunits of heteromeric ACCese(one BC,BCCP and β-CT,and two α-CTs) from cotton (Gossypium hirsutum cv.zhongmian 35)by RACE-PCR.The deduced amino acid sequence of these cDNAs shares high similarity with other reported heteromeric ACCese subunits.The phylogenetic analysis indicated that the different subunits of heteromeric ACCase were grouped in a similar pattern.Southern blot analysis revealed the milti-copy patterns of these heteromeric ACCase genes in cotton genome.Semi-quantitative RT-PCR demonstrated that heteromeric ACCese genes were constitutively expressed in all of the cotton tissues,but the transcripts accumulated at a relatively low level in roots.To our knowledge,this is the first report about characterization of the heteromeric ACCase genes in cotton.

  7. Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

    Institute of Scientific and Technical Information of China (English)

    Lie-Feng ZHANG; Qi RUI; Lang-Lai XU

    2005-01-01

    The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.

  8. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    Science.gov (United States)

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  9. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    Directory of Open Access Journals (Sweden)

    Yingmei Peng

    Full Text Available Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC while the other as bacteria-type pepcase (BTPC because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

  10. Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal

    Science.gov (United States)

    Abu-Elheiga, Lutfi; Matzuk, Martin M.; Kordari, Parichher; Oh, WonKeun; Shaikenov, Tattym; Gu, Ziwei; Wakil, Salih J.

    2005-01-01

    Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. We previously reported that ACC2 null mice are viable, and that ACC2 plays an important role in the regulation of fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I, a mitochondrial component of the fatty-acyl shuttle system. Herein, we used gene targeting to knock out the ACC1 gene. The heterozygous mutant mice (Acc1+/–) had normal fertility and lifespans and maintained a similar body weight to that of their wild-type cohorts. The mRNA level of ACC1 in the tissues of Acc1+/– mice was half that of the wild type; however, the protein level of ACC1 and the total malonyl-CoA level were similar. In addition, there was no difference in the acetate incorporation into fatty acids nor in the fatty acid oxidation between the hepatocytes of Acc1+/– mice and those of the wild type. In contrast to Acc2–/– mice, Acc1–/– mice were not detected after mating. Timed pregnancies of heterozygotes revealed that Acc–/– embryos are already undeveloped at embryonic day (E)7.5, they die by E8.5, and are completely resorbed at E11.5. Our previous results of the ACC2 knockout mice and current studies of ACC1 knockout mice further confirm our hypotheses that malonyl-CoA exists in two independent pools, and that ACC1 and ACC2 have distinct roles in fatty acid metabolism. PMID:16103361

  11. Core promoter acetylation is not required for high transcription from the phosphoenolpyruvate carboxylase promoter in maize

    Directory of Open Access Journals (Sweden)

    Horst Ina

    2009-12-01

    Full Text Available Abstract Background Acetylation of promoter nucleosomes is tightly correlated and mechanistically linked to gene activity. However, transcription is not necessary for promoter acetylation. It seems, therefore, that external and endogenous stimuli control histone acetylation and by this contribute to gene regulation. Photosynthetic genes in plants are excellent models with which to study the connection between stimuli and chromatin modifications because these genes are strongly expressed and regulated by multiple stimuli that are easily manipulated. We have previously shown that acetylation of specific histone lysine residues on the photosynthetic phosphoenolpyruvate carboxylase (Pepc promoter in maize is controlled by light and is independent of other stimuli or gene activity. Acetylation of upstream promoter regions responds to a set of other stimuli which include the nutrient availability of the plant. Here, we have extended these studies by analysing histone acetylation during the diurnal and circadian rhythm of the plant. Results We show that histone acetylation of individual lysine residues is removed from the core promoter before the end of the illumination period which is an indication that light is not the only factor influencing core promoter acetylation. Deacetylation is accompanied by a decrease in gene activity. Pharmacological inhibition of histone deacetylation is not sufficient to prevent transcriptional repression, indicating that deacetylation is not controlling diurnal gene regulation. Variation of the Pepc promoter activity during the day is controlled by the circadian oscillator as it is maintained under constant illumination for at least 3 days. During this period, light-induced changes in histone acetylation are completely removed from the core promoter, although the light stimulus is continuously applied. However, acetylation of most sites on upstream promoter elements follows the circadian rhythm. Conclusion Our results

  12. Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

    Science.gov (United States)

    Kandoi, Deepika; Mohanty, Sasmita; Govindjee; Tripathy, Baishnab C

    2016-12-01

    Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of (35)S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

  13. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

    Directory of Open Access Journals (Sweden)

    Marek M Galka

    Full Text Available Abscisic acid ((+-ABA is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC, x-ray crystallography and in silico modelling to identify putative (+-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP substrate. Functionally, (+-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM, but more potent inhibition of Rubisco activation (Ki of ~ 130 μM. Comparative structural analysis of Rubisco in the presence of (+-ABA with RuBP in the active site revealed only a putative low occupancy (+-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+-ABA binding site in the RuBP binding pocket. Overall we conclude that (+-ABA interacts with Rubisco. While the low occupancy (+-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

  14. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    Science.gov (United States)

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  15. Variations in ribulose 1,5-bisphosphate carboxylase protein levels, activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii.

    Science.gov (United States)

    Lanaras, T; Codd, G A

    1982-05-01

    Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.

  16. A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

    Institute of Scientific and Technical Information of China (English)

    Xiao-Fu Zhou; Peng-Da Ma; Ren-Hou Wang; Bo Liu; Xing-Zhi Wang

    2006-01-01

    A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunlt (rbcS) gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena,transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

  17. Design and synthesis of disubstituted (4-piperidinyl)-piperazine derivatives as potent acetyl-CoA carboxylase inhibitors.

    Science.gov (United States)

    Chonan, Tomomichi; Tanaka, Hiroaki; Yamamoto, Daisuke; Yashiro, Miyoko; Oi, Takahiro; Wakasugi, Daisuke; Ohoka-Sugita, Ayumi; Io, Fusayo; Koretsune, Hiroko; Hiratate, Akira

    2010-07-01

    Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the structure-based design and synthesis of a novel series of disubstituted (4-piperidinyl)-piperazine derivatives as ACC inhibitors. Our structure-based approach led to the discovery of the indole derivatives 13i and 13j, which exhibited potent in vitro ACC inhibitory activity.

  18. A conformational investigation of propeptide binding to the integral membrane protein γ-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry

    DEFF Research Database (Denmark)

    Parker, Christine H; Morgan, Christopher R; Rand, Kasper Dyrberg;

    2014-01-01

    Gamma (γ)-glutamyl carboxylase (GGCX) is an integral membrane protein responsible for the post-translational catalytic conversion of select glutamic acid (Glu) residues to γ-carboxy glutamic acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the mechanism of carboxylation and the rol...

  19. Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

    NARCIS (Netherlands)

    Tsuprun, V.L.; Boekema, E.J.; Samsonidze, T.G.; Pushkin, A.V.

    1991-01-01

    The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of 1

  20. Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

    Science.gov (United States)

    Bagley, Scott W; Southers, James A; Cabral, Shawn; Rose, Colin R; Bernhardson, David J; Edmonds, David J; Polivkova, Jana; Yang, Xiaojing; Kung, Daniel W; Griffith, David A; Bader, Scott J

    2012-02-03

    Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

  1. Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

    Institute of Scientific and Technical Information of China (English)

    WANG Rui-jian; YANG Xue-ying; ZHENG Liang-yu; YANG Ye; GAO Gui; CAO Shu-gui

    2008-01-01

    The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.

  2. Genetic Mutation of Vitamin K-dependent Gamma-glutamyl Car-boxylase Domain in Patients with Calcium Oxalate Urolithiasis

    Institute of Scientific and Technical Information of China (English)

    Jiankun QIAO; Tao WANG; Jun YANG; Jihong LIU; Xiaoxin GONG; Xiaolin GUO; Shaogang WANG; Zhangqun YE

    2009-01-01

    To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase (GGCX or VKDC) in patients with calcium oxalate urolithasis, renal cortex and peripheral blood sam-ples were obtained from severe hydronephrosis patients (with or without calculi), and renal tumor pa-tients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis (COU) by polymerase chain reaction (PCR) and denatured high pressure liquid chromatography (DHPLC), and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

  3. Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese Foxtail (Alopecurus japonicus.

    Directory of Open Access Journals (Sweden)

    Hongle Xu

    Full Text Available Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.

  4. Toward a better knowledge of the molecular evolution of phosphoenolpyruvate carboxylase by comparison of partial cDNA sequences.

    Science.gov (United States)

    Gehrig, H H; Heute, V; Kluge, M

    1998-01-01

    To get deeper insight into the evolution of phosphoenolpyruvate carboxylase we have identified PEPC fragments (about 1,100 bp) of another 12 plants species not yet investigated in this context. The selected plants include one Chlorophyta, two Bryophyta, four Pteridophyta, and five Spermatophyta species. The obtained phylogenetic trees on PEPC isoforms are the most complete ones up to now available. Independent of their manner of construction, the resulting dendrograms are very similar and fully consistent with the main topology as it is postulated for the evolution of the higher terrestrial plants. We found a distinct clustering of the PEPC sequences of the prokaryotes, the algae, and the spermatophytes. PEPC isoforms of the archegoniates are located in the phylogenetic trees between the algae and spermatophytes. Our results strengthen the view that the PEPC is a very useful molecular marker with which to visualize phylogenetic trends both on the metabolic and organismic levels.

  5. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    Science.gov (United States)

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  6. Rubisco and PEP carboxylase responses to changing irradiance in a Brazilian Cerrado tree species, Qualea grandiflora Mart. (Vochysiaceae).

    Science.gov (United States)

    Paulilo, M T; Besford, R T; Wilkins, D

    1994-02-01

    The activities of ribulose-1,5-bisphosphate carboxylase-oxygenase, Rubisco (E.C. 4.1.1.39) and phosphoenolpyruvate carboxylase, PEPc (E.C. 4.1.1.31), and concentrations of protein and chlorophyll were measured in extracts from cotyledons and first leaves of Qualea grandiflora Mart. (Vochysiaceae) seedlings after transfer from high-light (20 days at 320 micro mol m(-2) s(-1), PAR) to low-light (35 days at 120 micro mol m(-2) s(-1), PAR) conditions. When Tween 20 and glycerol were added to the extraction medium, Rubisco activities obtained for Qualea grandiflora were comparable to published values for several coniferous species and the broad-leaved species, Prunus avium L. Stella, grown in a similar light environment. Rubisco activity in cotyledons of Q. grandiflora grown in high light for 20 days and then transferred to low light for a further 35 days was similar to the activity in cotyledons of plants grown continuously in high light. However, the first leaf above the cotyledons showed a greater response to the change in irradiance; in high light, Rubisco activity of the first leaf was 1.8 times higher on a fresh weight basis and 2.7 times higher on an area basis than that of leaves transferred from high to low light. Fresh weight and chlorophyll concentration expressed on a unit leaf area basis were also higher in the high-light treatment. These responses to irradiance are indicative of a species adapted to growth in an unshaded habitat. The PEPc activity in leaves was 15% of Rubisco activity, which is typical of species with a C(3) photosynthetic pathway. The relatively slow growth rate of Q. grandiflora observed in these experiments could not be attributed to a low carboxylation capacity per unit leaf area.

  7. Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

    Science.gov (United States)

    Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

    2016-03-01

    Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

  8. 小麦导入磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的初步研究%Preliminary Study on Phosphoenolpyruvate Carboxylase (PEPCase) Gene Introduced into Wheat

    Institute of Scientific and Technical Information of China (English)

    张彬; 马建军; 贾栋

    2009-01-01

    [Objective] The aim of this study was to introduce Phosphoenolpyruvate Carboxylase (PEPCase) gene into common wheat Linyou 145. [Method] With the material of common wheat Linyou 145, Phosphoenolpyruvate Carboxylase (PEPCase) gene was introduced into wheat embryo callus by the agrobacterium-mediated transformation system, and then analyzed through successive selection with selective medium containg gygromycin to detect the gene at the molecular level. [Result] The hyg-resistant plants were obtained, and GUS histochemical staining showed the leaf of resistant plants was stained dark blue. The target bands appeared in PCR analysis. [Conclusion] Phosphoenolpyruvate Carboxylase (PEPCase) gene has been primarily introduced into the recipient material.

  9. Synthesis, Biological Evaluation and Molecular Docking Studies of Piperidinylpiperidines and Spirochromanones Possessing Quinoline Moieties as Acetyl-CoA Carboxylase Inhibitors

    Directory of Open Access Journals (Sweden)

    Tonghui Huang

    2015-09-01

    Full Text Available Acetyl-coenzyme A carboxylases (ACCs play critical roles in the regulation of fatty acid metabolism and have been targeted for the development of drugs against obesity, diabetes and other metabolic diseases. Two series of compounds possessing quinoline moieties were designed, synthesized and evaluated for their potential to inhibit acetyl-CoA carboxylases. Most compounds showed moderate to good ACC inhibitory activities and compound 7a possessed the most potent biological activities against ACC1 and ACC2, with IC50 values of 189 nM and 172 nM, respectively, comparable to the positive control. Docking simulation was performed to position compound 7a into the active site of ACC to determine a probable binding model.

  10. Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

    Science.gov (United States)

    Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

    2013-03-01

    Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations.

  11. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    Science.gov (United States)

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  12. Refolding and Purification of Yeast Acetyl-CoA Carboxylases CT Domain Expressed as Inclusion Bodies in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    YANG Xue-ying; TAO Jin; ZHENG Liang-yu; WANG Rui-jian; ZHAO Ke; CAO Shu-gui

    2009-01-01

    Acetyl-CoA carboxylase(ACCase) is a crucial enzyme in fatty acid synthesis, by regulating the first committed step in the process. Therefore, it is a potential target for the development of new compounds against obesity or as herbicides. The cDNA encoding yeast ACCase CT domains(YCTs) from Saccharomyces cerevisiae was amplified by RT-PCR and inserted into the vector PET28a(+) for bacterial expression of YCT fused to N-terminal His-tag(YCT-his6). YCTs-his6 was expressed in Escherichia coli BL21(DE3) Plys as inclusion bodies, which was solubilized in 8 mol/L urea. Ni-agarose chromatography was used to purify the inclusion bodies under denaturing condition. Correct refolding was achieved via systematic dialysis to remove the denaturant gently in the presence of 0.5 mmol/L Triton X-100. The low concentration Triton X-100 was included in the refolding buffer to increase the solubilization and enhance dimeric formation of refolding proteins. The activity of the refolded YCT-his6 was 1.2 U/mg as measured in a spectrophotometric assay using malonyl-CoA as the substrate. To our knowledge, it is the first time that the bioactive YCT-his6 has been expressed successfully in E. Coli and isolated from their inclusion bodies.

  13. Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants.

    Science.gov (United States)

    Lin, Lin; Luo, Zhaopeng; Yan, Fei; Lu, Yuwen; Zheng, Hongying; Chen, Jianping

    2011-08-01

    The P3 protein encoded by Shallot yellow stripe virus onion isolate (SYSV-O) interacted in the Yeast Two-hybrid (Y2H) system and in co-immunoprecipitation (Co-IP) assays with the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein that is encoded by the rbcL gene of its onion host. Dissection analysis by Y2H showed that the main part of SYSV P3 (amino acids 1-390) and onion RbcL (amino acids 1-137) were responsible for the interaction. The P3 proteins encoded by Onion yellow dwarf virus (OYDV), Soybean mosaic virus Pinellia isolate (SMV-P), and Turnip mosaic virus (TuMV) also interacted with RbcL, suggesting that a P3/RbcL interaction might exist generally for potyviruses. An interaction between P3 of these potyviruses and the small subunit of RubisCO (RbcS) was also demonstrated. Moreover, the P3N-PIPO protein encoded by a newly identified open reading frame embedded within the P3 cistron also interacted with both RbcL and RbcS. It is possible that the potyvirus P3 protein affects the normal functions of RubisCO which thus contributes to symptom development.

  14. Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

    Institute of Scientific and Technical Information of China (English)

    Ben-Hua JI; Hong-He TAN; Rong ZHOU; De-Mao JIAO; Yun-Gang SHEN

    2005-01-01

    Spraying a 1-2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.)Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%,respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes.

  15. 3D-QSAR and molecular docking analysis of (4-piperidinyl-piperazines as acetyl-CoA carboxylases inhibitors

    Directory of Open Access Journals (Sweden)

    Udghosh Singh

    2017-02-01

    Full Text Available Acetyl-CoA carboxylase (ACC is a crucial metabolic enzyme, which plays a vital role in fatty acid metabolism and obesity induced type 2 diabetes. Herein, we have performed 3D-QSAR and molecular docking analysis on a novel series of (4-piperidinyl-piperazines to design potent ACC inhibitors. This study correlates the ACC inhibitory activities of 68 (4-piperidinyl-piperazine derivatives with several stereo-chemical parameters representing steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The CoMFA and CoMSIA models exhibited excellent rncv2 values of 0.974 and 0.985, and rcv2 values of 0.671 and 0.693, respectively. CoMFA predicted rpred2 of 0.910 and CoMSIA predicted rpred2 of 0.963 showed that the predicted values were in good agreement with experimental values. Glide5.5 program was used to explore the binding mode of inhibitors inside the active site of ACC. We have accordingly designed novel ACC inhibitors by utilising the LeapFrog and predicted with excellent inhibitory activity in the developed models.

  16. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    Science.gov (United States)

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-09-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.

  17. Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span.

    Directory of Open Access Journals (Sweden)

    Kotaro Azuma

    Full Text Available Vitamin K is a fat-soluble vitamin that plays important roles in blood coagulation and bone metabolism. One of its functions is as a co-factor for γ-glutamyl carboxylase (Ggcx. Conventional knockout of Ggcx causes death shortly after birth in homozygous mice. We created Ggcx-floxed mice by inserting loxP sequences at the sites flanking exon 6 of Ggcx. By mating these mice with albumin-Cre mice, we generated Ggcx-deficient mice specifically in hepatocytes (Ggcx(Δliver/Δliver mice. In contrast to conventional Ggcx knockout mice, Ggcx(Δliver/Δliver mice had very low activity of Ggcx in the liver and survived several weeks after birth. Furthermore, compared with heterozygous mice (Ggcx(+/Δliver , Ggcx(Δliver/Δliver mice had shorter life spans. Ggcx(Δliver/Δliver mice displayed bleeding diathesis, which was accompanied by decreased activity of coagulation factors II and IX. Ggcx-floxed mice can prove useful in examining Ggcx functions in vivo.

  18. Chemical inhibition of acetyl-CoA carboxylase suppresses self-renewal growth of cancer stem cells

    Science.gov (United States)

    Corominas-Faja, Bruna; Cuyàs, Elisabet; Gumuzio, Juan; Bosch-Barrera, Joaquim; Leis, Olatz; Martin, Ángel G.; Menendez, Javier A.

    2014-01-01

    Cancer stem cells (CSC) may take advantage of the Warburg effect-induced siphoning of metabolic intermediates into de novo fatty acid biosynthesis to increase self-renewal growth. We examined the anti-CSC effects of the antifungal polyketide soraphen A, a specific inhibitor of the first committed step of lipid biosynthesis catalyzed by acetyl-CoA carboxylase (ACACA). The mammosphere formation capability of MCF-7 cells was reduced following treatment with soraphen A in a dose-dependent manner. MCF-7 cells engineered to overexpress the oncogene HER2 (MCF-7/HER2 cells) were 5-fold more sensitive than MCF-7 parental cells to soraphen A-induced reductions in mammosphere-forming efficiency. Soraphen A treatment notably decreased aldehyde dehydrogenase (ALDH)-positive CSC-like cells and impeded the HER2's ability to increase the ALDH+-stem cell population. The following results confirmed that soraphen A-induced suppression of CSC populations occurred through ACACA-driven lipogenesis: a.) exogenous supplementation with supraphysiological concentrations of oleic acid fully rescued mammosphere formation in the presence of soraphen A and b.) mammosphere cultures of MCF-7 cells with stably silenced expression of the cytosolic isoform ACACA1, which specifically participates in de novo lipogenesis, were mostly refractory to soraphen A treatment. Our findings reveal for the first time that ACACA may constitute a previously unrecognized target for novel anti-breast CSC therapies. PMID:25246709

  19. Transcritption regulation of soybean ribulose-1,5-bisphos-phate carboxylase small sub-unit gene by external factors

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Ribulose-1,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4-8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and 0.4% NaCl, respectively, is 2.5-3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accumulated with diurnal variation but easily influenced by light and low temperature.

  20. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    Directory of Open Access Journals (Sweden)

    Roger Huerlimann

    Full Text Available The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT, and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid and in two forms (homomeric and heteromeric. All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa and Chromista (Stramenopiles, Haptophyta and Cryptophyta have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO, Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta. These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was

  1. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  2. Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Parvy

    Full Text Available Fatty acid (FA metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC, the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

  3. A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase

    Science.gov (United States)

    Bozaquel-Morais, Bruno L.; Madeira, Juliana B.; Venâncio, Thiago M.; Pacheco-Rosa, Thiago; Masuda, Claudio A.; Montero-Lomeli, Monica

    2017-01-01

    Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were “transcriptional regulation”, “protein post-translational modifications” and “lipid metabolism”. Further investigation of the “transcriptional regulation” cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators. PMID:28076367

  4. Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system.

    Science.gov (United States)

    Parvy, Jean-Philippe; Napal, Laura; Rubin, Thomas; Poidevin, Mickael; Perrin, Laurent; Wicker-Thomas, Claude; Montagne, Jacques

    2012-01-01

    Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

  5. 依赖生物素的羧化酶的结构研究进展%Advances in structural studies of biotin-dependent carboxylases

    Institute of Scientific and Technical Information of China (English)

    樊晨; 向嵩

    2013-01-01

    依赖生物素的羧化酶羧化形式多样的底物分子,在多个代谢途径中发挥重要的功能.在它们催化的反应中,生物素充当羧基转运的载体,它们的Biotin Carboxylase(BC)和CarboxylTransferase(CT)结构域催化反应的两个步骤,生物素的羧化和羧基由生物素向底物分子的转移.近期一系列对它们结构的研究揭示了BC和CT结构域催化反应的机制,也为理解羧基在反应中的转运过程提供了线索,极大地深化了对这些酶功能机理的认识.对这方面研究的近期进展做一概述.%Biotin-dependent carboxylases carboxylate a wide range of molecules, playing important roles in several metabolic pathways. In the carboxylation reactions catalyzed by these enzymes, biotin acts as a carboxyl carrier, their Biotin Carboxylase (BC) and CarboxylTransferase (CT) domains catalyze two steps of the reaction, carboxylation of biotin and transfer of the carboxyl group from biotin to the substrate molecule. Recent structural studies provided significant insights into the mechanism of the reactions catalyzed by the BC and CT domains, and the carboxyl transportation process, greatly advanced the understanding of these enzymes' function. Here we briefly summarize recent progresses in this area.

  6. Alterations in barley ribulose-1,5-bisphosphate carboxylase/oxygenase activase gene expression during development and in response to illumination.

    Science.gov (United States)

    Rundle, S J; Zielinski, R E

    1991-08-05

    Two genes encode Rbu-P2-carboxylase activase in barley (RcaA and RcaB): RcaA encodes polypeptides of 46 and 42 kDa, which are generated by the alternatively spliced RcaA1 and RcaA2 mRNAs, respectively; RcaB encodes a 42-kDa polypeptide (Rundle, S. J., and Zielinski, R. E. (1991) J. Biol. Chem. 266, 4677-4685). In the cellular differentiation gradient of the first leaf of barley, the three Rca mRNAs accumulate differentially. RcaA1 and A2 mRNAs accumulate predominantly in the mature, most photosynthetically active regions of the leaf in a pattern that parallels accumulation of total Rbu-P2-carboxylase activase protein. However, the kinetics of accumulation of RcaA1 and RcaA2 mRNA differ slightly, indicating that either changes in RcaA pre-mRNA splicing or mRNA turnover occur during development. RcaB mRNA, in contrast, accumulates in the youngest and oldest cell populations at the base and tip of the leaf, respectively. In the mid-region of the leaf, the difference in accumulation between RcaA and RcaB mRNAs is largely attributable to differences in the rates of transcription of the two Rca genes. In this region of the leaf, the three Rca mRNAs accumulate differentially throughout the course of the diurnal cycle. Steady state levels of the three Rca mRNA species increase in parallel in response to increasing irradiance; these changes were accompanied by increased Rbu-P2-carboxylase activase protein accumulation.

  7. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    Energy Technology Data Exchange (ETDEWEB)

    Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  8. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    Science.gov (United States)

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-12-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

  9. The cyclic keto-enol insecticide spirotetramat inhibits insect and spider mite acetyl-CoA carboxylases by interfering with the carboxyltransferase partial reaction.

    Science.gov (United States)

    Lümmen, Peter; Khajehali, Jahangir; Luther, Kai; Van Leeuwen, Thomas

    2014-12-01

    Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda, as well as the spider mite (Tetranychus urticae) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families.

  10. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of β cells and reduction of pyruvate carboxylase expression.

    Science.gov (United States)

    Abd El Latif, Amira; El Bialy, Badr El Said; Mahboub, Hamada Dahi; Abd Eldaim, Mabrouk Attia

    2014-10-01

    Moringa oleifera Lam. contains many active ingredients with nutritional and medicinal values. It is commonly used in folk medicine as an antidiabetic agent. The present study was designed to investigate how an aqueous extract from the leaves of M. oleifera reveals hypoglycemia in diabetic rats. M. oleifera leaf extract counteracted the alloxan-induced diabetic effects in rats as it normalized the elevated serum levels of glucose, triglycerides, cholesterol, and malondialdehyde, and normalized mRNA expression of the gluconeogenic enzyme pyruvate carboxylase in hepatic tissues. It also increased live body weight gain and normalized the reduced mRNA expression of fatty acid synthase in the liver of diabetic rats. Moreover, it restored the normal histological structure of the liver and pancreas damaged by alloxan in diabetic rats. This study revealed that the aqueous extract of M. oleifera leaves possesses potent hypoglycemic effects through the normalization of elevated hepatic pyruvate carboxylase enzyme and regeneration of damaged hepatocytes and pancreatic β cells via its antioxidant properties.

  11. The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

    Science.gov (United States)

    Dalziel, Katie J; O'Leary, Brendan; Brikis, Carolyne; Rao, Srinath K; She, Yi-Min; Cyr, Terry; Plaxton, William C

    2012-04-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

  12. Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

    Science.gov (United States)

    Paulus, Judith Katharina; Förster, Kerstin; Groth, Georg

    2014-06-05

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds.

  13. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    Science.gov (United States)

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  14. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

    Science.gov (United States)

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-05-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

  15. Cloning and Characterization of a Pyruvate Carboxylase Gene from Penicillium rubens and Overexpression of the Genein the Yeast Yarrowia lipolytica for Enhanced Citric Acid Production.

    Science.gov (United States)

    Fu, Ge-Yi; Lu, Yi; Chi, Zhe; Liu, Guang-Lei; Zhao, Shou-Feng; Jiang, Hong; Chi, Zhen-Ming

    2016-02-01

    In this study, a pyruvate carboxylase gene (PYC1) from a marine fungus Penicillium rubens I607 was cloned and characterized. ORF of the gene (accession number: KM397349.1) had 3534 bp encoding 1177 amino acids with a molecular weight of 127.531 kDa and a PI of 6.20. The promoter of the gene was located at -1200 bp and contained a TATAA box, several CAAT boxes and a sequence 5'-SYGGRG-3'. The PYC1 deduced from the gene had no signal peptide, was a homotetramer (α4), and had the four functional domains. After expression of the PYC1 gene from the marine fungus in the marine-derived yeast Yarrowia lipolytica SWJ-1b, the transformant PR32 obtained had much higher specific pyruvate carboxylase activity (0.53 U/mg) than Y. lipolytica SWJ-1b (0.07 U/mg), and the PYC1 gene expression (133.8%) and citric acid production (70.2 g/l) by the transformant PR32 were also greatly enhanced compared to those (100 % and 27.3 g/l) by Y. lipolytica SWJ-1b. When glucose concentration in the medium was 60.0 g/l, citric acid (CA) concentration formed by the transformant PR32 was 36.1 g/l, leading to conversion of 62.1% of glucose into CA. During a 10-l fed-batch fermentation, the final concentration of CA was 111.1 ± 1.3 g/l, the yield was 0.93 g/g, the productivity was 0.46 g/l/h, and only 1.72 g/l reducing sugar was left in the fermented medium within 240 h. HPLC analysis showed that most of the fermentation products were CA. However, minor malic acid and other unknown products also existed in the culture.

  16. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    Science.gov (United States)

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues. PMID:27194739

  17. The effect of nitrogen limitation on acetyl-CoA carboxylase expression and fatty acid content in Chromera velia and Isochrysis aff. galbana (TISO).

    Science.gov (United States)

    Huerlimann, Roger; Steinig, Eike J; Loxton, Heather; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2014-06-15

    Lipids from microalgae have become a valuable product with applications ranging from biofuels to human nutrition. While changes in fatty acid (FA) content and composition under nitrogen limitation are well documented, the involved molecular mechanisms are poorly understood. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the FA synthesis and elongation pathway. Plastidial and cytosolic ACCases provide malonyl-CoA for de novo FA synthesis in the plastid and FA elongation in the endoplasmic reticulum, respectively. The present study aimed at investigating the expression of plastidial and cytosolic ACCase in Chromera velia and Isochrysis aff. galbana (TISO) and their impact on FA content and elongation level when grown under nitrogen-deplete conditions. In C. velia, plastidial ACCase was significantly upregulated during nitrogen starvation and with culture age, strongly correlating with increased FA content. Conversely, plastidial ACCase of I. aff. galbana was not differentially expressed in nitrogen-deplete cultures, but upregulated during the logarithmic phase of nitrogen-replete cultures. In contrast to plastidial ACCase, the cytosolic ACCase of C. velia was downregulated with culture age and nitrogen-starvation, strongly correlating with an increase in medium-chain FAs. In conclusion, the expression of plastidial and cytosolic ACCase changed with growth phase and nutrient status in a species-specific manner and nitrogen limitation did not always result in FA accumulation.

  18. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    Science.gov (United States)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  19. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

    Institute of Scientific and Technical Information of China (English)

    ZENG Yonghui; JIAO Nianzhi; CAI Haiyuan; CHEN Xihan; WEI Chaoling

    2004-01-01

    Phylogenetic diversity of Form I and Form Ⅱ ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer sets were designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries were constructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The results show that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while that of Form Ⅱ is higher at the inshore station where salinity is lower and productivity is higher. Several clusters of sequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for genetic diversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

  20. Light inhibition of mitochondrial respiration in a mutant of Chlamydomonas reinhardtii devoid of ribulose-1,5-bisphosphate carboxylase/oxygenase activity.

    Science.gov (United States)

    Gans, P; Rebeille, F

    1988-01-01

    The effect of light on mitochondrial respiration has been investigated in Chlamydomonas reinhardtii rcl-u-1-10-6C, a mutant devoid of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. No CO2 uptake was observed in the light, confirming that there was no Rubisco activity, but the CO2 evolution rate was diminished by 65 to 80%. This inhibition was ascribable to a decrease in the tricarboxylic acid cycle (Krebs cycle) activity. At the same time, O2 evolution associated with stimulation of the O2 uptake appears. Darkness or addition of DCMU fully reversed the effect of light, indicating that the inhibitory process is linked to photosystem activities. Levels of pyridine nucleotides (NAD(H) and NADP(H)) and adenine nucleotides (ATP and ADP), the most probable mediators of the interaction between photosynthesis and respiration, were measured in dark and in light. During a dark to light transition the level of NADPH increased significantly whereas the NAD(H) pool remained almost fully oxidized. The level of ADP was always extremely low. These results suggest that the inhibition of Krebs cycle activity is due to a competition for cytosolic ADP between chloroplastic photophosphorylations and oxidative phosphorylations.

  1. Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2.

    Science.gov (United States)

    Kampe, Kapil; Sieber, Jonas; Orellana, Jana Marina; Mundel, Peter; Jehle, Andreas Werner

    2014-02-15

    Type 2 diabetes is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are susceptible to saturated FFAs, which induce endoplasmic reticulum (ER) stress and podocyte death. Genome-wide association studies indicate that expression of acetyl-CoA carboxylase (ACC) 2, a key enzyme of fatty acid oxidation (FAO), is associated with proteinuria in type 2 diabetes. Here, we show that stimulation of FAO by aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR) or by adiponectin, activators of the low-energy sensor AMP-activated protein kinase (AMPK), protects from palmitic acid-induced podocyte death. Conversely, inhibition of carnitine palmitoyltransferase (CPT-1), the rate-limiting enzyme of FAO and downstream target of AMPK, augments palmitic acid toxicity and impedes the protective AICAR effect. Etomoxir blocked the AICAR-induced FAO measured with tritium-labeled palmitic acid. The beneficial effect of AICAR was associated with a reduction of ER stress, and it was markedly reduced in ACC-1/-2 double-silenced podocytes. In conclusion, the stimulation of FAO by modulating the AMPK-ACC-CPT-1 pathway may be part of a protective mechanism against saturated FFAs that drive podocyte death. Further studies are needed to investigate the potentially novel therapeutic implications of these findings.

  2. Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl-coenzyme A carboxylase.

    Science.gov (United States)

    Karatolos, N; Williamson, M S; Denholm, I; Gorman, K; ffrench-Constant, R; Nauen, R

    2012-06-01

    Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

  3. Electrophoretic assay for ribulose 1,5-bisphosphate carboxylase/oxygenase in guard cells and other leaf cells of Vicia faba L

    Energy Technology Data Exchange (ETDEWEB)

    Tarczynski, M.C.; Outlaw, W.H. Jr.; Arold, N.; Neuhoff, V.; Hampp, R. (Florida State Univ., Tallahassee (USA) Max-Planck-Institute fuer Experimentelle Medizin, Goettingen (West Germany) Universitaet Tuebingen (West Germany))

    1989-04-01

    The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.

  4. Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica.

    Science.gov (United States)

    Yovkova, Venelina; Otto, Christina; Aurich, Andreas; Mauersberger, Stephan; Barth, Gerold

    2014-03-01

    To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

  5. Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

    Science.gov (United States)

    Bogdanova, Vera S; Zaytseva, Olga O; Mglinets, Anatoliy V; Shatskaya, Natalia V; Kosterin, Oleg E; Vasiliev, Gennadiy V

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

  6. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Kung, Daniel W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Esler, William P. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Amor, Paul A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Bagley, Scott W. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Beysen, Carine [KineMed Inc., Emeryville, CA (United States); Carvajal-Gonzalez, Santos [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Doran, Shawn D. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Limberakis, Chris [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Mathiowetz, Alan M. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); McPherson, Kirk [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Price, David A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Ravussin, Eric [Louisiana State Univ., Baton Rouge, LA (United States); Sonnenberg, Gabriele E. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Southers, James A. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Sweet, Laurel J. [Pfizer Worldwide Research and Development, Cambridge, MA (United States); Turner, Scott M. [KineMed Inc., Emeryville, CA (United States); Vajdos, Felix F. [Pfizer Worldwide Research and Development, Cambridge, MA (United States)

    2014-12-26

    We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.

  7. The glossyhead1 allele of acc1 reveals a principal role for multidomain acetyl-coenzyme a carboxylase in the biosynthesis of cuticular waxes by Arabidopsis

    KAUST Repository

    Lu, Shiyou

    2011-09-23

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C 20:0 or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling. © 2011 American Society of Plant Biologists. All Rights Reserved.

  8. Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

    Science.gov (United States)

    Ding, Geng; Che, Ping; Ilarslan, Hilal; Wurtele, Eve S; Nikolau, Basil J

    2012-05-01

    3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

  9. Maternal obesity reduces milk lipid production in lactating mice by inhibiting acetyl-CoA carboxylase and impairing fatty acid synthesis.

    Directory of Open Access Journals (Sweden)

    Jessica L Saben

    Full Text Available Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC, and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln mice. Mammary gland levels of AMP-activated protein kinase (AMPK, which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.

  10. Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.

    Science.gov (United States)

    Xu, Wenxin; Zhou, You; Chollet, Raymond

    2003-05-01

    Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.

  11. Evaluation of pharmacokinetic/pharmacodynamic relationships of PD-0162819, a biotin carboxylase inhibitor representing a new class of antibacterial compounds, using in vitro infection models.

    Science.gov (United States)

    Ogden, Adam; Kuhn, Michael; Dority, Michael; Buist, Susan; Mehrens, Shawn; Zhu, Tong; Xiao, Deqing; Miller, J Richard; Hanna, Debra

    2012-01-01

    The present study investigated the pharmacokinetic/pharmacodynamic (PK/PD) relationships of a prototype biotin carboxylase (BC) inhibitor, PD-0162819, against Haemophilus influenzae 3113 in static concentration time-kill (SCTK) and one-compartment chemostat in vitro infection models. H. influenzae 3113 was exposed to PD-0162819 concentrations of 0.5 to 16× the MIC (MIC = 0.125 μg/ml) and area-under-the-curve (AUC)/MIC ratios of 1 to 1,100 in SCTK and chemostat experiments, respectively. Serial samples were collected over 24 h. For efficacy driver analysis, a sigmoid maximum-effect (E(max)) model was fitted to the relationship between bacterial density changes over 24 h and corresponding PK/PD indices. A semimechanistic PK/PD model describing the time course of bacterial growth and death was developed. The AUC/MIC ratio best explained efficacy (r(2) = 0.95) compared to the peak drug concentration (C(max))/MIC ratio (r(2) = 0.76) and time above the MIC (T>MIC) (r(2) = 0.88). Static effects and 99.9% killing were achieved at AUC/MIC values of 500 and 600, respectively. For time course analysis, the net bacterial growth rate constant, maximum bacterial density, and maximum kill rate constant were similar in SCTK and chemostat studies, but PD-0162819 was more potent in SCTK than in the chemostat (50% effective concentration [EC(50)] = 0.046 versus 0.34 μg/ml). In conclusion, basic PK/PD relationships for PD-0162819 were established using in vitro dynamic systems. Although the bacterial growth parameters and maximum drug effects were similar in SCTK and the chemostat system, PD-0162819 appeared to be more potent in SCTK, illustrating the importance of understanding the differences in preclinical models. Additional studies are needed to determine the in vivo relevance of these results.

  12. Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate.

    Science.gov (United States)

    Del Prete, Sonia; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T; Carginale, Vincenzo

    2016-01-15

    With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.

  13. Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria.

    Science.gov (United States)

    Park, Joonho; Khuu, Nicholas; Howard, Alexander S M; Mullen, Robert T; Plaxton, William C

    2012-07-01

    Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.

  14. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Shiro Maeda

    2010-02-01

    Full Text Available It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4x10(-6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33-1.96. The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8, odds ratio = 1.61, 95% Cl: 1.35-1.91. Rs2268388 was also associated with type 2 diabetes-associated end-stage renal disease (ESRD in European Americans (p = 6 x 10(-4, odds ratio = 1.61, 95% Cl: 1.22-2.13. Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

  15. Transcriptional regulation of acetyl-CoA carboxylase α isoforms in dairy ewes during conjugated linoleic acid induced milk fat depression.

    Science.gov (United States)

    Ticiani, E; Urio, M; Ferreira, R; Harvatine, K J; De Oliveira, D E

    2016-10-01

    Feeding trans-10, cis-12 CLA to lactating ewes reduces milk fat by down-regulating expression of enzymes involved in lipid synthesis in the mammary gland and increases adipose tissue lipogenesis. Acetyl-CoA carboxylase α (ACC-α) is a key regulated enzyme in de novo fatty acid synthesis and is decreased by CLA. In the ovine, the ACC-α gene is expressed from three tissue-specific promoters (PI, PII and PIII). This study evaluated promoter-specific ACC-α expression in mammary and adipose tissue of lactating cross-bred Lacaune/Texel ewes during milk fat depression induced by rumen-unprotected trans-10, cis-12 CLA supplement. In all, 12 ewes arranged in a completely randomized design were fed during early, mid and late lactation one of the following treatments for 14 days: Control (forage+0.9 kg of concentrate on a dry matter basis) and CLA (forage+0.9 kg of concentrate+27 g/day of CLA (29.9% trans-10, cis-12)). Mammary gland and adipose tissue biopsies were taken on day 14 for gene expression analysis by real-time PCR. Milk fat yield and concentration were reduced with CLA supplementation by 27%, 21% and 35% and 28%, 26% and 42% during early, mid and late lactation, respectively. Overall, our results suggest that trans-10, cis-12 CLA down-regulates mammary ACC-α gene expression by decreasing expression from PII and PIII in mammary gland and up-regulates adipose ACC-α gene expression by increasing expression from PI.

  16. Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

    Science.gov (United States)

    Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

    2014-03-01

    Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

  17. Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca(2+).

    Science.gov (United States)

    Qian, Baoyun; Li, Xia; Liu, Xiaolong; Chen, Pingbo; Ren, Chengang; Dai, Chuanchao

    2015-03-01

    We determined the effects of endogenous nitric oxide and Ca(2+) on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca(2+) involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

  18. Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

    Science.gov (United States)

    O'Leary, Brendan; Fedosejevs, Eric T; Hill, Allyson T; Bettridge, James; Park, Joonho; Rao, Srinath K; Leach, Craig A; Plaxton, William C

    2011-11-01

    This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.

  19. Continuous fat oxidation in acetyl–CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity

    Science.gov (United States)

    Choi, Cheol Soo; Savage, David B.; Abu-Elheiga, Lutfi; Liu, Zhen-Xiang; Kim, Sheene; Kulkarni, Ameya; Distefano, Alberto; Hwang, Yu-Jin; Reznick, Richard M.; Codella, Roberto; Zhang, Dongyan; Cline, Gary W.; Wakil, Salih J.; Shulman, Gerald I.

    2007-01-01

    Acetyl–CoA carboxylase 2 (ACC)2 is a key regulator of mitochondrial fat oxidation. To examine the impact of ACC2 deletion on whole-body energy metabolism, we measured changes in substrate oxidation and total energy expenditure in Acc2−/− and WT control mice fed either regular or high-fat diets. To determine insulin action in vivo, we also measured whole-body insulin-stimulated liver and muscle glucose metabolism during a hyperinsulinemic–euglycemic clamp in Acc2−/− and WT control mice fed a high-fat diet. Contrary to previous studies that have suggested that increased fat oxidation might result in lower glucose oxidation, both fat and carbohydrate oxidation were simultaneously increased in Acc2−/− mice. This increase in both fat and carbohydrate oxidation resulted in an increase in total energy expenditure, reductions in fat and lean body mass and prevention from diet-induced obesity. Furthermore, Acc2−/− mice were protected from fat-induced peripheral and hepatic insulin resistance. These improvements in insulin-stimulated glucose metabolism were associated with reduced diacylglycerol content in muscle and liver, decreased PKCθ activity in muscle and PKCε activity in liver, and increased insulin-stimulated Akt2 activity in these tissues. Taken together with previous work demonstrating that Acc2−/− mice have a normal lifespan, these data suggest that Acc2 inhibition is a viable therapeutic option for the treatment of obesity and type 2 diabetes. PMID:17923673

  20. Characterization of acetyl-CoA and propionyl-CoA carboxylases encoded by Leptospira interrogans serovar Lai: an initial biochemical study for leptospiral gluconeogenesis via anaplerotic CO2 assimilation

    Institute of Scientific and Technical Information of China (English)

    Nanqiu Peng; Yi Zhong; Qing Zhang; Mingyue Zheng; Wei Zhao; Hualiang Jiang; Chen Yang; Xiaokui Guo; Guoping Zhao

    2012-01-01

    Leptospira interrogans is the causative agent of leptospirosis.The in vitro growth of L.interrogans requires CO2 and a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases was suggested by genomic analysis to assimilate CO2.Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both acetyl-CoA carboxylase (ACC)and propionyl-CoA carboxylase (PCC) activities.The trisubunit holoenzyme (LA_2736-LA_2735 and LA_3803),although failed to be purified,was designated ACC based on its substrate preference toward acetyl-CoA.The partially purified bi-subunit holoenzyme (LA_2432-LA_2433) has a considerably higher activity against propionyi-CoA as the substrate than that of acetyl-CoA,and thus,designated PCC.Native polyacrylamide gel electrophoresis indicated that this PCC has a molecular mass of around 669 kDa,suggesting an α4β4 quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit (LA_2433) indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination.Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypropionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly.However,as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCulloughJohnson-Harris minimal medium,further systematic analysis is required to unveil the mechanism of gluconeogenesis via anaplerotic CO2 assimilation in Leptospira species.

  1. Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependent protein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds.

    Science.gov (United States)

    Hill, Allyson T; Ying, Sheng; Plaxton, William C

    2014-02-15

    The aim of the present study was to characterize the native protein kinase [BTPC (bacterial-type phosphoenolpyruvate carboxylase)-K (BTPC Ser451 kinase)] that in vivo phosphorylates Ser451 of the BTPC subunits of an unusual Class-2 PEP (phosphoenolpyruvate) carboxylase hetero-octameric complex of developing COS (castor oil seeds). COS BTPC-K was highly purified by PEG fractionation and hydrophobic size-exclusion anion-exchange and affinity chromatographies. BTPC-K phosphorylated BTPC strictly at Ser451 (Km=1.0 μM; pH optimum=7.3), a conserved target residue occurring within an intrinsically disordered region, as well as the protein histone III-S (Km=1.7 μM), but not a COS plant-type PEP carboxylase or sucrose synthase or α-casein. Its activity was Ca2+- (K0.5=2.7 μM) and ATP- (Km=6.6 μM) dependent, and markedly inhibited by trifluoperazine, 3-phosphoglycerate and PEP, but insensitive to calmodulin or 14-3-3 proteins. BTPC-K exhibited a native molecular mass of ~63 kDa and was soluble rather than membrane-bound. Inactivation and reactivation occurred upon BTPC-K's incubation with GSSG and then DTT respectively. Ser451 phosphorylation by BTPC-K inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP and 10 mM L-malate). Our collective results indicate a possible link between cytosolic Ca2+ signalling and anaplerotic flux control in developing COS.

  2. Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton QB protein: Phylogenetic implications

    Science.gov (United States)

    Reith, Michael; Cattolico, Rose Ann

    1986-01-01

    The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton QB protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa. Images PMID:16578794

  3. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

    Science.gov (United States)

    Houtz, Robert L.

    2001-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  4. Role of a novel I1781T mutation and other mechanisms in conferring resistance to acetyl-CoA carboxylase inhibiting herbicides in a black-grass population.

    Directory of Open Access Journals (Sweden)

    Shiv Shankhar Kaundun

    Full Text Available BACKGROUND: Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2. METHODOLOGY/PRINCIPAL FINDINGS: Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6 and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. CONCLUSION/SIGNIFICANCE: This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some

  5. Large structures at high resolution: the 1.6 A crystal structure of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol bisphosphate.

    Science.gov (United States)

    Andersson, I

    1996-05-31

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) from spinach is a hexadecamer (L8S8, Mr = 550,000) consisting of eight large (L, 475 residues) and eight small subunits (S, 123 residues). High-resolution data collection on crystals with large unit cells is not a trivial task due to the effect of radiation damage and the large number of overlapping reflections when conventional data collection methods are used. In order to minimise these effects, data on rubisco were collected with a giant Weissenberg camera at long crystal to image-plate distances at the synchrotron of the Photon Factory, Japan. Relative to conventional data sets, this experimental arrangement allowed a 20 to 30-fold reduction of the X-ray dose/exposure time for data collection. This paper describes the refined 1.6 A crystal structure of activated rubisco complexed with a transition state analogue, 2-carboxyarabinitol-bisphosphate. The crystallographic asymmetric unit contains an L4S4 unit, representing half of the molecule. The structure presented here is currently the highest resolution structure for any protein of comparable size. Refinement of the model was carried out by restrained least squares techniques without non-crystallographic symmetry averaging. The results show that all L and S subunits have identical three-dimensional structures, and their arrangement within the hexadecamer has no intrinsic asymmetry. A detailed analysis of the high-resolution maps identified 30 differences in the sequence of the small subunit, indicating a larger than usual heterogeneity for this nuclear encoded protein in spinach. No such differences were found in the sequence of the chloroplast encoded large subunit. The transition state analogue is in the cis conformation at the active site suggesting a key role for the carbamate of Lys201 in catalysis. Analysis of the active site around the catalytically essential magnesium ion further indicates that residues in the second liganding sphere of the metal

  6. MEDICA 16 inhibits hepatic acetyl-CoA carboxylase and reduces plasma triacylglycerol levels in insulin-resistant JCR: LA-cp rats.

    Science.gov (United States)

    Atkinson, Laura L; Kelly, Sandra E; Russell, James C; Bar-Tana, Jacob; Lopaschuk, Gary D

    2002-05-01

    Intracellular triacylglycerol (TG) content of liver and skeletal muscle contributes to insulin resistance, and a significant correlation exists between TG content and the development of insulin resistance. Because acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme for liver fatty acid biosynthesis and a key regulator of muscle fatty acid oxidation, we examined whether ACC plays a role in the accumulation of intracellular TG. We also determined the potential role of 5'-AMP-activated protein kinase (AMPK) in this process, since it can phosphorylate and inhibit ACC activity in both liver and muscle. TG content, ACC, and AMPK were examined in the liver and skeletal muscle of insulin-resistant JCR:LA-cp rats during the time frame when insulin resistance develops. At 12 weeks of age, there was a threefold elevation in liver TG content and a sevenfold elevation in skeletal muscle TG content. Hepatic ACC activity was significantly elevated in 12-week-old JCR:LA-cp rats compared with lean age-matched controls (8.75 +/- 0.53 vs. 3.30 +/- 0.18 nmol. min(-1). mg(-1), respectively), even though AMPK activity was also increased. The observed increase in hepatic ACC activity was accompanied by a 300% increase in ACC protein expression. There were no significant differences in ACC activity, ACC protein expression, or AMPK activity in the skeletal muscle of the 12-week JCR:LA-cp rats. Treatment of 12-week JCR:LA-cp rats with MEDICA 16 (an ATP-citrate lyase inhibitor) resulted in a decrease in hepatic ACC and AMPK activities, but had no effect on skeletal muscle ACC and AMPK. Our data suggest that alterations in ACC or AMPK activity in muscle do not contribute to the development of insulin resistance. However, increased liver ACC activity in the JCR:LA-cp rat appears to contribute to the development of lipid abnormalities, although this increase does not appear to occur secondary to a decrease in AMPK activity.

  7. Functions of plant phosphoenolpyruvate carboxylase and its applications for genetic engineering%植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用

    Institute of Scientific and Technical Information of China (English)

    魏绍巍; 黎茵

    2011-01-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is an important ubiquitous cytosol enzyme that fixes HCO3 together with phosphoenolpyruvate (PEP) and yields oxaloacetate that can be converted to intermediates of the citric acid cycle. In plant cells, PEPC participates in CO2 assimilation and other important metabolic pathways, and it has broad functions in different plant tissues. PEPC is also involved in the regulation of storage product synthesis and metabolism in seeds, such as affecting the metabolic fluxes from sugars/starch towards the synthesis of fatty acids or amino acids and proteins. In this review, we introduced the progress in classification, structure and regulation of PEPC in plant tissues. We discussed the potential applications of plant PEPCs in genetic engineering. The researches in functions and regulationmechanism of plant PEPCs will provide beneficial approaches to applications of plant PEPCs in high-yield crops breeding, energy crop and microbe genetic engineering.%植物磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)是广泛存在的一种细胞质酶,催化磷酸烯醇式丙酮酸(PEP)和HCO3-生成草酰乙酸(OAA),后者可转化生成三羧酸循环的多种中间产物.PEPC在植物细胞中参与植物的光合碳同化等重要代谢途径,并且在不同组织中具有多种生理功能.PEPC同时也参与调控植物种子的营养物质合成与代谢过程,控制糖类物质流向脂肪酸合成或蛋白质合成途径.以下介绍了植物PEPC的种类、蛋白质结构特点及其在植物组织中的调控方式,并重点论述了PEPC在生物基因工程中的应用方面的进展,随着对其功能机制和应用研究的深入,将有助于植物PEPC在高产优质农作物育种、能源植物和工业微生物等的开发利用等方面得到更好的发展与应用.

  8. Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene.

    Science.gov (United States)

    Kamimura, Kazuo; Okabayashi, Ai; Kikumoto, Mei; Manchur, Mohammed Abul; Wakai, Satoshi; Kanao, Tadayoshi

    2010-03-01

    Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

  9. Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas.

    Science.gov (United States)

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-08-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.

  10. Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

    Science.gov (United States)

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-01-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes. PMID:16885306

  11. 乙酰辅酶A羧化酶抑制剂的构效关系和抗性研究进展%Advances in structure properties and resistance of acetyl-CoA carboxylase inhibitors

    Institute of Scientific and Technical Information of China (English)

    衣克寒; 付颖; 叶非

    2012-01-01

    乙酰辅酶A羧化酶(ACCase)抑制剂是以乙酰辅酶A羧化酶为作用靶标的一类除草剂.这类除草剂通过抑制真核型乙酰辅酶A生成丙二酰辅酶A的羧化反应,进而抑制植物脂肪酸的合成,多用于苗后有选择性地防除一年生禾本科杂草.本文综述了该类除草剂的作用机理、构效关系及在应用中的抗性研究进展.%Acetyl-CoA carboxylase is one of the targets of ACCase inhibitor herbicides. A carboxylation reaction from an eukaryotic type of acetyl coenzyme A to malonyl coenzyme A can be inhibited by such herbicides, and then, fatty acid biosynthesis could be inhibited. It is used for a selective herbicide to control the annual postemer-gence weeds. The mechanism of action, construction and weed resistance were briefly summarized in this paper.

  12. Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid

    Energy Technology Data Exchange (ETDEWEB)

    Du, Z.; Aghoram, K.; Outlaw, W.H. Jr.

    1996-12-31

    Plants regulate water loss and CO{sub 2} gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard-cell phosphoenolpyruvate carboxylase, which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening. In this study, the authors have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (isolated guard cells) pre-loaded with {sub 32}PO{sub 4}, the authors induced stomatal opening and guard-cell malate accumulation by incubation with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 {micro}M abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA.

  13. Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

    Directory of Open Access Journals (Sweden)

    Nathan E Kreel

    Full Text Available Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363 found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

  14. Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle

    Directory of Open Access Journals (Sweden)

    Shi Xuanming

    2012-06-01

    Full Text Available Abstract Background The enzyme acetyl-CoA carboxylase-alpha (ACC-α is rate limiting for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA is dominantly active in lipogenic tissues. This promoter is in principal repressed but activated under favorable nutritional conditions. Previous analyses already coarsely delineated the repressive elements on the distal promoter but did not resolve the molecular nature of the repressor. Knowledge about the molecular functioning of this repressor is fundamental to understanding the nutrition mediated regulation of PIA activity. We analyzed here the molecular mechanism calibrating PIA activity. Results We finely mapped the repressor binding sites in reporter gene assays and demonstrate together with Electrophoretic Mobility Shift Assays that nuclear factor-Y (NF-Y and CCAAT/enhancer binding protein-β(C/EBPβ each separately repress PIA activity by binding to their cognate low affinity sites, located on distal elements of the promoter. Simultaneous binding of both factors results in strongest repression. Paradoxically, over expression of NFY factors, but also - and even more so - of C/EBPβ significantly activated the promoter when bound to high affinity sites on the proximal promoter. However, co-transfection experiments revealed that NF-Y may eventually diminish the strong stimulatory effect of C/EBPβ at the proximal PIA in a dose dependent fashion. We validated by chromatin immunoprecipitation, that NF-Y and C/EBP factors may physically interact. Conclusion The proximal promoter segment of PIA appears to be principally in an active state, since even minute concentrations of both, NF-Y and C/EBPβ factors can saturate the high affinity activator sites. Higher factor concentrations will saturate the low affinity repressive sites on the distal promoter resulting in reduced and calibrated promoter activity. Based on measurements of the mRNA concentrations of

  15. Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome.

    Science.gov (United States)

    Elsaied, Hosam; Kimura, Hiroyuki; Naganuma, Takeshi

    2002-06-01

    To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no

  16. Influence of the nitrate concentration and source in the incorporation of 14{sub C}O2 by the RuBP-carboxylase from wheat (triticum aestivum) and maize (zea mays); Influencia de la concentracion y fuente de nitrogeno en la incorporacion de 14{sub C}O2 por la RuBp-carboxilasa de trigo (triticum aewtivum) y maiz (zea mays)

    Energy Technology Data Exchange (ETDEWEB)

    Saez Angulo, R. M.; Gines Diaz, M. J.; Garcia Pineda, M. D.

    1982-07-01

    The effect of the concentration and source of nitrogen in the culture media has been studied regarding its influence in the activity of the RuBP-carboxylase from wheat and maize during the first month of development. Wheat and maize has been chosen as plants representatives of two different types of CO{sub 2} assimilation: C3 and M- respectively. Plants have been grown in hydroponic media and under temperature, humidity and nutrient salts control. A negative effect of NH{sub 4} has been observed in the enzymatic activity of wheat seedlings, being this effect more remarkable as NH{sub 4} concentration increases and as long the time of treatment. In our experimental conditions the most favorable source of nitrogen has been N0{sub 3}NH{sub 4}. The specific activity of the enzyme from wheat is about four times higher than in maize, even it decreases with time. This decreasing has not been observed in maize, with the exception of total absence of nitrogen in the media. We have not seen significant differences between the two photo periods which have been tested. Also, no differences have been found in the enzyme activities at the different NO{sub 3}NH{sub 4} concentrations assayed, and it seems that RuBP-carboxylase metabolism is only affected in the case of absolute stress. (Author) 20 refs.

  17. Effects of Sowing Date and Interplanting Patterns on Carboxylase Activity and Grain Yield of Summer Maize%播期与接茬方式对夏玉米羧化酶活性及产量的影响

    Institute of Scientific and Technical Information of China (English)

    党红凯; 李瑞奇; 李雁鸣; 李晓爽; 孟建

    2015-01-01

    旨为明确播期和接茬方式对夏玉米羧化酶活性及产量的影响,同时为确定夏玉米与小麦的适宜接茬方式和播种期提供研究依据。2008,2009年进行了4个播期的夏玉米田间试验,6月1,6,11日为“三密一稀”种植的麦田中套种,6月16日为15 cm等行距小麦收获后平播,分别用S06-01、S06-06、S06-11、S06-16表示。生育期间测定羧化酶活性。结果表明,随播期推迟,夏玉米基部第1~4叶的RuBPCase 和PEPCase 活性增强,PEPC/RuBPC 比值减小。棒3叶PEPCase活性随生育进程呈单峰变化,播期早的叶片展开虽早,PEPCase活性高峰出现也早,但后期下降幅度也较大,以致不同处理棒3叶生育期间PEPCase活性的平均值差异并不显著。随播期推迟,棒3叶同叶位叶片光合势与叶源量有减小的趋势,玉米成穗数有增加趋势。千粒质量变化与灌浆期温度密切相关而与播期关系不大,最终产量以S06-16处理较高。处理间经济系数也以S06-16最高或较高,说明产量形成不仅与光合物质生产能力有关,还与光合物质有效转运相关。综上可见,夏玉米平播既保证与套种相同或较高的产量,又便于小麦玉米生产的全程机械化,是较适宜的接茬方式。但为避开集中平播后的浇水高峰,在不早于小麦收获前9 d(6月6日以后)套播,可为夏收夏种期间的农事操作提供较为充足的时间。%In order to clarify the effect of sowing date and interplanting pattern on carboxylase activity and grain yield of summer maize and to provide research support for decision-making on interplanting pattern and sowing date,field experiments were conducted in the years 2008 and 2009.The experiment included four treatments of maize sowing dates,June 1,6,11 seeded inter-rows of previous wheat ,and June 16 directly-seeded after wheat har-vest,and the four treatments were expressed in S06-01,S06-06,S06

  18. 磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响%Effect of phosphoenolpyruvate carboxylase gene knock-out on physiological metabolism in Corynebacterium glutamicum V1

    Institute of Scientific and Technical Information of China (English)

    仇爱梅; 窦文芳; 李会; 许正宏

    2012-01-01

    [Objective] In order to optimize precursor supply for L-valine biosynthesis, a Corynebacterium glutamicum V1 mutant with phosphoenolpyruvate carboxylase gene (pepc) in-frame deletion was constructed through crossover PCR and homologous recombination. The effect of pepc knock-out on physiological characteristics of the mutant was investigated. [Methods] The upstream and downstream fragments of pepc were cloned from C. glutamicum V1 chromosome and ligated to integration vector. The mutant C. glutamicum V1-Δpepc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and enzymes activity measurement of pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH) and pyruvate kinase (PK). The mutant with pepc gene in-frame deletion was screened and confirmed by PCR and phosphoenolpyruvate carboxylase activity determination. [Results] The pepc knock-out resulted in L-argine accumulation to 7.48 g/L and no accumulation of L-valine, which accompanied by increase of PDH activity and PC activity in C. glutamicum V1-Δpepc. The knock-out of pepc gene affected the metabolism of the strain to some extent. [Conclusion] Blocking the anaplerotic pathway PEPC participated increased TCA cycle, leading to the increase of L-argine and decrease of amino acids with pyruvic acid as precursor , such as L-valine and alanine.%[目的]L-缬氨酸生物合成的前体物质是丙酮酸.为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1 (Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-△pepc,并研究pepc敲除后菌株生理特性的改变.[方法]运用交叉PCR方法得到pepc基因内部缺失的同源片段△pepc,并构建敲除质粒pK18mobsacB-△pepc.利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-△pepc

  19. Acetyl-coenzyme A Carboxylase: A Key Metabolic Enzyme of Fatty Acid and Progress of Its Gene Clone%乙酰辅酶A羧化酶:脂肪酸代谢的关键酶及其基因克隆研究进展

    Institute of Scientific and Technical Information of China (English)

    李洁琼; 郑世学; 喻子牛; 张吉斌

    2011-01-01

    Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism in most living organisms. In this article, structure, types, functions and inhibitors of ACC, as well as research status of ACC gene clone are systematically discussed. ACC is a multi-subunit enzyme in most prokaryotes, whereas it is a large, multi-domain enzyme in most eukaryotes. In addition, there are two special types found from Streptomyces coelicolor and Metallosphaera sedula. All of these types contain three key domains: Biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT). CT domain, as a candidate target, has been widely used for screening of plant herbicides and drug development against obesity, diabetes and other symptoms of the metabolic syndrome. The gene encoded ACC is also becoming an important target gene applied in the fields of transgenic oil plants and biodiesel. Previous studies showed thatβ-CT in plant plasmid was the limit factor of heteromeric ACC, and BCCP was a negative regulator of fatty acid synthesis. Lipid synthesis metabolism is a very complex network, especially feedback inhibition mechanism exists in it. As a result, cloning and expression of ACC gene may increase the activity of ACC in the host, but not necessarily could obviously promote the accumulation of fatty acid. Fig 2, Ref 52%乙酰辅酶A羧化酶( Acetyl-CoA carboxy lase,ACC)在脂肪酸合成和分解代谢中发挥着重要作用.系统介绍了该酶的结构与分类、生物学作用与应用、抑制剂的类型与作用机理以及基因克隆4个方面的进展.ACC在大多数原核生物中为多亚基型酶,而在大多数真核生物中为多功能型单亚基酶,在天蓝色链霉菌和古菌勤奋金属球菌中为另外两种特殊类型;但都具备3个关键的功能域,即生物素羧化酶(BC)、生物素羧基载体蛋白(BCCP)和羧基转移酶(CT).CT功能域作为潜在的靶标广泛应用于植物除草剂的筛选和哺乳

  20. Acetyl-CoA carboxylase expression in the liver of quail with hyperuricemia and abdominal obesity%乙酰辅酶A羧化酶在高尿酸血症合并腹型肥胖鹌鹑肝脏中的表达

    Institute of Scientific and Technical Information of China (English)

    林志健; 张冰; 刘小青

    2012-01-01

    The quail model of hyperuricemia combined with abdominal obesity was induced by high purine diet.Body weight in model group showed no change.Serum uric acid level in model group was increased significantly on 7,14,21,and 28 d( P<0.05 or P<0.01 ).Abdominal fat index in model group increased significantly on 28d.On 7 d and 28 d,serum free fatty acid level was increased significantly.Acetyl-CoA carboxylase( ACC ) protein expression in the liver of model quail was increased as shown by ELISA and immunohistochemisty ( P<0.05 or P<0.01 ),suggesting that the alteration of ACC expression contributes to the pathogenesis of hyperuricemia combined with abdominal obesity.%采用高嘌呤饮食诱导鹌鹑高尿酸血症合并腹型肥胖模型,造模期间模型组鹌鹑体重未见明显变化.模型组鹌鹑第7、14、21、28天血尿酸水平显著升高(P<0.05或P<0.01);第28天腹脂率显著升高;第7、28天血清游离脂肪酸水平显著升高;ELISA和免疫组化均显示第28天肝脏乙酰辅酶A羧化酶(ACC)蛋白表达显著升高(P<0.05或P<0.01),提示ACC表达的改变与高尿酸血症及腹型肥胖的形成有关.

  1. Study of the properties of Ribulose 1,5-biphosphate carboxylase/oxygenase from maize (Zea mays) and wheat (Triticum aestivum) by incorporation of 14{sub C}O2; Estudio de las propiedades de la Ribulosa-1,5-Difosfato Carboxilasa/Oxigenasa de maiz (Zea Mais) y de trigo (Triticum Aestivum), por incorporacion de CO{sub 2} marcado con 14{sub C}O2

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, M. D.; Saez, R. M.

    1982-07-01

    After a bibliographic review of the properties of RuBP-carboxylase/oxygenase, a methodology is described which allows the treatment of a large number of samples for the assay of the enzyme activity. 14{sup C}O{sub 3}HNa is used as a marker for the counting of the incorporated radioactivity as acid insoluble material. 14''CC{sub 2} from the labeled sodium bicarbonate is the species used by the enzyme both as an activator as well as a substrate. The following experiments are described and its results given: Determination of the optimal conditions for the activation of the enzyme; study of the kinetics of the catalytic action; effect of the Mg{sup 2} concentration and determination of the Km{sub (s)} from CO{sub 2} and ribulose 1,5-biphosphate; also determination of the optimum pH at different concentrations of CO{sub 2}2 and Mg{sup 2}. (Author) 64 refs.

  2. Effects of Glucose and Vitamin C Inhabitation on Activities of Acetyl-CoA Carboxylase,Fatty Acid Synthase and Carnitine Palmitoyltransferases Ⅰduring Embryo Development of Carassius auratus gibelio%葡萄糖、维生素 C浸泡对普安银鲫胚胎发育中乙酰辅酶 A羧化酶、脂肪酸合成酶及肉毒碱棕榈酰转移酶Ⅰ活性的影响

    Institute of Scientific and Technical Information of China (English)

    蒋左玉; 姚俊杰; 安苗; 熊铧龙; 朱忠胜

    2014-01-01

    In order to study the changes of activities of acetyl-CoA carboxylase ( ACC ) , fatty acid synthase ( FAS) , and carnitine palmitoyltransferaseⅠ ( CPTⅠ) , and the effects of glucose and vitamin C inhabitation on them during embryo development of Carassius auratus gibelio ( C. auratus) , glucose solution and vitamin C solutions with different concentrations were used for hatching. The concentrations of glucose were 0, 5, 10, 15 and 20 g/L, respectively, and the concentrations of vitamin C were 0, 20, 25, 30 and 35 mg/L, respec-tively. Membrane break time and hatching rate were recorded to decide the optimal concentrations of glucose and vitamin C. Solutions without addition ( control group) and with optimal concentrations of glucose ( glucose group) or vitamin C ( vitamin C group) were used for hatching, and the characteristics of changes of ACC, FAS and CPTⅠactivities were analyzed during embryo development. The results showed as follows:1) mem-brane break time was the shortest, and hatching rate was the highest when the concentrations of glucose and vi-tamin C were 15 g/d and 30 mg/L, respectively. 2) The specific activity and total activity of ACC, FAS and CPTⅠ showed increasing tends during embryo development of C. auratus. 3) The specific activities and total activities of ACC and FAS in glucose group were significantly higher than those in control group at mid-gas-trul, crystal appear and prehatching stages ( P<0.05) , and the specific activity an total activity of CPTⅠ was significantly higher than that in control group at crystal appear and prehatching stages ( P<0.05) . 4) The total activities of ASS and FAS in vitamin C group were significantly higher than those in control group ( P<0.05) . In conclusion, the inhabitation in solutions with appropriate concentrations of glucose ( 15 g/L) and vitamin C ( 30 mg/L) can promote synthesis and secretion of ACC, FAS and CPTⅠ during embryo development of C. auratus, and form new metabolic levels to

  3. Dithiothreitol decreases the thermal stability and unfolding cooperativity of ribulose-1, 5-bisphosphate carboxylase/oxygenase

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Plant rubisco consists of eight large subunits (55 kD) encoded by chloroplast gene and eight small subunits (15 kD) encoded by nuclear gene. There are abundant cysteine residues that do not form disulfide bonds in native rubisco. Differential scanning calorimetry has been used to study some plant rubisco and suggested an irreversible two-state denaturation due to the high cooperativity in subunits. By comparing the data from circular dichroism, fluorescence, differential scanning calorimetry, SDS electrophoresis, and activity assays in the absence or presence of DTT, we suggest that the formation of disulfide bonds in subunits during the early thermal unfolding may increase the thermal stability and the thermal unfolding cooperativity of rubisco.

  4. Biotin deficiency in the rat as a model for reduced pyruvate carboxylase activity

    NARCIS (Netherlands)

    Schrijver, Jacobus

    1978-01-01

    The investigations described in this thesis are a contribution to the study of Leigh's disease (Subacute Necrotizing Encephalomyelopathy, SNE). SNE resembles in neuropathology Wernicke's encephalopathy, which is caused by thiamine deficiency. The scope and the purpose of the present study is given i

  5. Brain glycogenolysis, adrenoceptors, pyruvate carboxylase, Na(+),K(+)-ATPase and Marie E. Gibbs' pioneering learning studies.

    Science.gov (United States)

    Hertz, Leif; Xu, Junnan; Song, Dan; Du, Ting; Yan, Enzhi; Peng, Liang

    2013-01-01

    The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present "perspective" suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially β-adrenergic or (2) even small increases in extracellular K(+) concentration above its normal resting level. It discusses (1) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; (2) the established role of K(+)-stimulated glycogenolysis for K(+) uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K(+) homeostasis in the brain in vivo; and (3) the plausible role of transmitter-induced glycogenolysis, stimulating Na(+),K(+)-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K(+) concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).

  6. Brain Glycogenolysis, Adrenoceptors, Pyruvate Carboxylase, Na+,K+-ATPase and Marie E. Gibbs’ Pioneering Learning Studies

    Directory of Open Access Journals (Sweden)

    Leif eHertz

    2013-04-01

    Full Text Available The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., JCBFM, in press. According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present ‘perspective’ suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either i activation of astrocytic receptors, perhaps especially beta-adrenergic, or ii even small increases in extracellular K+ concentration above its normal resting level. It discusses i the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; ii the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and iii the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP.

  7. Brain glycogenolysis, adrenoceptors, pyruvate carboxylase, Na+,K+-ATPase and Marie E. Gibbs' pioneering learning studies

    OpenAIRE

    Hertz, Leif; Xu, Junnan; SONG, DAN; Du, Ting; Yan, Enzhi; Peng, Liang

    2013-01-01

    The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present “perspective” suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially β-adrener...

  8. Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

    Science.gov (United States)

    Hazra, Suratna; Henderson, J. Nathan; Liles, Kevin; Hilton, Matthew T.; Wachter, Rebekka M.

    2015-01-01

    In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min−1, the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light. PMID:26283786

  9. Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

    Directory of Open Access Journals (Sweden)

    Samy Selim

    2016-03-01

    Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000.

  10. Is L-Carnitine Supplementation Beneficial in 3-Methylcrotonyl-CoA Carboxylase Deficiency?

    DEFF Research Database (Denmark)

    Thomsen, Jákup Andreas; Lund, Allan Meldgaard; Olesen, Jess Have

    2015-01-01

    and muscle tissue with and without L-carnitine supplementation to evaluate the current treatment strategy of not recommending L-carnitine supplementation to Faroese 3-MCCd patients. Methods: Blood and urine samples and muscle biopsies were collected from patients at inclusion and at 3 months. Eight patients...... received L-carnitine supplementation when recruited; five did not. Included patients who received supplementation were asked to stop L-carnitine, the others were asked to initiate L-carnitine supplementation during the study. Symptoms were determined by review of hospital medical records and questionnaires...... carnitine levels, 6.9 (SD 1.4) μmol/L and 785 (SD 301) nmol/g wet weight, respectively. L-Carnitine supplementation increased muscle and plasma carnitine levels to a low-normal range, 25.5 (SD 10.9) μmol/L and 1,827 (SD 523) nmol/g wet weight, p

  11. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian;

    2015-01-01

    Corynebacterium glutamicum, a Gram-positive bacterium used for the production of various biochemicals, is naturally a biotin auxotroph. We introduced the biotin genes from Bacillus subtilis on a plasmid, pBIO, into a lysine-producing derivative (termed AHP-3) that has been described previously......, and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... was cultivated without biotin, indicating a suboptimal intracellular concentration of biotin. In an attempt to locate the potential bottleneck, we added pimelic acid, an early biotin precursor, and found that growth rate could be restored fully, which demonstrates that the bottleneck is in pimeloyl-CoA (or...

  12. Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase: product inhibition, cooperativity, and magnesium activation.

    Science.gov (United States)

    Hazra, Suratna; Henderson, J Nathan; Liles, Kevin; Hilton, Matthew T; Wachter, Rebekka M

    2015-10-01

    In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light.

  13. Sterol Regulatory Element Binding Protein 1a Regulates Hepatic Fatty Acid Partitioning by Activating Acetyl Coenzyme A Carboxylase 2

    OpenAIRE

    Im, Seung-Soon; Hammond, Linda E.; Yousef, Leyla; Nugas-Selby, Cherryl; Shin, Dong-Ju; Seo, Young-Kyo; Fong, Loren G.; Young, Stephen G.; Osborne, Timothy F.

    2009-01-01

    We generated a line of mice in which sterol regulatory element binding protein 1a (SREBP-1a) was specifically inactivated by insertional mutagenesis. Homozygous mutant mice were completely viable despite expressing SREBP-1a mRNA below 5% of normal, and there were minimal effects on expression of either SREBP-1c or -2. Microarray expression studies in liver, where SREBP-1a mRNA is 1/10 the level of the highly similar SREBP-1c, demonstrated that only a few genes were affected. The only downregu...

  14. Electron transport, pep carboxylase activity, and maximal net co2 assimilation exhibit coordinated and proportional decline with loss of hydraulic conductance during water stress in Zea mays

    Science.gov (United States)

    Efforts to improve the photosynthetic performance of species are presently focused on leaf-level traits (e.g., quantum efficiency, mesophyll osmoregulation, stress protein regulation). Here, we emphasize that efforts to improve plant performance in arid environments would benefit from also consider...

  15. Study on the Efficacy of Some Current Herbicides for Control of Resistant and Susceptible Canarygrass (Phalaris spp. Biotypes to Acetyl CoA Carboxylase (ACCase Inhibitors

    Directory of Open Access Journals (Sweden)

    e Zand

    2011-02-01

    Full Text Available Abstract Two separate greenhouse experiments were conducted in the greenhouse facilities of the Iranian Plant Protection Research Institute, Tehran, to study the efficacy of some herbicides to control of resistant and susceptible P. minor and P. paradoxa biotypes. In each experiment, resistant and susceptible biotypes were treated separately by 19 herbicide treatments. Treatments included 10 ACCase inhibitors, 6 Acetolactate Synthase (ALS inhibitors, prosulfocarb, flamprop-M-isopropyl, isoproturon plus diflufenican and a non-sprayed control. To evaluate the effects of treatments, different characteristics including percent damage based on EWRC scores at 15 and 30 days after spraying, percentage of survived plants after spraying relative to before spraying, and percentage of dry weight and wet weight of individual plants relative to control were studied. Results showed that the susceptible biotypes of P. minor were best controlled by clodinafop propargyl and pinoxaden at 450 ml/ha while pinoxaden at 450 ml/ha and cycloxydim were best options for control of the resistant biotype. Among ALS inhibitors, iodosulfuron plus mesosulfuron could control susceptible and resistant biotypes of P. minor very effectively and semi-satisfactory, respectively. Iodosulfuron plus mesosulfuron and sulfosulfuron plus metsulfuron could remarkably reduce the wet weight of individual plants compared to control so that the plants were not damaging any more. Among other herbicides, isoproturon plus diflufenican could control the susceptible and resistant biotypes semi-satisfactory and very effectively, respectively. Keywords: Herbicide resistance, ACCase inhibitors, ALS inhibitors

  16. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Maeda, Shiro; Kobayashi, Masa-aki; Araki, Shin-ichi

    2010-01-01

    It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A ca......It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl...... among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8), odds ratio = 1.61, 95% Cl: 1.35-1.91). Rs2268388 was also associated with type 2 diabetes......-associated end-stage renal disease (ESRD) in European Americans (p = 6 x 10(-4), odds ratio = 1.61, 95% Cl: 1.22-2.13). Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including...

  17. Phylogeny and evolutionary history of Leymus (Triticeae; Poaceae based on a single-copy nuclear gene encoding plastid acetyl-CoA carboxylase

    Directory of Open Access Journals (Sweden)

    Ding Cun-Bang

    2009-10-01

    Full Text Available Abstract Background Single- and low- copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of polyploid taxa. Leymus is a polyploid genus with a diverse array of morphology, ecology and distribution in Triticeae. The genomic constitution of Leymus was assigned as NsXm, where Ns was presumed to be originated from Psathyrostachys, while Xm represented a genome of unknown origin. In addition, little is known about the evolutionary history of Leymus. Here, we investigate the phylogenetic relationship, genome donor, and evolutionary history of Leymus based on a single-copy nuclear Acc1 gene. Results Two homoeologues of the Acc1 gene were isolated from nearly all the sampled Leymus species using allele-specific primer and were analyzed with those from 35 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1 Leymus is closely related to Psathyrostachys, Agropyron, and Eremopyrum; (2 Psathyrostachys juncea is an ancestral Ns-genome donor of Leymus species; (3 the Xm genome in Leymus may be originated from an ancestral lineage of Agropyron and Eremopyrum triticeum; (4 the Acc1 sequences of Leymus species from the Qinghai-Tibetan plateau are evolutionarily distinct; (5 North America Leymus species might originate from colonization via the Bering land bridge; (6 Leymus originated about 11-12MYA in Eurasia, and adaptive radiation might have occurred in Leymus during the period of 3.7-4.3 MYA and 1.7-2.1 MYA. Conclusion Leymus species have allopolyploid origin. It is hypothesized that the adaptive radiation of Leymus species might have been triggered by the recent upliftings of the Qinghai-Tibetan plateau and subsequent climatic oscillations. Adaptive radiation may have promoted the rapid speciation, as well as the fixation of unique morphological characters in Leymus. Our results shed new light on our understanding of the origin of Xm genome, the polyploidization events and evolutionary history of Leymus that could account for the rich diversity and ecological adaptation of Leymus species.

  18. Mechanism and Significance of Post-Translational Modifications in the Large (LS) and Small (SS) Subunits of Ribulose-1,5 Bisphosphate Carboxylase/Oxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Houtz, Robert, L.

    2012-11-09

    This project focused on a molecular and biochemical characterization of the protein methyltransferases responsible for methylation of the LS and SS in Rubisco, and the associated functional consequences accompanying these modifications. Our results provided some of the most informative structural and mechanistic understandings of SET domain protein methyltransferases. These results also positioned us to provide the first unambiguous assignment of the kinetic reaction mechanism for SET-domain protein methyltransferases, and to design and engineer an alternative substrate for Rubisco LSMT, enabling substrate specificity and functional significance studies. We demonstrated that the minimal substrate recognized by Rubisco LSMT is free lysine as well as monomethyllysine, an observation corroborated both by structural analyses as well as enzymatic activity and subsequent product distribution analyses. Ternary complexes between Rubisco LSMT and free lysine compared to complexes with monomethyllysine demonstrated that the structural basis for multiple methyl group additions is a consequence of hydrogen-bond driven spatial shifts in the amino group of Lys-14, which maintains the direct in-line geometry necessary for SN2 nucleophilic attack. The structural observations are also consistent with the previous proposal that the multiplicity of methyl group additions takes place through a processive mechanism, with successive methyl group additions to an enzyme protein complex which does not disassociate prior to the formation of trimethyllysine. This mechanism has important implications, since the regulation of gene expression by SET domain histone methyltransferases is not only dependent on site-specific lysine methylation, but also the degree of methylation. We examined the kinetic reaction mechanism for three different types of SET domain protein methyltransferases, each under conditions supporting mono-, di-, or trimethyllysine formation corroborated by product analyses. Additionally, the tight initial binding of Rubisco LSMT to Rubisco also allowed us to design a novel immobilized complex between Rubisco and Rubisco LSMT, which allowed for an unambiguous demonstration of the requirement for trimethyllysine formation prior to disassociation of the Rubisco LSMT:Rubisco complex, and therefore proof of the processive mechanism for methyl group transfer. These kinetic studies also demonstrated that an important factor has been overlooked in all kinetic analyses of SET domain protein methyltransferases reported to date. This factor is the influence of the low turnover number for SET domain protein methyltransferases and how, relative to the time-frame of kinetic enzyme assays, this can generate changes in kinetic profiles shifting reciprocal plot patterns from random/ordered bi-bi to the real kinetic reaction mechanism plots of ping-pong. Although the ternary complexes of Rubisco LSMT with S-Adenosylhomocysteine and lysine and monomethyllysine were informative in regard to reaction mechanism, they were not helpful in identifying the mechanism used by Rubisco LSMT for determining substrate specificity. We were unsuccessful at obtaining ternary complexes of Rubisco LSMT with bound synthetic polypeptide substrates, as has been reported for several histone methyltransferases. However, we were able to model a polypeptide sequence corresponding to the N-terminal region of the LS of Rubisco into the apparent substrate binding cleft in Rubisco LSMT. Knowledge of the determinants of polypeptide substrate specificity are important for identifying possible alternate substrates, as well as the possibility of generating more desirable substrates amenable to site-directed mutagenesis experiments unlike Rubisco. We determined that Rubisco LSMT is capable of methylating synthetic polypeptide mimics of the N-terminal region of the LS, both free as well as conjugated to keyhole limpet hemacyanin, but with considerable less efficiency than intact holoenzyme.

  19. NCBI nr-aa BLAST: CBRC-HSAP-09-0006 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-HSAP-09-0006 ref|ZP_00960995.1| pyruvate carboxylase [Roseovarius nubinhibens ...ISM] gb|EAP76566.1| pyruvate carboxylase [Roseovarius nubinhibens ISM] ZP_00960995.1 1.2 28% ...

  20. NCBI nr-aa BLAST: CBRC-PABE-10-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-10-0001 ref|ZP_00960995.1| pyruvate carboxylase [Roseovarius nubinhibens ...ISM] gb|EAP76566.1| pyruvate carboxylase [Roseovarius nubinhibens ISM] ZP_00960995.1 3.5 26% ...

  1. NCBI nr-aa BLAST: CBRC-PTRO-10-0009 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-10-0009 ref|ZP_00960995.1| pyruvate carboxylase [Roseovarius nubinhibens ...ISM] gb|EAP76566.1| pyruvate carboxylase [Roseovarius nubinhibens ISM] ZP_00960995.1 2.1 27% ...

  2. Solar ultraviolet radiation affects the activity of ribulose-1, 5-bisphosphate carboxylase-oxygenase and the composition of photosynthetic and xanthophyll cycle pigments in the intertidal green alga Ulva lactuca L.

    NARCIS (Netherlands)

    Bischof, K; Krabs, G; Wiencke, C; Hanelt, D

    2002-01-01

    The effect of solar UV radiation on the physiology of the intertidal green macroalga Ulva lactuca L. was investigated. A natural Ulm community at the shore of Helgoland was covered with screening foils, excluding UV-B or UV-B + UV-A from the solar spectrum. In the sampled material, changes in the ac

  3. Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

    OpenAIRE

    Blazejak, Anna; Kuever, Jan; Erséus, Christer; Amann, Rudolf; Dubilier, Nicole

    2006-01-01

    Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluore...

  4. Cloning and sequence analysis of citrate synthase and phosphoenolpyruvate carboxylase in the root of ‘Sour pummelo' (Citrus grandis)%酸柚根系CS和PEPC基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    杨林通; 林郑和; 陈立松

    2012-01-01

    Total RNA was extracted from root of ' sour pummelo' ( Citnus grandis) by hot borate method. The sequence of conserved region, 3' RACE product and 5' RACE product of citrate synthase (CS) and phosphoenolpyruvate cariboxylase (PEPC) genes were obtained by RT-PCR and RACE. The full-length cDNA of CS was 1217 bp, which contained a 1413 bp open reading frame (ORF) encoding 472 amino acids with a calculated molecular mass of 52.487 ku and an isoelectric point of 6.9, a 67 bp 5'-untranslated region (UTR) and a277 bp 3'-UTR. An alignment of deduced amino acid sequence of CS gene from ' sour pummelo' with other plants showed ihey shared high homeology (85.4% -99.6%). The full-length cDNA of PEPC was 3307, which contained a 2604 bp ORF encoding 868 amino acids with a calculated molecular mass of 99.569 ku and an isoelectric point of 6.68, a 431 bp 5'-UTR and a 269 bp 3'-UTR. An alignment of deduced amino acid sequence of PEPC gene from sour pummelo with other plants showed they shared high homeology (85.8% -95.7%).%以酸柚(Citrus grandis)根系为材料,利用热硼酸法提取了根系总RNA,并逆转录成cDNA,利用PCR和RACE技术相继得到柠檬酸合酶基因(CS)和磷酸烯醇式丙酮酸羧化酶基因(PEPC)的保守区、3′端和5′端.酸柚根系CS基因全长1760bp,开放读码框有1413bp,编码472个氨基酸,氨基酸序列相对分子质量为52.487 ku,等电点为6.9,亲水指数为-0.199;5′端非编码区为67 bp,3′端非编码区为277 bp;推导的氨基酸经序列比对,发现与其他物种具有很高的同源性(85.4% -99.6%).酸柚根系PEPC基因全长3307bp,开放读码框有2604 bp,编码868个氨基酸,氨基酸序列相对分子质量为99.569ku,等电点为6.68,亲水指数为-0,398;5′端非编码区为431 bp,3′端非编码区为269 bp,推导的氨基酸经序列比对,发现与其他物种具有很高的同源性(85.8% - 95.7%).初步确定克隆到的为酸柚根系CS和PEPC基因,登陆Genbank,登陆号分别为HQ537481和HQ537482.

  5. AcEST: DK958123 [AcEST

    Lifescience Database Archive (English)

    Full Text Available _id B3DYN2 Definition tr|B3DYN2|B3DYN2_METI4 Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) OS=Methylacidiphilum inferno...ccus xan... 35 4.7 >tr|B3DYN2|B3DYN2_METI4 Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) OS=Methylacidiphilum inferno

  6. AcEST: BP916957 [AcEST

    Lifescience Database Archive (English)

    Full Text Available uvate carboxylase OS=Thermosynechococcus vulcanus Align length 75 Score (bit) 32.3 E-value 1.1 Report BLASTX...s naphthalen... 29 9.3 >sp|P0A3X6|CAPP_THEVL Phosphoenolpyruvate carboxylase OS=Thermosynechococcus vulcanus

  7. AcEST: BP912293 [AcEST

    Lifescience Database Archive (English)

    Full Text Available oenolpyruvate carboxylase OS=Thermosynechococcus vulcanus Align length 55 Score (bit) 30.4 E-value 2.8 Repor... OS=Bartonella... 29 6.3 >sp|P0A3X6|CAPP_THEVL Phosphoenolpyruvate carboxylase OS=Thermosynechococcus vulcan

  8. Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group.

    Science.gov (United States)

    Chonan, Tomomichi; Wakasugi, Daisuke; Yamamoto, Daisuke; Yashiro, Miyoko; Oi, Takahiro; Tanaka, Hiroaki; Ohoka-Sugita, Ayumi; Io, Fusayo; Koretsune, Hiroko; Hiratate, Akira

    2011-03-01

    Novel (4-piperidinyl)-piperazine derivatives were synthesized and evaluated as ACC1/2 non-selective inhibitors. Optimization of the substituents on the nitrogen of the piperidine ring led to the identification of the fluorine substituted tert-butoxycarbonyl group. Advanced analog, 1,1,1-trifluoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (12c) showed potent inhibitory activities in enzyme-assay and cell-based assays. Compound 12c also exhibited reduction of hepatic de novo fatty acid synthesis in rats after oral administration.

  9. Apoptosis of prostate cancer stem cells induced by αacetyl-CoA-carboxylase gene silencing mediated by siRNA%siRNA抑制α乙酰辅酶A羧化酶基因表达促进前列腺癌干细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    杨波; 赵德明; 刘辉; 刘峰; 郝继东; 王伟峰; 万建省

    2013-01-01

    目的:研究ACC-αRNA干扰对前列腺癌干细胞增殖的影响。方法设计并合成ACC-α基因的小干扰RNA(siRNA),转染前列腺癌干细胞系,通过Western blot检测RNA干扰对ACC-α表达的影响,并使用MTT法检测RNA干扰对细胞增殖的影响,使用荧光染色法分析前列腺癌干细胞的凋亡状况。结果 siRNA干扰96 h后可见ACC-α表达显著下调,转染96 h后前列腺癌干细胞增殖状况显著下降,通过荧光染色观察发现,siRN A干扰造成癌细胞的大量凋亡。结论 siRNA 干扰 ACC-α可以通过诱导凋亡显著的抑制前列腺癌干细胞的增殖, ACC-α是前列腺癌潜在的治疗靶点。%Objective To investigate the role of ACC-α siRNA interference in prostate stem cells proliferation . Methods The siRNA of ACC-αand transfect prostate stem cell were designed and synthetized .The effect of siRNA on ACC-αexpression was detected by western blot .MTT assay was used to detect the prostate stem cell proliferation and the apoptosis of prostate stem cells were evaluated by fluorescent staining .Results The expression of ACC-αwas significantly lower and the propagation of prostate cancer stem cells decreased obviously after 96 h transfection . There was a large number of cancer cell apoptosis under fluorescence microscopy by siRNA interference . Conclusion siRNA interference ACC-αcan suppress prostate proliferation through inducing cell apoptosis ,which was a potential therapeutic target of prostate cancer .

  10. 运用CRISPR/Cas系统敲除大肠杆菌磷酸烯醇式丙酮酸羧化酶基因及其对脂肪酸代谢的影响%Knocking out phosphoenolpyruvate carboxylase gene by CRISPR/Cas and its influence on fatty acid metabolism in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    夏军; 郑明刚; 王玲; 孙承君; 郑立; 祝建波

    2016-01-01

    [目的]CRISPR/Cas是目前基因编辑的一个重要的新兴技术,拟通过该技术,对大肠杆菌进行快速、高效的基因编辑,获取脂肪酸代谢工程菌.[方法]利用一个二元载体构建出CRISPR/Cas偶联λ-Red重组酶的系统,重叠PCR扩增出敲除的Donor,最后用电转的方式对Escherichia coli MG1655脂肪酸代谢相关基因进行快速编辑.同时,采用气相色谱-质谱(GC-MS)对脂肪酸进行定性及定量分析.[结果]获得了大肠杆菌△PEPC(partial)、△PEPC、△PEPC(partial)-△FadD、△PEPC-△FadD以及△FadD突变体菌株.各缺失体菌株脂肪酸含量均比野生型要高,最高的增长了3.7%,但是敲除ppc获得的脂肪酸增长量并没有预期的高.同时在脂肪酸组成上,各菌株均含有11:0、12:0、13:0、14:0、15:0、16:0、17:1、17:0、18:0,并且16:0,17:0,18:0占主要组成部分.[结论]运用该技术可以快速、高效地对大肠杆菌基因进行编辑,是大肠杆菌工程菌株构建的一个新思路,对其他工程菌的构建提供了一定的理论基础.

  11. AcEST: BP915822 [AcEST

    Lifescience Database Archive (English)

    Full Text Available r_hit_id Q6Y9M7 Definition tr|Q6Y9M7|Q6Y9M7_9FILI Ribulose bisphosphate carboxylase large chain (Fragment) OS=Anogramma...9 6e-78 >tr|Q6Y9M7|Q6Y9M7_9FILI Ribulose bisphosphate carboxylase large chain (Fragment) OS=Anogramma leptop...FILI Ribulose bisphosphate carboxylase large chain (Fragment) OS=Anogramma guatemalensis GN=rbcL PE=3 SV=1 L

  12. Recombinant micro-organism for use in method with increased product yield

    NARCIS (Netherlands)

    Van Maris, A.J.A.; Pronk, J.T.; Guadalupe Medina, V.G.; Wisselink, H.W.

    2014-01-01

    The invention relates to a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-1,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). The invention

  13. Reference: CACTFTPPCA1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available CACTFTPPCA1 Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M, Streubel M, West...he promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell. 16:1077-1090(2004) PubMed: 15100398 ...

  14. Main: 1JQO [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1JQO トウモロコシ Corn Zea mays L. Phosphoenolpyruvate Carboxylase 1 Name=Pep1; Synonyms=...FQAYTLKRIRDPNFKVTPQPPLSKEFADENKPAGLVKLNPASEYPPGLEDTLILTMKGIAAGMQNTG corn_1JQO.jpg ...

  15. Generation of Diversity in Streptococcus mutans Genes Demonstrated by MLST

    OpenAIRE

    Thuy Do; Gilbert, Steven C.; Douglas Clark; Farida Ali; Clarissa C Fatturi Parolo; Marisa Maltz; Russell, Roy R.; Peter Holbrook; Wade, William G.; David Beighton

    2010-01-01

    Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), r...

  16. The Role of Tumor Metastases Suppressor Gene, Drg-1, in Breast Cancer

    Science.gov (United States)

    2007-03-01

    ceramide level in the tumor cells, and this increase was abrogated by acetyl-CoA carboxylase inhibitor. In addition, carnitine palmitoyltransferase-1...Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 Ag/mL streptomycin, 100 units/mL...acetyl-CoA carboxylase inhibitor), fumonisin B1 (ceramide synthase inhibitor), etomoxir [ carnitine palmitoyltransferase-1 (CPT-1) inhibitor], and C2

  17. Regulation of immunological and inflammatory functions by biotin.

    Science.gov (United States)

    Kuroishi, Toshinobu

    2015-12-01

    Biotin is a water-soluble B-complex vitamin and is well-known as a co-factor for 5 indispensable carboxylases. Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of carboxylases and other proteins, whereas biotinidase catalyzes the release of biotin from biotinylated peptides. Previous studies have reported that nutritional biotin deficiency and genetic defects in either HLCS or biotinidase induces cutaneous inflammation and immunological disorders. Since biotin-dependent carboxylases involve various cellular metabolic pathways including gluconeogenesis, fatty acid synthesis, and the metabolism of branched-chain amino acids and odd-chain fatty acids, metabolic abnormalities may play important roles in immunological and inflammatory disorders caused by biotin deficiency. Transcriptional factors, including NF-κB and Sp1/3, are also affected by the status of biotin, indicating that biotin regulates immunological and inflammatory functions independently of biotin-dependent carboxylases. An in-vivo analysis with a murine model revealed the therapeutic effects of biotin supplementation on metal allergies. The novel roles of biotinylated proteins and their related enzymes have recently been reported. Non-carboxylase biotinylated proteins induce chemokine production. HLCS is a nuclear protein involved in epigenetic and chromatin regulation. In this review, comprehensive knowledge on the regulation of immunological and inflammatory functions by biotin and its potential as a therapeutic agent is discussed.

  18. AcEST: DK944008 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 1. 5' end sequence. DK944008 CL2Contig2 Show DK944008 Clone id YMU02A01NGRL0004_L01 Library YMU02 Length 753... Definition Adiantum capillus-veneris mRNA. clone: YMU02A01NGRL0004_L01. 5' end sequence. Accession DK944008...rams, Nucleic Acids Res. 25:3389-3402. Query= DK944008|Adiantum capillus-veneris mRNA, clone: YMU02A01NGRL00...hosphate carboxylase small ch... 180 8e-45 sp|P24007|RBS_PYRPY Ribulose bisphosph...ate carboxylase small chai... 177 4e-44 sp|Q40004|RBS_HORVU Ribulose bisphosphate carboxylase small chai...

  19. Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

    Science.gov (United States)

    He, Lan; Hamm, J Austin; Reddy, Alex; Sams, David; Peliciari-Garcia, Rodrigo A; McGinnis, Graham R; Bailey, Shannon M; Chow, Chi-Wing; Rowe, Glenn C; Chatham, John C; Young, Martin E

    2016-06-01

    Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCβ, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

  20. Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture.

    Science.gov (United States)

    Abu Laban, Nidal; Selesi, Draženka; Rattei, Thomas; Tischler, Patrick; Meckenstock, Rainer U

    2010-10-01

    Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX).

  1. Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions.

    Science.gov (United States)

    Moroney, James V; Jungnick, Nadine; Dimario, Robert J; Longstreth, David J

    2013-11-01

    This review presents an overview of the two ways that cyanobacteria, algae, and plants have adapted to high O2 and low CO2 concentrations in the environment. First, the process of photorespiration enables photosynthetic organisms to recycle phosphoglycolate formed by the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Second, there are a number of carbon concentrating mechanisms that increase the CO2 concentration around Rubisco which increases the carboxylase reaction enhancing CO2 fixation. This review also presents possibilities for the beneficial modification of these processes with the goal of improving future crop yields.

  2. Biotin Carboxyl Carrier Protein in Barley Chloroplast Membranes

    DEFF Research Database (Denmark)

    Kannangara, C. G.; Jense, C J

    1975-01-01

    Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained by solubil......Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained...

  3. Effects of High-fat Diet and Treadmill Exercise on AMP-activated Protein Kinase (AMPK)/Acetyl-CoA Carboxylase (ACC) Signaling Pathway and Fatty Acid Translocase CD36 Protein Content in Rat Gastrocnemius Muscle%高脂膳食和跑台运动对雄性大鼠腓肠肌腺苷酸活化蛋白激酶/乙酰辅酶A羧化酶信号通路和膜蛋白脂肪酸转位酶蛋白含量的影响

    Institute of Scientific and Technical Information of China (English)

    张云丽; 娄淑杰

    2015-01-01

    目的 探讨高脂膳食和8周有氧耐力运动对大鼠腓肠肌腺苷酸活化蛋白激酶(AMPK)/乙酰辅酶A羧化酶(ACC)信号通路和膜蛋白脂肪酸转位酶CD36含量的影响.方法 建立营养性肥胖大鼠模型,并随机分为肥胖安静组(OC组)和肥胖运动组(OE组),另设普通饲料安静组(NC组)和普通饲料运动组(NE组).运动干预结束后检测腓肠肌AMPKα、p-AMPKa ACC、 p-ACC和膜蛋白CD36的蛋白水平.结果 1)NE组与NC、OE及OC组相比,p-AMPKα的蛋白水平显著升高(P<0.01);OC组p-AMPKα的蛋白水平显著低于NC组(P<0.01).2)OC组p-ACC蛋白水平显著低于NC组(P<0.01);OE组p-ACC的蛋白水平显著高于OC组(P<0.01).3)NE组与NC、OE及OC组相比,OC组与NC组相比,膜蛋白CD36含量均无显著性变化(P>0.05).结论 1)运动可改善高脂膳食引起的AMPK/ACC信号通路障碍.2)运动对体质量不同大鼠p-ACC蛋白水平的影响存在差异.3)腓肠肌膜蛋白CD36的含量并没有伴随AMPK/ACC信号通路的激活或抑制而发生显著变化.

  4. Ethylmalonic aciduria is associated with an amino acid variant of short chain acyl-coenzyme A dehydrogenase

    DEFF Research Database (Denmark)

    Corydon, M J; Gregersen, N; Lehnert, W

    1996-01-01

    metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general...

  5. The promoter-terminator of chrysanthemunm rbcS1 directs very high expression levels in plants

    NARCIS (Netherlands)

    Outchkourov, N.S.; Peters, J.; Jong, de J.; Rademakers, W.; Ongsma, M.A.

    2003-01-01

    Transgenic plants are increasingly used as production platforms for various proteins, yet protein expression levels in the range of the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase have not yet been achieved by nuclear transformation. Suitable gene regulatory 5' and 3' elements

  6. Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.).

    Science.gov (United States)

    Evans, J R

    1983-06-01

    Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP(2)) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO(2) assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP(2) carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP(2) carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO(2), the intercellular partial pressure of CO(2) was always such that rate of assimilation appeared colimited by RuP(2) carboxylation and RuP(2) regeneration capacity.The initial slope of rate of CO(2) assimilation against intercellular partial pressure of CO(2) varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO(2) diffusion in the wall and liquid phase which causes a drop in CO(2) partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

  7. Regulation of methanol oxidation and carbon dioxide fixation in Xanthobacter strain 25a grown in continuous culture

    NARCIS (Netherlands)

    Croes, L.M.; Meijer, Wilhelmus; Dijkhuizen, L.

    1991-01-01

    The regulation of C1-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be

  8. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

    Science.gov (United States)

    In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

  9. Detecting ALS and ACCase herbicide tolerant accession of Echinochloa oryzoides (Ard.) Fritsch. in rice (Oryza sativa L.) fields

    DEFF Research Database (Denmark)

    Altop, Emine Kaya; Mennan, Husrev; Streibig, Jens Carl

    2014-01-01

    -sodium) and acetyl CoA carboxylase (cyhalofob-butyl) inhibiting herbicides. Comparison of 95% lower confidence intervals of ED90 derived from log-logistic dose-response curves, and twice the recommended field rates of the herbicides showed some, but not distinct separation of susceptible and tolerant accessions. We...

  10. Regulatory enzymes of mitochondrial beta-oxidation as targets for treatment of the metabolic syndrome

    NARCIS (Netherlands)

    Schreurs, M.; Kuipers, F.; van der Leij, F. R.

    2010-01-01

    P>Insulin sensitizers like metformin generally act through pathways triggered by adenosine monophosphate-activated protein kinase. Carnitine palmitoyltransferase 1 (CPT1) controls mitochondrial beta-oxidation and is inhibited by malonyl-CoA, the product of acetyl-CoA carboxylase (ACC). The adenosine

  11. AcEST: BP918715 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ) Value tr|Q9AXG0|Q9AXG0_GOSHI Ribulose-1,5-bisphosphate carboxylase/oxy... 58 3e-07 tr|Q8VYX9|Q8VYX9_PEA Ultraviolet...NEDSIKQGTF 318 EQENVKRVQLA+KYL+EAAL EANEDSI +GTF Sbjct: 364 EQENVKRVQLADKYLSEAALGEANEDSINRGTF 396 >tr|Q8VYX9|Q8VYX9_PEA Ultraviolet

  12. Fatty acid biosynthesis. VIII. The fate of malonyl-CoA in fatty acid biosynthesis by purified enzymes from lactating-rabbit mammary gland

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1971-01-01

    - 1. We have investigated the formation and utilization of malonyl-CoA in fatty acid synthesis catalysed by preparations of partially purified acetyl-CoA carboxylase and purified fatty acid synthetase from lactating-rabbit mammary gland. - 2. Carboxylation of [1-14C]acetyl-CoA was linked to fatty...

  13. Dietary vitamin K and therapeutic warfarin alter susceptibility to vascular calcification in experimental chronic kidney disease

    Science.gov (United States)

    The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease (CVD), with vascular calcification (VC) being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This gamma-glutamyl carboxylase substrate is an essential ...

  14. AcEST: BP917133 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 775_CUPTR Putative PROPIONYL-COA CARBOXYLASE (BETA SUBUNIT) PROTEIN OS=Cupriavidus taiwan...TA SUBUNIT) PROTEIN OS=Cupriavidus taiwanensis (strain R1 / LMG 19424) GN=RALTA_A2850 PE=4 SV=1 Length = 533

  15. Photorespiration.

    Science.gov (United States)

    Rao, K. K.; Hall, D. O.

    1982-01-01

    Topics in this discussion of photorespiration (light-dependent oxygen consumption and carbon dioxide evolution from leaves) include: (1) the biochemistry of photorespiration; (2) ribulose biphosphate carboxylase and glycollate synthesis; (3) metabolism of glycollate; (4) plants lacking photorespiratory systems; and (5) advantages of…

  16. Main: MNF1ZMPPC1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MNF1ZMPPC1 S000251 11-Oct-1999 (last modified) kehi MNF1 binding site in maize (Z.m....) Ppc1 (phosphoenolpyruvate carboxylase) gene promoter; Involved in light induction; MNF1; Ppc1; light; leaf; shoot; maize (Zea mays) GTGCCCTT ...

  17. Reference: MNF1ZMPPC1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MNF1ZMPPC1 Morishima A Identification of preferred binding sites of a light-inducib...le DNA-binding factor (MNF1) within 5'-upstream sequence of C4-type phosphoenolpyruvate carboxylase gene in maize Plant Mol Biol 38:633-646 (1998) PubMed: 9747808; ...

  18. Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

    DEFF Research Database (Denmark)

    Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

    2016-01-01

    conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis...

  19. Rubisco activity and regulation as targets for crop improvement

    Science.gov (United States)

    Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction wit...

  20. GenBank blastx search result: AK103974 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103974 001-016-F05 U23145.1 Rhodobacter capsulatus Calvin cycle carbon dioxide fi...ose-1,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pentose-5-pho

  1. GenBank blastx search result: AK062574 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062574 001-047-A01 U23145.1 Rhodobacter capsulatus Calvin cycle carbon dioxide fi...ose-1,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pentose-5-pho

  2. GenBank blastx search result: AK061772 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061772 001-039-C12 U23145.1 Rhodobacter capsulatus Calvin cycle carbon dioxide fi...ose-1,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pentose-5-pho

  3. GenBank blastx search result: AK288454 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288454 J090035A12 U23145.1 RCU23145 Rhodobacter capsulatus Calvin cycle carbon di...II ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pento

  4. GenBank blastx search result: AK110716 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110716 002-170-D07 U23145.1 Rhodobacter capsulatus Calvin cycle carbon dioxide fi...ose-1,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pentose-5-pho

  5. Studies on Dasyaceae. 3. Towards a phylogeny of the Dasyaceae (Ceramiales, Rhodophyta), based on comparative rbcL gene sequences and morphology

    NARCIS (Netherlands)

    de Jong, Y.S D M; van der Wurff, A.W G; Stam, W.T.; Olsen, J.L.

    1998-01-01

    Phylogenetic analyses of the Dasyaceae based on sequence analysis of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and 42 morphological characters are presented. Comparative sequence analysis confirms the general view of the Ceramiaceae as a primitive, paraphyletic grou

  6. GenBank blastx search result: AK243187 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243187 J100039E11 AF203480.1 AF203480 Lycopersicon esculentum cultivar R11-12 (35...S::Pto in Rio Grande x Money Maker)' phosphoenolpyruvate carboxylase kinase mRNA, partial cds. PLN 1e-41 1 ...

  7. GenBank blastx search result: AK241003 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241003 J065053E13 AF203480.1 AF203480 Lycopersicon esculentum cultivar R11-12 (35...S::Pto in Rio Grande x Money Maker)' phosphoenolpyruvate carboxylase kinase mRNA, partial cds. PLN 1e-14 1 ...

  8. GenBank blastx search result: AK243656 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243656 J100088L22 AF203480.1 AF203480 Lycopersicon esculentum cultivar R11-12 (35...S::Pto in Rio Grande x Money Maker)' phosphoenolpyruvate carboxylase kinase mRNA, partial cds. PLN 2e-53 1 ...

  9. Retinitis pigmentosa, cutis laxa, and pseudoxanthoma elasticum-like skin manifestations associated with GGCX mutations

    NARCIS (Netherlands)

    Kariminejad, Ariana; Bozorgmehr, Bita; Najafi, Abdolhamid; Khoshaeen, Atefeh; Ghalandari, Maryam; Najmabadi, Hossein; Kariminejad, Mohamad H; Vanakker, Olivier M; Hosen, Mohammad J; Malfait, Fransiska; Quaglino, Daniela; Florijn, Ralph J; Bergen, Arthur A B; Hennekam, Raoul C

    2014-01-01

    Gamma-glutamyl carboxylase (GGCX) mutations have been reported in patients with a pseudoxanthoma elasticum (PXE)-like phenotype, loose redundant skin, and multiple vitamin K-dependent coagulation factor deficiencies. We report on the clinical findings and molecular results in 13 affected members of

  10. Oxygen-18 incorporation into malic acid during nocturnal carbon dioxide fixation in crassulacean acid metabolism plants: a new approach to estimating in vivo carbonic anhydrase activity

    Energy Technology Data Exchange (ETDEWEB)

    Holtum, J.A.M.; Summons, R.; Roeske, C.A.; Comins, H.N.; O' Leary, M.H.

    1984-01-01

    Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with TC YO2, then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the ( TC) malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance or by mass spectrometry. Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum, 40 for Kalanchoee daigremontiana, and 100 or greater for Bryophyllum tubiflorum, Kalanchoee serrata, and Kalanchoae tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature. 37 references, 2 figures, 8 tables.

  11. Acc homoeoloci and the evolution of wheat genomes

    Science.gov (United States)

    We analyzed the DNA sequences of BACs from many wheat libraries containing the Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, to gain understanding of the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Mor...

  12. Regulation of Photosystem II Electron transport by Bicarbonate

    NARCIS (Netherlands)

    Rensen, van J.J.S.

    2012-01-01

    In oxygenic photosynthesis, carbon dioxide is fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and further reduced to carbohydrates. However, CO2, in the form of carbonate or bicarbonate, is also directly involved in the “light reactions” through structural and regulatory roles wit

  13. A novel expression cassette for the efficient visual selection of transformed tissues in florists' chrysanthemum (Chrysanthemum morifolium Ramat.).

    NARCIS (Netherlands)

    Mao, J.; Stoopen, G.M.; Jongsma, M.A.; Wang, C.Y.

    2011-01-01

    Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green flu

  14. 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 ...

  15. Main: CACTFTPPCA1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available CACTFTPPCA1 S000449 19-August-2004 (last modified) kehi Tetranucleotide (CACT) is a...ion of the phosphoenolpyruvate carboxylase (ppcA1) of the C4 dicot F. trinervia; Y=T/C; mesohpyll; CACT; Flaveria trinervia YACT ...

  16. Proteolysis during ensilage of forages varying in soluble sugar content.

    Science.gov (United States)

    Davies, D R; Merry, R J; Williams, A P; Bakewell, E L; Leemans, D K; Tweed, J K

    1998-02-01

    The effect of contrasting concentrations of water-soluble carbohydrates of herbage on silage fermentation and composition was examined using grass with high [250 g/kg of dry matter (DM)] concentrations of water-soluble carbohydrates and grass and clover with low (66 g/kg of DM) concentrations of water-soluble carbohydrates. Herbages were ensiled untreated, after inoculation with lactic acid bacteria, or after treatment with formic acid. Good quality silages were produced from herbage with high concentrations of water-soluble carbohydrates, regardless of treatment, and all pH values were below 3.7 after 90 d of ensilage. However, the silage formed from inoculated herbage had a significantly lower concentration of ammonia N and a significantly higher proportion of residual ribulose-1,5-bisphosphate carboxylase compared with the other two silages. Fast protein liquid chromatography (Pharmacia, Uppsala, Sweden) was used to measure ribulose-1,5-bisphosphate carboxylase, and measurement of true plant protein fractions in herbage and silage showed benefits over traditional measurements such as the measurement of N and ammonia N. Herbages with low concentrations of water-soluble carbohydrates produced inferior quality silages that had lower ribulose-1,5-bisphosphate carboxylase contents and higher ammonia N contents, regardless of treatment; few significant differences were observed among treatments. Under good ensiling conditions, when available water-soluble carbohydrate is adequate, the use of inoculants can improve fermentation characteristics and increase the ribulose-1,5-bisphosphate carboxylase content of silages. However, when the herbage has low concentrations of water-soluble carbohydrates, even in inoculated herbages, lactic acid bacteria may follow a heterofermentative pathway instead of a homofermentative pathway, which can result in a decrease in silage quality and a reduction in intact ribulose-1,5-bisphosphate carboxylase.

  17. Effects of exogenous spermidine on photosynthetic capacity and expression of Calvin cycle genes in salt-stressed cucumber seedlings.

    Science.gov (United States)

    Shu, Sheng; Chen, Lifang; Lu, Wei; Sun, Jin; Guo, Shirong; Yuan, Yinhui; Li, Jun

    2014-11-01

    We investigated the effects of exogenous spermidine (Spd) on growth, photosynthesis and expression of the Calvin cycle-related genes in cucumber seedlings (Cucumis sativus L.) exposed to NaCl stress. Salt stress reduced net photosynthetic rates (PN), actual photochemical efficiency of PSII (ΦPSII) and inhibited plant growth. Application of exogenous Spd to salinized nutrient solution alleviated salinity-induced the inhibition of plant growth, together with an increase in PN and ΦPSII. Salinity markedly reduced the maximum carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Vcmax), the maximal velocity of RuBP regeneration (Jmax), triose-phosphate utilization capacity (TPU) and carboxylation efficiency (CE). Spd alleviated the negative effects on CO2 assimilation induced by salt stress. Moreover, Spd significantly increased the activities and contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and fructose-1,6-biphosphate aldolase (ALD; aldolase) in the salt-stressed cucumber leaves. On the other hand, salinity up-regulated the transcriptional levels of ribulose-1,5-bisphosphate (RCA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribrokinase (PRK) and down-regulated the transcriptional levels of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS), ALD, triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate phosphatase (FBPase) and 3-phosphoglyceric acid kinase (PGK). However, Spd application to salt-stressed plant roots counteracted salinity-induced mRNA expression changes in most of the above-mentioned genes. These results suggest that Spd could improve photosynthetic capacity through regulating gene expression and activity of key enzymes for CO2 fixation, thus confers tolerance to salinity on cucumber plants.

  18. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Knudsen, J

    1997-01-01

    or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis...... by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase....... (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations...

  19. Phosphoenolpyruvate carboxykinase in cherry (Prunus avium L.) fruit during development.

    Science.gov (United States)

    Walker, Robert P; Battistelli, Alberto; Moscatello, Stefano; Chen, Zhi-Hui; Leegood, Richard C; Famiani, Franco

    2011-11-01

    In this study the abundance and location of phosphoenolpyruvate carboxykinase (PEPCK) was determined in the flesh and skin of the sweet cherry (Prunus avium L.) cultivar Durone Nero II during development. PEPCK was not present in young fruit but appeared in both tissues as the fruit increased in size. In these there was no net dissimilation of malic acid, which accounts for the bulk of their organic acid contents when PEPCK was present. To assist in understanding the function of PEPCK, the abundance of a number of other enzymes was determined. These enzymes were aspartate aminotransferase (AspAT), glutamine synthetase (GS), phosphoenolpyruvate carboxylase (PEPC), pyruvate, orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). A potential role for PEPCK in the regulation of pH and the utilization of malate in gluconeogenesis in the flesh and skin of cherries is presented.

  20. Elementary Mode Analysis for the Rational Design of Efficient Succinate Conversion from Glycerol by Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhen Chen

    2010-01-01

    Full Text Available By integrating the restriction of oxygen and redox sensing/regulatory system, elementary mode analysis was used to predict the metabolic potential of glycerol for succinate production by E. coli under either anaerobic or aerobic conditions. It was found that although the theoretical maximum succinate yields under both anaerobic and aerobic conditions are 1.0 mol/mol glycerol, the aerobic condition was considered to be more favorable for succinate production. Although increase of the oxygen concentration would reduce the succinate yield, the calculation suggests that controlling the molar fraction of oxygen to be under 0.65 mol/mol would be beneficial for increasing the succinate productivity. Based on the elementary mode analysis, the rational genetic modification strategies for efficient succinate production under aerobic and anaerobic conditions were obtained, respectively. Overexpressing the phosphoenolpyruvate carboxylase or heterogonous pyruvate carboxylase is considered to be the most efficient strategy to increase the succinate yield.

  1. Metabolic responses of tropical trees to ozone pollution.

    Science.gov (United States)

    Chapla, J; Kamalakar, J A

    2004-07-01

    Plants fumigated with 40ppbv, 80ppbv and 120ppbv concentrations of O3 exhibited significant reduction in total chlorophyll content, RuBP carboxylase activity and net photosynthesis. The reduction in total chlorophyll activity ranged from 12 to 36% in Bauhinia variegata, 11 to 35% in Ficus infectoria and 3 to 26% in Pongamia pinnata on fumigation with O3, while the RuBP carboxylase activity was reduced by 10 to 32% in Bauhinia variegata, 10 to 23% in Ficus infectoria and 9 to 15% in Pongamia pinnata. The net photosynthesis was also reduced by 6 to 26% in B. variegata, 16 to 39% in F. infectoria and 7 to 31% in P. pinnata on fumigation with 03. The relative higher sensitivity of tropical trees to O3 suggests that the ambient air quality standards in tropical tree areas need to be stringent to prevent vegetation from air pollution.

  2. AcEST: DK958095 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST39A01NGRL0030_B16 642 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0030_B16. 5' end sequence. DK95...8095 CL17Contig1 Show DK958095 Clone id TST39A01NGRL0030_B16 Library TST39 Length 64...2 Definition Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0030_B16. 5' end sequence. Accession DK958095...base search programs, Nucleic Acids Res. 25:3389-3402. Query= DK958095|Adiantum c...e carboxylase/oxygenase ... 348 2e-95 sp|P23489|RCA_CHLRE Ribulose bisphosphate carboxylase/oxygenase ... 33

  3. Crystal structure of Spot 14, a modulator of fatty acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Colbert, Christopher L.; Kim, Chai-Wan; Moon, Young-Ah; Henry, Lisa; Palnitkar, Maya; McKean, William B.; Fitzgerald, Kevin; Deisenhofer, Johann; Horton, Jay D.; Kwon, Hyock Joo (Alnylam Pharm.); (UTSMC)

    2011-09-06

    Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 {angstrom} and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.

  4. Differential responses of photosynthetic parameters of pigeonpea and amaranth leaf discs to SO{sub 2}

    Energy Technology Data Exchange (ETDEWEB)

    Saraswathi, J.; Rao, K.V.M. [Andhra University, Visakhapatnam (India). Dept. of Botany

    1995-08-01

    The reduction in chlorophyll (Chl) and protein contents and the increase in amino acid content in leaf discs in response to aqueous SO{sub 2} exposure under continuous irradiance were more expressed in Amaranthus paniculatus (C-4 plant) than in Cajantus cajan (C-3 plant). The content of SH-compounds increased more in pigenonpea than in amaranth leaf discs in response to SO{sub 2}. Aqueous SO{sub 2} exposure also reduced the CO{sub 2} fixation and ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities in leaf discs of both plant species. The differences in sensitivity of these plants to SO{sub 2} were related to their conversion efficiency of SO{sub 2} to less toxic substances and sulphydryl compounds.

  5. Peroxisome proliferator-activated receptor alpha (PPARalpha) protects against oleate-induced INS-1E beta cell dysfunction by preserving carbohydrate metabolism

    DEFF Research Database (Denmark)

    Frigerio, F; Brun, T; Bartley, C;

    2009-01-01

    AIMS/HYPOTHESIS: Pancreatic beta cells chronically exposed to fatty acids may lose specific functions and even undergo apoptosis. Generally, lipotoxicity is triggered by saturated fatty acids, whereas unsaturated fatty acids induce lipodysfunction, the latter being characterised by elevated basal...... enzyme pyruvate carboxylase. PPARalpha overproduction increased both beta-oxidation and fatty acid storage in the form of neutral triacylglycerol, revealing overall induction of lipid metabolism. These observations were substantiated by expression levels of associated genes. CONCLUSIONS...

  6. S14 as a Therapeutic Target in Breast Cancer

    Science.gov (United States)

    2006-08-01

    tumor lipogenesis and the availability of LPL in the breast cancer cell “metabolic microenvironment”. Box upper right: Hydrolysis of triglyceride by...metastases prompted a new hypothesis regarding the interaction of tumor lipogenesis, the availability of the enzyme lipoprotein lipase in the tumor...hitchcock.org (W.B. Kinlaw). 1 Currently Walter Reed U.S. Army Medical Center.expression of S14 and acetyl CoA-carboxylase, the rate- determining enzyme of long

  7. Photorespiration

    OpenAIRE

    Peterhansel, Christoph; Horst, Ina; Niessen, Markus; Blume, Christian; Kebeish, Rashad; Kürkcüoglu, Sophia; Kreuzaler, Fritz

    2010-01-01

    Photorespiration is initiated by the oxygenase activity of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RUBISCO), the same enzyme that is also responsible for CO2 fixation in almost all photosynthetic organisms. Phosphoglycolate formed by oxygen fixation is recycled to the Calvin cycle intermediate phosphoglycerate in the photorespiratory pathway. This reaction cascade consumes energy and reducing equivalents and part of the afore fixed carbon is again released as CO2. Because of this, ph...

  8. Physiological evidence for plasticity in glycolate/glycerate transport during photorespiration

    OpenAIRE

    Walker, Berkley J.; South, Paul F.; Ort, Donald R.

    2016-01-01

    Photorespiration recycles fixed carbon following the oxygenation reaction of Ribulose, 1–5, carboxylase oxygenase (Rubisco). The recycling of photorespiratory C2 to C3 intermediates is not perfectly efficient and reduces photosynthesis in C3 plants. Recently, a plastidic glycolate/glycerate transporter (PLGG1) in photorespiration was identified in Arabidopsis thaliana, but it is not known how critical this transporter is for maintaining photorespiratory efficiency. We examined a mutant defici...

  9. Photorespiration: metabolic pathways and their role in stress protection\\ud

    OpenAIRE

    Wingler, A; P.J. Lea; Quick, W.P.; Leegood, R C

    2000-01-01

    Photorespiration results from the oxygenase reaction catalysed by ribulose-1,5-bisphosphate carboxylase/\\ud oxygenase. In this reaction glycollate-2-phosphate is produced and subsequently metabolized in the\\ud photorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate. During this metabolic\\ud process, CO2 and NH3 are produced and ATP and reducing equivalents are consumed, thus\\ud making photorespiration a wasteful process. However, precisely because of this ine¤cien...

  10. Photorespiration: metabolic pathways and their role in stress protection.

    OpenAIRE

    Wingler, A; P.J. Lea; Quick, W.P.; Leegood, R C

    2000-01-01

    Photorespiration results from the oxygenase reaction catalysed by ribulose-1,5-bisphosphate carboxylase/oxygenase. In this reaction glycollate-2-phosphate is produced and subsequently metabolized in the photorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate. During this metabolic process, CO2 and NH3 are produced and ATP and reducing equivalents are consumed, thus making photorespiration a wasteful process. However, precisely because of this inefficiency, photore...

  11. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2...

  12. Disease: H00181 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available ala T, Randolph A, Coelho D, Fowler B, Valle D, Baumgartner MR 3-Methylcrotonyl-C...ing. Hum Mutat 26:164 (2005) PMID:15868465 Baumgartner MR Molecular mechanism of dominant expression in 3-me...thylcrotonyl-CoA carboxylase deficiency. J Inherit Metab Dis 28:301-9 (2005) PMID:11181649 Baumgartner... MR, Almashanu S, Suormala T, Obie C, Cole RN, Packman S, Baumgartner ER, Valle D The m

  13. [Description of the photosynthetic apparatus of Fucus vesiculosus L. in early embryogenesis].

    Science.gov (United States)

    Tarakhovskaia, E R; Maslov, Iu I

    2005-01-01

    Dynamics of some photosynthetic parameters was studied in gametes, zygotes, and embryos of kelp Fucus vesiculosus L. The following indices were determined at different stages of early development of the seaweed: the contents of pigments and ribulose-1,5-bisphosphate carboxylase/oxygenase, the rates of photosynthesis and dark respiration, and the activities of photosystems I and II. The dynamics of photosynthetic apparatus activity in zygotes and embryos of F. vesiculosus proved to reflect the main physiological processes of its early development.

  14. Malonyl-CoA Decarboxylase (MCD) as a Potential Therapeutic Target for Breast Cancer

    Science.gov (United States)

    2010-05-01

    of detection, using the ATP Bioluminescence Kit CLS II (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer’s protocol and read on a... ATP levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we s ynthesized a s mall m olecule i nhibitor o f M CD, 5...Figure 1). Acetyl-CoA carboxylase (ACC), the rate limiting enzyme of fatty acid synthesis, produces malonyl-CoA from the ATP dependent carboxylation of

  15. Disease: H01182 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available tive and the biotin is not recycled. Patients often exhibit feeding or breathing difficulties, skin rash, al...opecia, hypotonia and seizures. Biotin treatment can ameliorate or prevent sympto...ms. Inherited metabolic disease hsa00780(686) Biotin metabolism hsa04977(686) Vitamin digestion and absorpti...on BTD [HSA:686] [KO:K01435] Biotin [DR:D00029] Early-onset multiple carboxylase deficiency is described in

  16. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    Science.gov (United States)

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

  17. Measurement of 2-carboxyarabinitol 1-phosphate in plant leaves by isotope dilution. [Spinacea oleracea; Triticum aestivum; Arabidopsis thaliana; Maize; Phaseolus vulgaris; Petunia hybrida

    Energy Technology Data Exchange (ETDEWEB)

    Moore, B.D.; Kobza, J.; Seemann, J.R. (Univ. of Nevada, Reno (United States))

    1991-05-01

    The level of 2-carboxyarabinitol 1-phosphate (CA1P) in leaves of 12 species was determined by an isotope dilution assay. {sup 14}C-labeled standard was synthesized from (2-{sup 14}C)carboxyarabinitol 1,5-bisphosphate using acid phosphatase, and was added at the initial point of leaf extraction. Leaf CA1P was purified and its specific activity determined. CA1P was found in dark-treated leaves of all species examined, including spinach (Spinacea oleracea), wheat (Triticum aestivum), Arabidopsis thaliana, and maize (Zea mays). The highest amounts were found in bean (Phaseolus vulgaris) and petunia (Petunia hybrida), which had 1.5 to 1.8 moles CA1P per mole ribulose 1,5-bisphosphate carboxylase catalytic sites. Most species had intermediate amounts of CA1P (0.2 to 0.8 mole CA1P per mole catalytic sites). Such intermediate to high levels of CA1P support the hypothesis that CA1P functions in many species as a light-dependent regulator of ribulose 1,5-bisphosphate carboxylase activity and whole leaf photosynthetic CO{sub 2} assimilation. However, CA1P levels in spinach, wheat, and A. thaliana were particularly low (less than 0.09 mole CA1P per mole catalytic sites). In such species, CA1P does not likely have a significant role in regulating ribulose 1,5-bisphosphate carboxylase activity, but could have a different physiological role.

  18. Assessment of photosynthesis regulation in mixotrophically cultured microalga Chlorella sorokiniana

    Energy Technology Data Exchange (ETDEWEB)

    Li, Tingting; Kirchhoff, Helmut; Gargouri, Mahmoud; Feng, Jie; Cousins, Asaph B.; Pienkos, Philip T.; Gang, David R.; Chen, Shulin

    2016-11-01

    Mixotrophic growth of microalgae offers great potential as an efficient strategy for biofuel production. In this study, photosynthetic regulation of mixotrophically cultured Chlorella sorokiniana cells was systematically evaluated. Mixotrophic cells in the exponential growth phase showed the highest photosynthetic activity, where maximum photosynthetic O2 evolution was approximately 3- and 4-fold higher than cells in the same phase grown photoautotrophically in 1% CO2 (in air) and air, respectively. Additionally, characteristic chlorophyll fluorescence parameters demonstrated that no limitation in electron transport downstream of PSII was detected in mixotrophic cells. Up-regulation of photosynthetic activity was associated with high total ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylase activity and expression level of phosphoribulokinase (PRK). After 3 days, photosynthetic O2 evolution of mixotrophic cells that went to the stationary phase, was strongly reduced, with reduced photochemical efficiency and reorganization of the PSII complex. Simultaneously, enzymatic activity for Rubisco carboxylase and mRNA levels of Rubisco and PRK diminished. Importantly, there was almost no non-photochemical quenching for mixotrophic cells, whether grown in log or stationary phase. A decline in the quantum efficiency of PSII and an oxidized plastoquinone pool (PQ pool) was observed under N-depleted conditions during mixotrophic growth. These results demonstrate that photosynthesis is regulated differently in mixotrophically cultured C. sorokiniana cells than in cells grown under photoautotrophic conditions, with a particularly strong impact by nitrogen levels in the cells.

  19. Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

    Energy Technology Data Exchange (ETDEWEB)

    Mark Eiteman

    2007-07-31

    This report describes results toward developing a process to sequester CO{sub 2} centered on the enzymes PEP carboxylase and pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The study reports on strain development and process development. In the area of strain development, knockouts in genes which divert carbon from the enzymatic steps involved in CO{sub 2} consumption were completed, and were shown not to affect significantly the rate of CO{sub 2} sequestration and succinic acid generation. Furthermore, the pyc gene encoding for pyruvate carboxylase proved to be unstable when integrated onto the chromosome. In the area of process development, an optimal medium, pH and base counterion were obtained, leading to a sequestration rate as great as 800 mg/Lh. Detailed studies of gas phase composition demonstrated that CO{sub 2} composition has a significant affect on CO{sub 2} sequestration, while the presence of 'toxic' compounds in the gas, including NO{sub 2}, CO and SO{sub 2} did not have a detrimental effect on sequestration. Some results on prolonging the rate of sequestration indicate that enzyme activities decrease with time, suggesting methods to prolong enzyme activity may benefit the overall process.

  20. Enhanced tolerance to drought in transgenic rice plants overexpressing C4 photosynthesis enzymes

    Institute of Scientific and Technical Information of China (English)

    Jun-Fei; Gu; Ming; Qiu; Jian-Chang; Yang

    2013-01-01

    Maize-specific pyruvate orthophosphate dikinase(PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase(PCK). The wild-type(WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments,well-watered(WW), moderate drought(MD) and severe drought(SD), were imposed from 9d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities,biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines(PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%,20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments,respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase(PEPC) and carbonic anhydrase(CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to droughttolerance breeding via overexpression of C4enzymes in rice.

  1. Acinetobacter baumannii Coordinates Urea Metabolism with Metal Import To Resist Host-Mediated Metal Limitation

    Directory of Open Access Journals (Sweden)

    Lillian J. Juttukonda

    2016-09-01

    Full Text Available During infection, bacterial pathogens must adapt to a nutrient metal-limited environment that is imposed by the host. The innate immune protein calprotectin inhibits bacterial growth in vitro by chelating the divalent metal ions zinc (Zn2+, Zn and manganese (Mn2+, Mn, but pathogenic bacteria are able to cause disease in the presence of this antimicrobial protein in vivo. One such pathogen is Acinetobacter baumannii, a Gram-negative bacterium that causes pneumonia and bloodstream infections that can be complicated by resistance to multiple antibiotics. A. baumannii inhibition by calprotectin is dependent on calprotectin Mn binding, but the mechanisms employed by A. baumannii to overcome Mn limitation have not been identified. This work demonstrates that A. baumannii coordinates transcription of an NRAMP family Mn transporter and a urea carboxylase to resist the antimicrobial activities of calprotectin. This NRAMP family transporter facilitates Mn accumulation and growth of A. baumannii in the presence of calprotectin. A. baumannii is found to utilize urea as a sole nitrogen source, and urea utilization requires the urea carboxylase encoded in an operon with the NRAMP family transporter. Moreover, urea carboxylase activity is essential for calprotectin resistance in A. baumannii. Finally, evidence is provided that this system combats calprotectin in vivo, as deletion of the transporter impairs A. baumannii fitness in a mouse model of pneumonia, and this fitness defect is modulated by the presence of calprotectin. These findings reveal that A. baumannii has evolved mechanisms to subvert host-mediated metal sequestration and they uncover a connection between metal starvation and metabolic stress.

  2. Adaptation responses in C4 photosynthesis of maize under salinity.

    Science.gov (United States)

    Omoto, Eiji; Taniguchi, Mitsutaka; Miyake, Hiroshi

    2012-03-15

    The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.

  3. Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears.

    Science.gov (United States)

    Shimozuru, Michito; Nagashima, Akiko; Tanaka, Jun; Tsubota, Toshio

    2016-01-01

    Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period.

  4. Enhanced tolerance to drought in transgenic rice plants overexpressing C4 photosynthesis enzymes

    Directory of Open Access Journals (Sweden)

    Jun-Fei Gu

    2013-12-01

    Full Text Available Maize-specific pyruvate orthophosphate dikinase (PPDK was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase (PCK. The wild-type (WT cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments, well-watered (WW, moderate drought (MD and severe drought (SD, were imposed from 9 d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities, biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines (PPDK and PCK were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by 45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%, 20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments, respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase (PEPC and carbonic anhydrase (CA in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4 photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to drought-tolerance breeding via overexpression of C4 enzymes in rice.

  5. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes.

    Science.gov (United States)

    Priore, Paola; Siculella, Luisa; Gnoni, Gabriele Vincenzo

    2014-07-01

    Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

  6. Dicty_cDB: VSJ136 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available *** SEQUENCING IN PROGRESS ***, 1 ordered piece. 46 0.17 1 AF244346 |AF244346.1 Sargassum polycystum ribulos...products. 46 0.17 1 AF244338 |AF244338.1 Sargassum patens ribulose-1,5-bisphosphate carboxylase/oxygenase la...ds; chloroplast genes for chloroplast products. 46 0.17 1 AF076689 |AF076689.1 Sargassum...168O8, *** SEQUENCING IN PROGRESS ***, 2 unordered pieces. 46 0.17 1 AF244342 |AF244342.1 Sargassum sp. stra... for chloroplast products. 46 0.17 1 AF244336 |AF244336.1 Sargassum echinocarpum strain nep95 ribulose-1,5-b

  7. (Anthracen-9-yl(piperidin-1-ylmethanone

    Directory of Open Access Journals (Sweden)

    Hua-You Hu

    2008-11-01

    Full Text Available The title compound, C20H19NO, is a substructure of CP-640186, a potent inhibitor of mammalian acetyl-coenzyme A carboxylases. In the crystal structure, the amide group forms a dihedral angle of 87.0 (1° with the plane of the anthracene unit and the piperidine ring adopts a chair conformation. Molecules are arranged into layers parallel to (100 and adjacent anthracene units within layers form dihedral angles of 13.2 (1°. C—H...O interactions from the piperidine rings to the C=O group of the amide are observed between layers.

  8. Ectomycorrhizal association of three Lactarius species with Carpinus and Quercus trees in a Mexican montane cloud forest.

    Science.gov (United States)

    Lamus, Valentina; Montoya, Leticia; Aguilar, Carlos J; Bandala, Victor M; Ramos, David

    2012-01-01

    Ectomycorrhizal (EM) fungi are being monitored in the Santuario del Bosque de Niebla in the central region of Veracruz (eastern Mexico). Based on the comparison of DNA sequences (ITS rDNA) of spatiotemporally co-occurring basidiomes and EM root tips, we discovered the EM symbiosis of Lactarius indigo, L. areolatus and L. strigosipes with Carpinus caroliniana, Quercus xalapensis and Quercus spp. The host of the EM tips was identified by comparison of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL). Descriptions coupled with photographs of ectomycorrhizas and basidiomes are presented.

  9. Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

    Science.gov (United States)

    Boone-Villa, Daniel; Aguilera-Méndez, Asdrubal; Miranda-Cervantes, Adriana; Fernandez-Mejia, Cristina

    2015-10-01

    Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 μmol/mL; supplemented: 0.33±0.08 μmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

  10. Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise

    DEFF Research Database (Denmark)

    Roepstorff, Carsten; Halberg, Nils; Hillig, Thore

    2005-01-01

    conditions (P acetyl-CoA and acetylcarnitine were increased (P carnitine was decreased (P ...-activated protein kinase (a2-AMPK) was increased twice as much in L-CHO as in H-CHO (P acetyl-CoA carboxylase (ACC)ß Ser221 phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both...... of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation....

  11. Astrocytic control of biosynthesis and turnover of the neurotransmitters glutamate and GABA

    DEFF Research Database (Denmark)

    Schousboe, Arne; Bak, Lasse Kristoffer; Waagepetersen, Helle S

    2013-01-01

    . Astrocytes play a pivotal role in the maintenance of the neurotransmitter pools of glutamate and GABA since only these cells express pyruvate carboxylase, the enzyme required for de novo synthesis of the two amino acids. Such de novo synthesis is obligatory to compensate for catabolism of glutamate and GABA...... that GS is exclusively expressed in astrocytes. It should be kept in mind that the operation of the cycle is associated with movement of ammonia nitrogen between the two cell types and different mechanisms which can mediate this have been proposed. This review is intended to delineate the above mentioned...

  12. Computational analysis of the oscillatory dynamics in the processes of CO₂ assimilation and photorespiration.

    Science.gov (United States)

    Dubinsky, Andrey Yu; Ivlev, Alexander A

    2011-02-01

    The computational analysis of the model system consisting of the processes of CO₂ assimilation and photorespiration shows the appearance of sustained oscillations in the system which might reflect their presence in photosynthesizing cells. Concentrations of CO₂ and O₂ oscillate in opposite phases causing Rubisco switching continuously between the carboxylase (CO₂ assimilation) and the oxygenase (photorespiration) reactions. The results of modeling are consistent with carbon isotopic and other observed data. They show that the oscillation period varies from about 1s to 3s depending on the values of parameters taken. Too high concentrations of O₂ suppress the oscillations.

  13. Recent developments in photorespiration research.

    Science.gov (United States)

    Bauwe, Hermann

    2010-04-01

    Photorespiration is the light-dependent release of CO(2) initiated by Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) during oxygenic photosynthesis. It occurs during the biochemical reactions of the photorespiratory C(2) cycle, which is an ancillary metabolic process that allows photosynthesis to occur in oxygen-containing environments. Recent research has identified the genes for many plant photorespiratory enzymes, allowing precise functional analyses by reverse genetics. Similar studies with cyanobacteria disclosed the evolutionary origin of photorespiratory metabolism in these ancestors of plastids.

  14. Influence of pH on the /sup 14/C-labelling pattern after photosynthesis of suspended leaf slices and isolated mesophyll cells from chenopodium album in NaH/sup 14/CO/sub 3/

    Energy Technology Data Exchange (ETDEWEB)

    Baumann, G.; Guenther, G. (Paedagogische Hochschule Karl Liebknecht, Potsdam (German Democratic Republic). Sektion Chemie/Biologie)

    1983-01-01

    Photosynthetic fixation of /sup 14/C from solutions of NaH/sup 14/CO/sub 3/ (at constant concentrations of free CO/sub 2/) by suspended leaf slices or isolated mesophyll cells from Chenopodium album is increased with increasing pH. Above all, the incorporation of radioactivity into amino acids and malate is stimulated. A direct uptake of HCO/sub 3/ ions and its fixation by PEP carboxylase is suggested. Isolated mesophyll cells showed at pH 7.3 a higher rate of photosynthesis than at pH 5.0.

  15. Interaction of the nitrogen regulatory protein GlnB (PII) with biotin carboxyl carrier protein (BCCP) controls Acetyl-CoA levels in the cyanobacterium Synechocystis sp. PCC 6803

    OpenAIRE

    Waldemar Hauf; Katharina Schmid; Edileusa Cristina Marques Gerhardt; Luciano Fernandes Huergo; Karl Forchhammer

    2016-01-01

    The family of PII signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that PII proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of E. coli ACCase, this intera...

  16. Influence of age on leptin induced skeletal muscle signaling

    DEFF Research Database (Denmark)

    Guadalupe Grau, Amelia; Larsen, Steen; Guerra, Borja

    2014-01-01

    Age associated fat mass accumulation could be due to dysregulation of leptin signaling in skeletal muscle. Thus, we investigated total protein expression and phosphorylation levels of the long isoform of the leptin receptor (OB-Rb), and leptin signaling through Janus Kinase 2 (JAK2)/signal...... transducer and activator of transcription 3 (STAT3), insulin receptor substrate 1 (IRS-1), AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC), combined with the leptin signaling inhibitors suppressor of cytokine signaling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in human...

  17. Regulation of photosynthetic carbon fixation on the ocean margins. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Paul, J.H.

    1997-06-01

    The US Department of Energy is concerned with the fate of energy-related materials, including carbon dioxide, in the marine environment. Using laboratory studies, as well as field studies, an attempt was made to understand the molecular regulation of photosynthetic carbon reduction. The objectives were: to determine the mechanism of regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in phytoplankton in response to changes in light fields; and to determine regulation of (RuBPCase) in response to light under nutrient deprivation.

  18. Carbon isotope geochemistry and geobiology

    Science.gov (United States)

    Desmarais, D.

    1985-01-01

    Carbon isotope fractionation values were used to understand the history of the biosphere. For example, plankton analyses confirmed that marine extinctions at the end of the Cretaceous period were indeed severe (see Hsu's article in Sundquist and Broeker, 1984). Variations in the isotopic compositions of carbonates and evaporitic sulfates during the Paleozoic reflect the relative abundances of euxinic (anoxic) marine environments and organic deposits from terrestrial flora. The carbon isotopic composition of Precambrian sediments suggest that the enzyme ribulose bisphosphate carboxylase has existed for perhaps 3.5 billion years.

  19. Identification of hepatic biomarkers for physiological imbalance of dairy cows in early and mid lactation using proteomic technology

    DEFF Research Database (Denmark)

    Moyes, Kasey; Bendixen, Emøke; Codrea, Marius Cosmin;

    2013-01-01

    either the greatest (PI) or least (normal; N) degree of PI and were used for isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative profiling in liver using liquid chromatography-tandem mass spectrometry. We identified pyruvate carboxylase and isocitrate dehydrogenase...... as potential hepatic biomarkers for PI for cows during early lactation and alcohol dehydrogenase-4 and methylmalonate-semialdehyde dehydrogenase for cows in mid lactation. This preliminary study identified new biomarkers in liver for PI and provided a better understanding of the differences in coping...

  20. [Construction and fermentation control of reductive TCA pathway for malic acid production in Saccharomyces cerevisiae].

    Science.gov (United States)

    Yan, Daojiang; Wang, Caixia; Zhou, Jiemin; Liu, Yilan; Yang, Maohua; Xing, Jianmin

    2013-10-01

    Malic acid is widely used in food, and chemical industries. Through overexpressing pyruvate carboxylase and malate dehydrogenase in pdc1-deficient Saccharomyces cerevisiae, malic acid was successfully produced through the reductive TCA pathway. No malic acid was detected in wild type Saccharomyces cerevisiae, however, 45 mmol/L malic acid was produced in engineered strain, and the concentration of byproduct ethanol also reduced by 18%. The production of malic acid enhanced 6% by increasing the concentration of Ca2+. In addition, the final concentration reached 52.5 mmol/L malic acid by addition of biotin. The increasing is almost 16% higher than that of the original strain.

  1. Extracting DNA from submerged pine wood.

    Science.gov (United States)

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

    A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

  2. Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Menzel, G; Harloff, H-J; Jung, C

    2003-01-01

    Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

  3. Nitrosomonas communis strain YNSRA, an ammonia-oxidizing bacterium, isolated from the reed rhizoplane in an aquaponics plant.

    Science.gov (United States)

    Tokuyama, Tatsuaki; Mine, Atsusi; Kamiyama, Kaoru; Yabe, Ryuichi; Satoh, Kazuo; Matsumoto, Hirotoshi; Takahashi, Reiji; Itonaga, Koji

    2004-01-01

    An ammonia-oxidizing bacterium (strain YNSRA) was isolated from the rhizoplane of the reed (Phragmites communis) used in an aquaponics plant which is a wastewater treatment plant. Strain YNSRA was identified as Nitrosomonas communis by taxonomic studies. The hydroxylamine-cytochrome c reductase (HCR) of strain YNSRA was found to have a higher activity (25.60 u/mg) than that of Nitrosomonas europaea ATCC25978T (8.94 u/mg). Ribulose-1,5-bisphosphate carboxylase (RubisCO) activity was detected at very low levels in strain YNSRA, whereas strain ATCC25978T had definite activity.

  4. Enhanced succinate production from glycerol by engineered Escherichia coli strains.

    Science.gov (United States)

    Li, Qing; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-10-01

    In this study, an engineered strain Escherichia coli MLB (ldhA(-)pflB(-)) was constructed for production of succinate from glycerol. The succinate yield was 0.37mol/mol in anaerobic culture, however, the growth and glycerol consumption rates were very slow, resulting in a low succinate level. Two-stage fermentation was performed in flasks, and the succinate yield reached 0.93mol/mol, but the succinate titer was still low. Hence, overexpression of malate dehydrogenase, malic enzyme, phosphoenolpyruvate (PEP) carboxylase and PEP carboxykinase (PCK) from E. coli, and pyruvate carboxylase from Corynebacterium glutamicum in MLB was investigated for improving succinate production. Overexpression of PCK resulted in remarkable enhancement of glycerol consumption and succinate production. In flask experiments, the succinate concentration reached 118.1mM, and in a 1.5-L bioreactor the succinate concentration further increased to 360.2mM. The highest succinate yield achieved 0.93mol/mol, which was 93% of the theoretical yield, in the anaerobic stage.

  5. Proline biosynthesis genes and their regulation under salinity stress in the euryhaline copepod Tigriopus californicus.

    Science.gov (United States)

    Willett, Christopher S; Burton, Ronald S

    2002-08-01

    Diverse organisms regulate concentrations of intracellular organic osmolytes in response to changes in environmental salinity or desiccation. In marine crustaceans, accumulation of high concentrations of proline is a dominant component of response to hyperosmotic stress. In the euryhaline copepod Tigriopus californicus, synthesis of proline from its metabolic precursor glutamate is tightly regulated by changes in environmental salinity. Here, for the first time in a marine invertebrate, the genes responsible for this pathway have been cloned and characterized. The two proteins display the sequence features of homologous enzymes identified from other eukaryotes. One of the cloned genes, delta1-pyrroline-5-carboxylase reductase (P5CR), is demonstrated to have the reductase enzyme activity when expressed in proline-auxotroph bacteria, while the second, delta1-pyrroline-5-carboxylase synthase (P5CS), does not rescue proline-auxotroph bacteria. In contrast to results from higher plants, neither levels of P5CS nor P5CR mRNAs increase in response to salinity stress in T. californicus. Hence, regulation of proline synthesis during osmotic stress in T. californicus is likely mediated by some form of post-transcriptional regulation of either P5CS or P5CR. Understanding the regulation this pathway may elucidate the mechanisms limiting the salinity ranges of marine taxa.

  6. Overproduction of fatty acids in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Li, Xiaowei; Guo, Daoyi; Cheng, Yongbo; Zhu, Fayin; Deng, Zixin; Liu, Tiangang

    2014-09-01

    The long hydrocarbon fatty acyl chain is energy rich, making it an ideal precursor for liquid transportation fuels and high-value oleo chemicals. As Saccharomyces cerevisiae has many advantages for industrial production compared to Escherichia coli. Here, we attempted to engineer Saccharomyces cerevisiae for overproduction of fatty acids. First, disruption of the beta-oxidation pathway, elimination of the acyl-CoA synthetases, overexpression of different thioesterases and acetyl-CoA carboxylase ACC1, and engineering the supply of precursor acetyl-CoA. The engineered strain XL122 produced more than 120 mg/L of fatty acids. In parallel, we inactivated ADH1, the dominant gene for ethanol production, to redirect the metabolic flux to fatty acids synthesis. The engineered strain DG005 produced about 140 mg/L fatty acids. Additionally, Acetyl-CoA carboxylase was identified as a critical bottleneck of fatty acids synthesis in S. cerevisiae with a cell-free system. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 were further identified. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step. All these efforts, particularly the discovery of the limiting step are critical for developing a "cell factory" for the overproduction of fatty acids by using type I fatty acids synthase in yeast or other fungi.

  7. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    Science.gov (United States)

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  8. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  9. Observations of the uptake of carbonyl sulfide (COS by trees under elevated atmospheric carbon dioxide concentrations

    Directory of Open Access Journals (Sweden)

    L. Sandoval-Soto

    2012-02-01

    Full Text Available Global change affects ecosystems to adapt to elevated atmospheric concentrations of carbon dioxide (CO2. We understand that carbonyl sulfide (COS, a trace gas which is involved in building up the stratospheric sulfate aerosol layer, is taken up by vegetation with the same triad of the enzmyes which are metabolizing the CO2, i.e. Ribulose-1,5-bisphosphate Carboxylase-Oxygenase (Rubisco, Phosphoenolpyruvate Carboxylase (PEP-Co and carbonic anhydrase (CA. Therefore, we discuss a physiological/biochemical adaptation of these enzymes to affect the sink strength of vegetation for COS. We investigated the adaption of two European tree species, Fagus sylvatica and Quercus ilex, grown inside chambers under elevated CO2 and determined the exchange characteristics and the content of CA after a 1–2 yr period of adaption from 350 ppm to 800 ppm CO2. We could demonstrate that the COS compensation point, the CA activity and the deposition velocities may change and cause a decrease of the COS uptake by plant ecosystems. As a consequence, the atmospheric COS level may rise leading to higher input of this trace gas into the stratosphere and causing a higher energy reflection by the stratospheric sulfur aerosol into space, thus counteracting the direct radiative forcing by the tropospheric COS.

  10. [Carbon isotope (13C/12C) effect of photorespiration in photosynthetic organisms. Evidence for existence, probable mechanism].

    Science.gov (United States)

    Ivlev, A A

    2002-01-01

    Experimental evidence in favor of the new phenomenon predicted for photosynthesizing organisms, the fractionation of carbon isotopes in photorespiration is presented. A possible mechanism of this process is discussed. The fractionation of carbon in isotopes photorespiration occurs in the oxygenase phase of the functioning of ribulosebisphosphate carboxylase/oxygenase (rubisco), the key enzyme of photosynthesis, which is capable to act as carboxylase and oxygenase. Which function of the enzyme is active depends on CO2/O2 concentration ratio, which periodically changes in a cell. The key reaction in the mechanism of carbon isotope fractionation in photorespiration is glycine decarboxylation, which results in the splitting and removal from the cell of CO2 enriched with 12C and the accumulation of 13C photorespiratory carbon flow. The coupling of photorespiration and CO2 photoassimilation gives rise to two isotopically different carbon flows, which fill up separate carbohydrate pools, which are the sources of carbon in the following syntheses in the dark phase of photosynthesis. This enables one to identify, from the carbon isotope ratio of metabolites, their involvement in the photorespiratory and assimilatory carbon flows, to investigate the pathways of carbon metabolism, and to estimate more thoroughly the biosynthetic role of photorespiration.

  11. Changes in C-N metabolism under elevated CO2 and temperature in Indian mustard (Brassica juncea L.): an adaptation strategy under climate change scenario.

    Science.gov (United States)

    Seth, Chandra Shekhar; Misra, Virendra

    2014-11-01

    The present study was performed to investigate the possible role of carbon (C) and nitrogen (N) metabolism in adaptation of Indian mustard (Brassica juncea L.) growing under ambient (370 ± 15 ppm) and elevated CO2 (700 ± 15 ppm), and jointly in elevated CO2 and temperature (30/22 °C for day/night). The key enzymes responsible for C-N metabolism were studied in different samples of Brassica juncea L. collected from ambient (AMB), elevated (ELE) and ELExT growth conditions. Total percent amount of C and N in leaves were particularly estimated to establish a clear understanding of aforesaid metabolism in plant adaptation. Furthermore, key morphological and physiological parameters such as plant height, leaf area index, dry biomass, net photosynthetic rate, stomatal conductance, transpiration, total protein and chlorophyll contents were also studied in relation to C/N metabolism. The results indicated that the C-metabolizing enzymes, such as (ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, NAD-malic enzyme, NADP-malic enzyme and citrate synthase) and the N-metabolizing enzymes, such as (aspartate amino transferase, glutamine synthetase, nitrate reductase and nitrite reductase) showed significantly (P ELExT > AMB growth conditions. This is also evident by significant (P adaptation in Brassica juncea L. against elevated CO2 and temperature prevailing in climate change scenarios.

  12. Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

    Energy Technology Data Exchange (ETDEWEB)

    Mark A. Eiteman

    2005-11-01

    This report describes results toward developing a process to sequester CO{sub 2} centered on the enzyme pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The first phase of this research has focused on strain development and on process development. Progress in strain development has been made in three areas. The gene encoding for alcohol dehydrogenase has been ''knocked out'' of the bacteria, and thereby eliminating the synthesis of the by-product ethanol. The gene for glucokinase has been overexpressed in the production strain with the goal of faster utilization of glucose (and hence CO{sub 2}). Efforts have continued toward integrating pyruvate carboxylase gene (pyc) onto the E. coli chromosome. Progress in process development has come in conducting several dozen fermentation experiments to find a defined medium that would be successful for the growth of the bacteria, while permitting a high rate of CO{sub 2} utilization in a subsequent prolonged production phase. Using this defined medium, the strains that continue to be constructed are being compared for CO{sub 2} utilization, so that we may understand the factors that govern the biological sequestration process.

  13. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

    Science.gov (United States)

    Al-Haideri, Halah; White, Michael A; Kelly, David J

    2016-02-01

    Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.

  14. Tobacco guard cells fix CO2 by both Rubisco and PEPcase while sucrose acts as a substrate during light-induced stomatal opening.

    Science.gov (United States)

    Daloso, Danilo M; Antunes, Werner C; Pinheiro, Daniela P; Waquim, Jardel P; Araújo, Wagner L; Loureiro, Marcelo E; Fernie, Alisdair R; Williams, Thomas C R

    2015-11-01

    Transcriptomic and proteomic studies have improved our knowledge of guard cell function; however, metabolic changes in guard cells remain relatively poorly understood. Here we analysed metabolic changes in guard cell-enriched epidermal fragments from tobacco during light-induced stomatal opening. Increases in sucrose, glucose and fructose were observed during light-induced stomatal opening in the presence of sucrose in the medium while no changes in starch were observed, suggesting that the elevated fructose and glucose levels were a consequence of sucrose rather than starch breakdown. Conversely, reduction in sucrose was observed during light- plus potassium-induced stomatal opening. Concomitant with the decrease in sucrose, we observed an increase in the level as well as in the (13) C enrichment in metabolites of, or associated with, the tricarboxylic acid cycle following incubation of the guard cell-enriched preparations in (13) C-labelled bicarbonate. Collectively, the results obtained support the hypothesis that sucrose is catabolized within guard cells in order to provide carbon skeletons for organic acid production. Furthermore, they provide a qualitative demonstration that CO2 fixation occurs both via ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPcase). The combined data are discussed with respect to current models of guard cell metabolism and function.

  15. Fe deficiency induced changes in rice (Oryza sativa L.) thylakoids.

    Science.gov (United States)

    Wang, Yuwen; Xu, Chao; Li, Kang; Cai, Xiaojie; Wu, Min; Chen, Guoxiang

    2017-01-01

    Iron deficiency is an important abiotic stress that limits productivity of crops all over the world. We selected a hybrid rice (Oryza sativa L.), LYPJ, which is super high-yield and widely cultured in China, to investigate changes in the components and structure of thylakoid membranes and photosynthetic performance in response to iron deficiency. Our results demonstrated that photosystem I (PSI) is the primary target for iron deficiency, while the changes in photosystem II (PSII) are important for rebuilding a balance in disrupted energy utilization and dissipation caused by differential degradation of photosynthetic components. The result of immunoblot analysis suggested that the core subunit PsaA declined drastically, while PsbA remained relatively stable. Furthermore, several organizational changes of the photosynthetic apparatus were found by BN-PAGE, including a marked decrease in the PSI core complexes, the Cytb 6 /f complex, and the trimeric form of the LHCII antenna, consistent with the observed unstacking grana. The fluorescence induction analysis indicated a descending PSII activity with energy dissipation enhanced markedly. In addition, we proposed that the crippled CO2 assimilation could be compensated by the enhanced of phosphoenolpyruvate carboxylase (PEPC), which is suggested by the decreased ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and photosynthetic efficiency.

  16. The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

    Science.gov (United States)

    Picossi, Silvia; Flores, Enrique; Herrero, Antonia

    2015-09-01

    Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph.

  17. New Insights into the Nitrogen Form Effect on Photosynthesis and Photorespiration

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Under high light conditions, ammonium nutrition has a negative effect on plant growth. This suggests that the adverse effects of ammonium nutrition on plant growth may be related to carbon gain, photosynthesis, and photorespiration.However, there is no consistent evidence of a specific mechanism that could explain the plant growth reduction under ammonium supply. It is generally accepted that during the light reaction, a surplus of nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) is produced, which is not completely used during the assimilation of CO2. Nitrate reduction in the leaf represents an additional sink for NADPH that is not available to ammonium-grown plants. Nitrate and ammonium nutrition may use different pathways for NADPH consumption, which leads to differences in photosynthesis and photorespiration. The morphological (i.e., cell size, mesophyll thickness, and chloroplast volume) and enzymic (i.e.,ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase), and glutamine synthetase/glutamate synthetase (GS/GOGAT)) differences that develop when plants are treated with either nitrate or ammonium nitrogen forms are related to photosynthesis and photorespiration. The differences in photorespiration rate for plants treated with nitrate or ammonium are related to the conversion of citrate to 2-oxoglutarate (2-OG) and photorespiratory CO2 refixation.

  18. Geniposide regulates glucose-stimulated insulin secretion possibly through controlling glucose metabolism in INS-1 cells.

    Directory of Open Access Journals (Sweden)

    Jianhui Liu

    Full Text Available Glucose-stimulated insulin secretion (GSIS is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM. Although the KATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.

  19. Activities of carboxylating enzymes in the CAM species Opuntia ficus-indica grown under current and elevated CO2 concentrations.

    Science.gov (United States)

    Israel, A A; Nobel, P S

    1994-06-01

    Responses of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase) to an elevated atmospheric CO2 concentration were determined along with net CO2 uptake rates for the Crassulacean acid metabolism species Opuntia ficus-indica growing in open-top chambers. During the spring 13 months after planting, total daily net CO2 uptake of basal and first-order daughter cladodes was 28% higher at 720 than at 360 μl CO2 l(-1). The enhancement, caused mainly by higher CO2 assimilation during the early part of the night, was also observed during late summer (5 months after planting) and the following winter. The activities of Rubisco and PEPCase measured in vitro were both lower at the elevated CO2 concentration, particularly under the more favorable growth conditions in the spring and late summer. Enzyme activity in second-order daughter cladodes increased with cladode age, becoming maximal at 6 to 10 days. The effect ofelevated CO2 on Rubisco and PEPCase activity declined with decreasing irradiance, especially for Rubisco. Throughout the 13-month observation period, O. ficus-indica thus showed increased CO2 uptake when the atmospheric CO2 concentration was doubled despite lower activities of both carboxylating enzymes.

  20. Mammalian ACSF3 protein is a malonyl-CoA synthetase that supplies the chain extender units for mitochondrial fatty acid synthesis.

    Science.gov (United States)

    Witkowski, Andrzej; Thweatt, Jennifer; Smith, Stuart

    2011-09-30

    The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.

  1. The genomic region of rbcLS in Synechococcus sp. PCC 7942 contains genes involved in the ability to grow under low CO2 concentration and in chlorophyll biosynthesis.

    Science.gov (United States)

    Ronen-Tarazi, M; Lieman-Hurwitz, J; Gabay, C; Orus, M I; Kaplan, A

    1995-08-01

    Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.

  2. Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

    Science.gov (United States)

    Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

    2016-04-01

    Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.

  3. Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

    Energy Technology Data Exchange (ETDEWEB)

    Mark A. Eiteman

    2006-07-31

    This report describes results toward developing a process to sequester CO{sub 2} centered on the enzyme pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The phases of research have included strain development and process development. Though we continue to work on one important component of strain development, the research has principally focused on process development. In the previous year we constructed several strains which would serve as templates for the CO{sub 2} sequestration, including the knock-out of genes involved in the formation of undesirable byproducts. This project period the focus has been on the integration of the pyruvate carboxylase gene (pyc) onto the E. coli chromosome. This has proven to be a difficult task because of relatively low expression of the gene and resulting low enzyme activity when only one copy of the gene is present on the chromosome. Several molecular biology techniques have been applied, with some success, to improve the level of protein activity as described herein. Progress in process development has come as a result of conducting numerous fermentation experiments to select optimal conditions for CO{sub 2} sequestration. This process-related research has progressed in four areas. First, we have clarified the range of pH which results in the optimal rate of sequestration. Second, we have determined how the counterion used to control the pH affects the sequestration rate. Third, we have determined how CO{sub 2} gas phase composition impacts sequestration rate. Finally, we have made progress in determining the affect of several potential gaseous impurities on CO{sub 2} sequestration; in particular we have completed a study using NO{sub 2}. Although the results provide significant guidance as to process conditions for CO{sub 2} sequestration and succinate production, in some cases we do not yet understand

  4. Photosynthesis of C3, C3–C4, and C4 grasses at glacial CO2

    Science.gov (United States)

    Pinto, Harshini; Sharwood, Robert E.; Tissue, David T.; Ghannoum, Oula

    2014-01-01

    Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3–C4 grass grown under ambient (400 μl l–1) and glacial (180 μl l–1) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3–C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3–C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3–C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3–C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3–C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments. PMID:24723409

  5. Photosynthesis of C3, C3-C4, and C4 grasses at glacial CO2.

    Science.gov (United States)

    Pinto, Harshini; Sharwood, Robert E; Tissue, David T; Ghannoum, Oula

    2014-07-01

    Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 μl l(-1)) and glacial (180 μl l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.

  6. Influences of dietary biotin and avidin on growth, survival, deficiency syndrome and hepatic gene expression of juvenile Nile tilapia Oreochromis niloticus.

    Science.gov (United States)

    Sarker, Pallab Kumer; Yossa, Rodrigue; Karanth, Santhosh; Ekker, Marc; Vandenberg, Grant W

    2012-08-01

    This study was undertaken to assess the interactive effects of dietary biotin and avidin on growth, feed conversion, survival and deficiency syndrome of tilapia and to determine the influence of dietary biotin deficiency on the expression of key genes related to biotin metabolism in tilapia. Six iso-nitrogenous and iso-energetic diets based on a common purified basal diet (vitamin-free casein as the protein source) were prepared for this study. The six dietary groups were 0 g avidin with 0 mg biotin (A0B0), 0 g avidin with 0.06 mg biotin/kg diet (A0B1), four avidin-supplemented diets incorporating at a incremental concentrations 0.25, 0.5, 1.0 and 2.0 g/kg diet with 0.06 mg biotin/kg diet (A15B1, A30B1, A60B1 and A120B1). Fish were hand-fed three times a day to apparent satiation for 12 weeks. Each diet was fed to three replicate groups of fish. Fish were kept in glass aquaria in a recirculating aquaculture system under standardized environmental conditions. Growth was significantly higher in fish that received the biotin-supplemented diet (A0B1), compared to diets lacking biotin or supplemented with avidin. Tilapia fed higher concentration of avidin-supplemented diets (A60B1 and A120B1) showed significant growth depression and displayed severe deficiency syndromes such as lethargy, anorexia, circular swimming and convulsions, which ultimately lead to death. There was a strong proportional linear relationship between the avidin content of the diet and feed conversion ratio, FCR (y = 0.43x + 0.135; r = 0.960; P protein efficiency ratio, PER (y = -0.309x + 2.195; r = 0.961; P levels of biotinidase, pyruvate carboxylase, propionyl-CoA carboxylase-A and propionyl-CoA carboxylase-B transcripts were noted in fish fed all graded level of avidin-supplemented diets. A broken-line analysis indicated that feeding tilapia a diet with 44.5 times more avidin than the dietary biotin requirement can induce deficiency syndromes including retarded growth, when

  7. Propionic acidemia associated with visual hallucinations.

    Science.gov (United States)

    Shuaib, Taghreed; Al-Hashmi, Nadia; Ghaziuddin, Mohammad; Megdad, Eman; Abebe, Dejene; Al-Saif, Amr; Doubi, Alaa; Aldhalaan, Hesham; Abouzied, Mohei Eldin; Al-Owain, Mohammed

    2012-06-01

    Propionic acidemia, an autosomal recessive disorder, is a common form of organic aciduria resulting from the deficiency of propionyl-CoA carboxylase. It is characterized by frequent and potentially lethal episodes of metabolic acidosis often accompanied by hyperammonemia. A wide range of brain abnormalities have been reported in propionic acidemia. We report recurrent visual hallucinations in 2 children with propionic acidemia. Four visual hallucination events were observed in the 2 patients. Three episodes were preceded by an intercurrent illness, and 2 were associated with mild metabolic decompensation. The 2 events in one patient were associated with a seizure disorder with abnormal electroencephalogram. Brain magnetic resonance imaging showed abnormal basal ganglia and faint temporo-occipital swelling bilaterally. This is probably the first report of visual hallucinations in propionic acidemia and should alert the treating clinicians to look for visual hallucinations in patients with organic acidurias, especially in an unusually anxious child.

  8. Body weight management effect of burdock (Arctium lappa L.) root is associated with the activation of AMP-activated protein kinase in human HepG2 cells.

    Science.gov (United States)

    Kuo, Daih-Huang; Hung, Ming-Chi; Hung, Chao-Ming; Liu, Li-Min; Chen, Fu-An; Shieh, Po-Chuen; Ho, Chi-Tang; Way, Tzong-Der

    2012-10-01

    Burdock (Arcticum lappa L.) root is used in folk medicine and also as a vegetable in Asian countries. In the present study, burdock root treatment significantly reduced body weight in rats. To evaluate the bioactive compounds, we successively extracted the burdock root with ethanol (AL-1), and fractionated it with n-hexane (AL-2), ethyl acetate (AL-3), n-butanol (AL-4), and water (AL-5). Among these fractions, AL-2 contained components with the most effective hypolipidemic potential in human hepatoma HepG2 cells. AL-2 decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) by stimulating AMP-activated protein kinase (AMPK) through the LKB1 pathway. Three active compounds were identified from the AL-2, namely α-linolenic acid, methyl α-linolenate, and methyl oleate. These results suggest that burdock root is expected to be useful for body weight management.

  9. Improving cell growth and lipid accumulation in green microalgae Chlorella sp. via UV irradiation.

    Science.gov (United States)

    Liu, Shuyu; Zhao, Yueping; Liu, Li; Ao, Xiyong; Ma, Liyan; Wu, Minghong; Ma, Fang

    2015-04-01

    Microalgae with high biomass and high lipid content are the ideal feedstock for biodiesel production. To obtain such microalgae, ultraviolet (UV) irradiation was applied to Chlorella sp. to induce mutagenesis. The growth characteristics, total nitrogen (TN), and biochemical compositions of the control and UV mutation strains were analyzed. Compared to the control strain, the biomass for the UV mutation strain was 7.6 % higher and it presented a higher growth rate. The lipid content of the UV mutation strain showed different levels of increase and reached the maximum value of 28.1 % on day 15. Furthermore, the lipid productivity of the UV mutation strain showed a desired increase. The nitrogen consumption and Acetyl-CoA carboxylase (ACC) activity contributed to the lipid production by UV. All these results indicate that UV mutagenesis is an efficient method to improve probability for using Chlorella sp. as the potential raw material for biodiesel production.

  10. Dicty_cDB: Contig-U15036-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available uence 14020 from Patent WO20090... 220 3e-55 CP000614_2701( CP000614 |pid:none) Burkholderia vietnamiensis G...-56 CP001157_119( CP001157 |pid:none) Azotobacter vinelandii DJ, compl... 221 1e-55 CP000438_5745( CP000438 |pid:none) Pseudomona...zole carboxylase ... 158 1e-36 AL939115_7( AL939115 |pid:none) Streptomyces coelicolor A3(2) comp... 158 1e-...0 3e-06 3 >( BJ437546 ) Dictyostelium discoideum cDNA clone:ddv34i15, 3' end, single read. Length = 756 Scor...FM180568_455( FM180568 |pid:none) Escherichia coli 0127:H6 E2348/6... 150 3e-34 CP001063_404( CP001063 |pid:none) Shigella boydii

  11. The antisense expression of AhPEPC1 increases seed oil production in peanuts (Arachis hypogaea L.

    Directory of Open Access Journals (Sweden)

    L. Pan

    2016-12-01

    Full Text Available Although phosphoenolpyruvate carboxylases (PEPCs are reported to be involved in fatty acid accumulation, nitrogen assimilation, and salt and drought stresses, knowledge regarding PEPC gene functions is still limited, particularly in peanuts (Arachis hypogaea L.. In this study, the antisense expression of the peanut PEPC isoform 1 (AhPEPC1 gene increased the lipid content by 5.7%–10.3%. This indicated that AhPEPC1 might be related to plant lipid accumulation. The transgenic plants underwent more root elongation than the wild-type under salinity stress. Additionally, the specific down regulation of the AhPEPC1 gene improved the salt tolerance in peanuts. This is the first report on the role of PEPC in lipid accumulation and salt tolerance in peanuts.

  12. Physical activity is associated with retained muscle metabolism in human myotubes challenged with palmitate

    DEFF Research Database (Denmark)

    Green, C J; Bunprajun, T; Pedersen, B K

    2013-01-01

    in satellite cells challenged with palmitate. Although the benefits of physical activity on whole body physiology have been well investigated, this paper presents novel findings that both diet and exercise impact satellite cells directly. Given the fact that satellite cells are important for muscle maintenance......  The aim of this study was to investigate whether physical activity is associated with preserved muscle metabolism in human myotubes challenged with saturated fatty acids. Human muscle satellite cells were isolated from sedentary or active individuals and differentiated into myocytes in culture......) serine(307) compared to myocytes from active individuals. Despite equal lipid accumulation following palmitate treatment, myocytes from sedentary individuals exhibited delayed acetyl coenzyme A carboxylase phosphorylation compared to the active group. Myocytes from sedentary individuals had significantly...

  13. A versatile Escherichia coli strain for identification of biotin transporters and for biotin quantification.

    Science.gov (United States)

    Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

    2014-01-01

    Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays.

  14. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships.

    Science.gov (United States)

    Tabita, F Robert; Satagopan, Sriram; Hanson, Thomas E; Kreel, Nathan E; Scott, Stephanie S

    2008-01-01

    There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

  15. AMPK Activation Affects Glutamate Metabolism in Astrocytes

    DEFF Research Database (Denmark)

    Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H

    2015-01-01

    on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic...... acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK...

  16. AcEST: DK950350 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST38A01NGRL0008_G19 684 Adiantum capillus-veneris mRNA. clone: TST38A01NGRL0008_G1...9. 5' end sequence. DK950350 CL17Contig1 Show DK950350 Clone id TST38A01NGRL0008_G19 Library TST38 Length 68...4 Definition Adiantum capillus-veneris mRNA. clone: TST38A01NGRL0008_G19. 5' end sequence. Accession DK950350...tein database search programs, Nucleic Acids Res. 25:3389-3402. Query= DK950350|A...oxylase/oxygenase ... 286 9e-77 sp|O98997|RCA_PHAAU Ribulose bisphosphate carboxylase/oxygenase ... 281 2e-75 sp|Q42450

  17. Silky bent grass resistance to herbicides: one year of monitoring in Belgium.

    Science.gov (United States)

    Henriet, F; Bodson, B; Morales, R Meza

    2013-01-01

    Silky bent grass (Apera spica-venti (L.) P. Beauv.) is a common weed of cereal crops widely spread in Northern and Easthern Europe (Germany, Czech Republic,...), Northern Asia, Siberia and Canada. Up to now, no resistant case has been detected in Belgium but some chemical weeding failures have been observed in Wallonia fields. During summer 2011, 37 seed samples of Apera spica-venti were collected in Wallonia and submitted to resistance tests in controlled conditions. Three modes of action were tested: acetyl coenzyme-A carboxylase inhibitors (pinoxaden and cycloxydim), acetolactate synthase inhibitors (mesosulfuron+iodosulfu-ron, pyroxsulam and sulfometuron) and photosynthesis inhibitors (isoproturon). One susceptible standard population was included in the test in order to validate it and to permit wild populations classification according to "R" rating system developed by Moss et al (2007). Most of populations were susceptible but some populations showed resistance to at least one of the three tested modes of action.

  18. Crystallization and Characterization of Galdieria sulphuraria RUBISCO in Two Crystal Forms: Structural Phase Transition Observed in P21 Crystal Form

    Directory of Open Access Journals (Sweden)

    Boguslaw Stec

    2007-10-01

    Full Text Available We have isolated ribulose-1,5-bisphosphate-carboxylase/oxygenase (RUBISCOfrom the red algae Galdieria Sulphuraria. The protein crystallized in two different crystalforms, the I422 crystal form being obtained from high salt and the P21 crystal form beingobtained from lower concentration of salt and PEG. We report here the crystallization,preliminary stages of structure determination and the detection of the structural phasetransition in the P21 crystal form of G. sulphuraria RUBISCO. This red algae enzymebelongs to the hexadecameric class (L8S8 with an approximate molecular weight 0.6MDa.The phase transition in G. sulphuraria RUBISCO leads from two hexadecamers to a singlehexadecamer per asymmetric unit. The preservation of diffraction power in a phasetransition for such a large macromolecule is rare.

  19. Cell Membrane Softening in Cancer Cells

    Science.gov (United States)

    Schmidt, Sebastian; Händel, Chris; Käs, Josef

    Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

  20. Synthesis and biological activity of novel tiliroside derivants.

    Science.gov (United States)

    Qin, Nan; Li, Chun-Bao; Jin, Mei-Na; Shi, Li-Huan; Duan, Hong-Quan; Niu, Wen-Yan

    2011-10-01

    A series of new tiliroside derivatives were synthesized and characterized by analytical (1)H NMR, (13)C NMR and mass spectrometry. All of the compounds were evaluated for anti-diabetic properties in vitro using HepG2 cells. Compounds 3c, 3d, and 3i-l caused significant enhancements in glucose consumption by insulin-resistant HepG2 cells compared with control cells and cells that were exposed to metformin (an anti-diabetic drug). Moreover, compound 3l significantly activated adenosine 5'-monophosphate-activated protein kinase activity and reduced acetyl-CoA carboxylase activity. Thus, the tiliroside derivative 3l offers potential to be developed as a new approach for treating type II diabetes.

  1. Organic acid-tolerant microorganisms and uses thereof for producing organic acids

    Science.gov (United States)

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-05-06

    Organic acid-tolerant microorganisms and methods of using same. The organic acid-tolerant microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid. Further modifications to the microorganisms such as increasing expression of malonyl-CoA reductase and/or acetyl-CoA carboxylase provide or increase the ability of the microorganisms to produce 3HP. Methods of generating an organic acid with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers include replacing acsA or homologs thereof in cells with genes of interest and selecting for the cells comprising the genes of interest with amounts of organic acids effective to inhibit growth of cells harboring acsA or the homologs.

  2. Diversity of Bacterial Photosymbionts in Lubomirskiidae Sponges from Lake Baikal

    Directory of Open Access Journals (Sweden)

    Nina V. Kulakova

    2014-01-01

    Full Text Available Sponges are permanent benthos residents which establish complex associations with a variety of microorganisms that raise interest in the nature of sponge-symbionts interactions. A molecular approach, based on the identification of the 16S rRNA and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes, was applied to investigate diversity and phylogeny of bacterial phototrophs associated with four species of Lubomirskiidae in Lake Baikal. The phylogeny inferred from both genes showed three main clusters of Synechococcus associated with Baikalian sponges. One of the clusters belonged to the cosmopolitan Synechococcus rubescens group and the two other were not related to any of the assigned phylogenetic groups but placed as sister clusters to S. rubescens. These results expanded the understanding of freshwater sponge-associated photoautotroph diversity and suggested that the three phylogenetic groups of Synechococcus are common photosynthetic symbionts in Lubomirskiidae sponges.

  3. The use of Pyr-mutants to modify pyrimidine metabolism in lactococci

    DEFF Research Database (Denmark)

    Hansen, Steen Lyders Lerche; Martinussen, Jan; Hammer, Karin

    1998-01-01

    The specific engineering of organisms used for startercultures has become an effective way of improving and developing new products in the dairy industry. In order to obtain strains with specific characteristics, it is imperative to have a good understanding of the central biochemical pathways...... . Any growing organism needs nucleotides in order to be able to synthesise DNA, RNA and several co-enzymes. This demand can be met in two ways 1. By de novo synthesis of nucleotides, or 2. By exploiting nucleotides, nucleosides and nucleobases arising from degradation of DNA or RNA or taken up from......-synthetase catalyses synthesis of CTP from UTP. We have cloned the pyrG gene of Lactococcus lactis subsp. cremoris MG1363 and constructed a pyrG mutant that requires cytidine for growth. Furthermore a mutant carrying a chromosomal deletion of the de novo gene pyrF, encoding orotidylat carboxylase, has been constructed...

  4. The use of pyr-mutations to modify pyrimidine pools in Lactococci

    DEFF Research Database (Denmark)

    Hansen, Steen Lyders Lerche; Martinussen, Jan; Hammer, Karin

    1999-01-01

    The specific engineering of organisms used for startercultures has become an effective way of improving and developing new products in the dairy industry. In order to obtain strains with specific characteristics, it is imperative to have a good understanding of the central biochemical pathways...... . Any growing organism needs nucleotides in order to be able to synthesise DNA, RNA and several co-enzymes. This demand can be met in two ways 1. By de novo synthesis of nucleotides, or 2. By exploiting nucleotides, nucleosides and nucleobases arising from degradation of DNA or RNA or taken up from......-synthetase catalyses synthesis of CTP from UTP. We have cloned the pyrG gene of Lactococcus lactis subsp. cremoris MG1363 and constructed a pyrG mutant that requires cytidine for growth. Furthermore a mutant carrying a chromosomal deletion of the de novo gene pyrF, encoding orotidylat carboxylase, has been constructed...

  5. Photorespiration and the potential to improve photosynthesis.

    Science.gov (United States)

    Hagemann, Martin; Bauwe, Hermann

    2016-12-01

    The photorespiratory pathway, in short photorespiration, is an essential metabolite repair pathway that allows the photosynthetic CO2 fixation of plants to occur in the presence of oxygen. It is necessary because oxygen is a competing substrate of the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase, forming 2-phosphoglycolate that negatively interferes with photosynthesis. Photorespiration very efficiently recycles 2-phosphoglycolate into 3-phosphoglycerate, which re-enters the Calvin-Benson cycle to drive sustainable photosynthesis. Photorespiration however requires extra energy and re-oxidises one quarter of the 2-phosphoglycolate carbon to CO2, lowering potential maximum rates of photosynthesis in most plants including food and energy crops. This review discusses natural and artificial strategies to reduce the undesired impact of air oxygen on photosynthesis and in turn plant growth.

  6. Photorespiration: current status and approaches for metabolic engineering.

    Science.gov (United States)

    Maurino, Veronica G; Peterhansel, Christoph

    2010-06-01

    Photorespiration results from the oxygenase reaction catalysed by ribulose-1,5-bisphosphate carboxylase/oxygenase and serves as a carbon recovery system. It comprises enzymatic reactions distributed in chloroplasts, peroxisomes and mitochondria. The recent discovery of a cytosolic bypass and the requirement of complex formation between some photorespiratory proteins added additional levels of complexity to the known pathway. Photorespiration may have evolved in both, C(3) and C(4) plants, to prevent an accumulation of toxic levels of glycolate. Moreover, it is suggested that photorespiration evolved in cyanobacteria before the origin of chloroplasts. Synthetic detours, reminiscent of secondary photorespiratory pathways naturally occurring in cyanobacteria, were installed in Arabidopsis thaliana to bypass photorespiration. An enrichment of CO(2) in the chloroplast and positive effects on plant growth raised the question why these pathways have been lost from higher plants.

  7. ACACβ gene (rs2268388) and AGTR1 gene (rs5186) polymorphism and the risk of nephropathy in Asian Indian patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Shah, Viral N; Cheema, Balneek Singh; Sharma, Rajni;

    2013-01-01

    Patients with type 2 diabetes (T2DM) are usually obese and concurrent obesity results into activation of the renin-angiotensin-system (RAS) which is a risk factor for diabetic nephropathy (DN). Gene-gene interaction between acetyl-coenzymeA carboxylase beta (ACACβ) gene, which is involved in fatty....... We also found significant epistatic interactions between these two genes. TT genotypes of ACACβ gene and CC genotype of AGTR1 gene confers the risk of DN and both genes had significant epistatic interaction in Asian Indian patients with T2DM....... acid metabolism and angiotensin II receptors (AGTR1) gene, which mediates RAS proteins actions on renal tissue, polymorphism with DN have not been studied earlier. The present study was designed with the aim to examine the association of an ACACβ (rs2268388) and AGTR1 (rs5186) gene polymorphism...

  8. Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

    Directory of Open Access Journals (Sweden)

    Andrew J. Bowling

    2014-04-01

    Full Text Available Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH, originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC and phosphoenolpyruvate carboxykinase (PEPCK. Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

  9. Heavy atom isotope effects on enzymatic reactions

    Science.gov (United States)

    Paneth, Piotr

    1994-05-01

    The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

  10. AcEST: DK959542 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST39A01NGRL0004_P16 758 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0004_P16. 5' end sequence. DK95...9542 CL2Contig2 Show DK959542 Clone id TST39A01NGRL0004_P16 Library TST39 Length 758... Definition Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0004_P16. 5' end sequence. Accession DK959542...ation of protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= DK959542|Adiantum capillu...oxylase small chai... 181 5e-45 sp|P17673|RBS_TRIRP Ribulose bisphosphate carboxylase small chai... 180 8e-45 sp|P10795

  11. Substrate overload: Glucose oxidation in human myotubes conquers palmitate oxidation through anaplerosis

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    To date, two cardinal principles govern oxidation of glucose and fatty acids in skeletal muscle; exogenous fatty acid reduces glucose oxidation and glucose reduces fatty acid oxidation. Both glucose and palmitate (PA) oxidation was increased by increasing their concentration and inhibited...... by increasing concentrations of the other in human myotubes established from healthy, lean subjects exposed to acute stepwise increases in glucose and PA levels. At high substrate levels; PA oxidation was reduced while release of acid soluble metabolites was increased and, both glucose oxidation and release...... of citrate was increased which could be abolished by phenylacetic acid (inhibitor of pyruvate carboxylase (PC)). The present data challenges above preconceptions. Although they operate at low-moderate substrate levels additional two principles determine substrate oxidation at higher substrate concentrations...

  12. Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Matthews, V B; Åström, Maj-Brit; Chan, M H S

    2009-01-01

    C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser...... kinase (p44/42 Thr(202)/Tyr(204)) phosphorylation in these muscles. In addition, phosphorylation of ACCbeta was markedly elevated in the Bdnf electroporated muscles. CONCLUSIONS/INTERPRETATION: These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing......AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2...

  13. AcEST: DK943743 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU02A01NGRL0003_M20 581 Adiantum capillus-veneris mRNA. clone: YMU02A01NGRL0003_M2...0. 5' end sequence. DK943743 CL2Contig2 Show DK943743 Clone id YMU02A01NGRL0003_M20 Library YMU02 Length 581... Definition Adiantum capillus-veneris mRNA. clone: YMU02A01NGRL0003_M20. 5' end sequence. Accession DK943743...se search programs, Nucleic Acids Res. 25:3389-3402. Query= DK943743|Adiantum capillus-veneris mRNA, clone: ...|RBS_HELAN Ribulose bisphosphate carboxylase small chai... 178 2e-44 sp|Q43832|RBS2_SPIOL Ribulose bisphosph

  14. The role of the glucose-sensing transcription factor carbohydrate-responsive element-binding protein pathway in termite queen fertility

    DEFF Research Database (Denmark)

    Sillam-Dussès, David; Hanus, Robert; Thomas-Poulsen, Michael

    2016-01-01

    Termites are among the few animals that themselves can digest the most abundant organic polymer, cellulose, into glucose. In mice and Drosophila, glucose can activate genes via the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to induce glucose utilization and de...... novo lipogenesis. Here, we identify a termite orthologue of ChREBP and its downstream lipogenic targets, including acetyl-CoA carboxylase and fatty acid synthase. We show that all of these genes, including ChREBP, are upregulated in mature queens compared with kings, sterile workers and soldiers...... in eight different termite species. ChREBP is expressed in several tissues, including ovaries and fat bodies, and increases in expression in totipotent workers during their differentiation into neotenic mature queens. We further show that ChREBP is regulated by a carbohydrate diet in termite queens...

  15. Metabolic engineering of Torulopsis glabrata for malate production.

    Science.gov (United States)

    Chen, Xiulai; Xu, Guoqiang; Xu, Nan; Zou, Wei; Zhu, Pan; Liu, Liming; Chen, Jian

    2013-09-01

    The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5gL(-1) malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.

  16. Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin-Benson-Bassham reductive pentose phosphate cycle in Rhodobacter sphaeroides and Rhodopseudomonas palustris.

    Science.gov (United States)

    Laguna, Rick; Tabita, F Robert; Alber, Birgit E

    2011-02-01

    Rhodobacter sphaeroides ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deletion strain 16 was capable of photoheterotrophic growth with acetate, while Rhodopseudomonas palustris RubisCO-deletion strain 2040 could not grow under these conditions. The reason for this difference lies in the fact that Rba. sphaeroides and Rps. palustris use different pathways for acetate assimilation, the ethylmalonyl-CoA pathway, and glyoxylate-bypass cycle, respectively. The ethylmalonyl-CoA pathway is distinct from the glyoxylate cycle as one molecule of CO(2) and one molecule of HCO(3) (-) per three molecules of acetyl-CoA are co-assimilated to form two malate molecules. The glyoxylate cycle directly converts two acetyl-CoA molecules to malate. Each pathway, therefore, also dictates at what point, CO(2) and reductant are consumed, thereby determining the requirement for the Calvin-Benson-Bassham reductive pentose phosphate cycle.

  17. Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains.

    Science.gov (United States)

    Oba, Takahiro; Kusumoto, Kenichi; Kichise, Yuki; Izumoto, Eiji; Nakayama, Shunichi; Tashiro, Kosuke; Kuhara, Satoru; Kitagaki, Hiroshi

    2014-08-01

    Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production.

  18. Effects and Mechanisms of P and K Nutrients on Yield and Protein Content of Fodder Rice

    Institute of Scientific and Technical Information of China (English)

    TANG Xiang-ru; YU Tie-qiao

    2002-01-01

    Effects and mechanisms of P and K nutrients on yield and protein content of Weiyou 56, a fodder hybrid rice combination, were studied through pot experiment and biochemical analysis. The results showed that the increase of P and K nutrients enhanced the activities of PEP carboxylase (PEPC), glutamine synthase (GS) and sucrose phosphate synthase (SPS) in leaves, sucrose synthase (SS), ADP glucose pyrophosphorylase (ADPGP) and GS in grains, and the chlorophyll content in leaves, soluble sugar and starch content in grains,protein N and total N content in leaves and grains. Howerer, they decreased soluble sugar content in leaves and led to an increase of protein content in brown rice, biomass, grain yield and harvest index. Excessive P nutrients slightly reduced SPS and ADPG activity in leaves and grains respectively.

  19. Modeling the coverage of an AFM tip by enzymes and its application in nanobiosensors.

    Science.gov (United States)

    Amarante, Adriano M; Oliveira, Guedmiller S; Bueno, Carolina C; Cunha, Richard A; Ierich, Jéssica C M; Freitas, Luiz C G; Franca, Eduardo F; Oliveira, Osvaldo N; Leite, Fábio L

    2014-09-01

    A stochastic simulation of adsorption processes was developed to simulate the coverage of an atomic force microscope (AFM) tip with enzymes represented as rigid polyhedrons. From geometric considerations of the enzyme structure and AFM tip, we could estimate the average number of active sites available to interact with substrate molecules in the bulk. The procedure was exploited to determine the interaction force between acetyl-CoA carboxylase enzyme (ACC enzyme) and its substrate diclofop, for which steered molecular dynamics (SMD) was used. The theoretical force of (1.6±0.5) nN per enzyme led to a total force in remarkable agreement with the experimentally measured force with AFM, thus demonstrating the usefulness of the procedure proposed here to assist in the interpretation of nanobiosensors experiments.

  20. Differential gene expression between cross-fertilized and self-fertilized kernels during the early stages of seed development in maize

    Institute of Scientific and Technical Information of China (English)

    FanrongMeng; ZhongfuNi; LiminWu; QixinSun

    2005-01-01

    In maize, cross-fertilization advantage was observed during the early stage of embryo development and grain filling. In this study, we performed screening of differentially expressed genes between maize cross-fertilized and self-fertilized kernels at 5, 10, and 15 days after pollination (DAP) by differential display technique. The results indicated that the patterns of gene expression altered obviously in crossfertilized kernels as compared to self-fertilized kernels. Both quantitative and qualitative differences were observed. Four differentially expressed cDNAs were cloned and sequenced, their expression patterns were confirmed by reverse-Northern blot. Sequence analysis and database search revealed that two of them were new sequences; the other two were methyl-binding domain protein (MBD) andphos phoenolpyruvate carboxylase (PEPC), respectively, and it is concluded that the altered gene expression may be responsible for theobserved maize heterosis.

  1. DCP — EDRN Public Portal

    Science.gov (United States)

    Des-gamma-carboxy prothrombin (DCP) is a non-functional precursor of prothrombin. Prothrombin, produced by the liver, serves a critical role in normal hemostasis. Functional prothrombin contains several gamma-carboxy-glutamic acid (Gla) residues that are produced as the result of post-translational modification of glutamic acid residues mediated by vitamin K dependent gamma-glutamyl carboxylase. The formation of Gla residues can be impaired in patients with vitamin K deficiency or in patients receiving oral anticoagulant therapy. DCP lacks thrombotic activity and has been shown in multiple studies to be present in the serum of patients with HCC. DCP arises from an acquired defect in the post-translational carboxylation of the pro-thrombin precursor in malignant hepatocytes.

  2. DNA identification of Salvia divinorum samples.

    Science.gov (United States)

    Murphy, Terence M; Bola, Gurpreet

    2013-01-01

    Salvia divinorum (diviner's sage) is a plant in the mint family that produces an hallucinogenic compound, salvinorin A. The plant is used, often by chewing or smoking, as a "recreational" drug source and is regulated or banned in several states and countries. We describe a simple DNA technique, polymerase chain reaction of the ribulose bisphosphate carboxylase large subunit (rbcL) gene, that can distinguish S. divinorum leaf pieces from pieces of tobacco or cannabis. We have also found DNA sequences adjacent to the chloroplast leucine transfer RNA (trnL) gene that are specific to S. divinorum and distinguish it from other horticulturally popular Salvia species. We report some significant differences between the S. divinorum trnL sequences we determined and those now published in GenBank.

  3. AcEST: DK946100 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 6. 5' end sequence. DK946100 CL2Contig2 Show DK946100 Clone id YMU02A01NGRL0011_H06 Library YMU02 Length 574... Definition Adiantum capillus-veneris mRNA. clone: YMU02A01NGRL0011_H06. 5' end sequence. Accession DK946100...ch programs, Nucleic Acids Res. 25:3389-3402. Query= DK946100|Adiantum capillus-veneris mRNA, clone: YMU02A0...boxylase small cha... 179 7e-45 sp|P10053|RBS_PINTH Ribulose bisphosphate carboxylase small chai... 178 2e-4...FDAKRQVQVSGFLVQKPS 571 K YPE F+R++GFD KRQVQ F+ KP+ Sbjct: 146 AKKAYPEYFVRIIGFDNKR

  4. The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels

    KAUST Repository

    Tong, Winghang

    2011-09-01

    Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells. © 2011 Elsevier Inc.

  5. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

    DEFF Research Database (Denmark)

    Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

    2012-01-01

    The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice......)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose...

  6. Exercise-induced regulation of key factors in substrate choice and gluconeogenesis in mouse liver

    DEFF Research Database (Denmark)

    Knudsen, Jakob Grunnet; Biensø, Rasmus Sjørup; Hassing, Helle Adser;

    2015-01-01

    As the demand for hepatic glucose production increases during exercise, regulation of liver substrate choice and gluconeogenic activity becomes essential. The aim of the present study was to investigate the effect of a single exercise bout on gluconeogenic protein content and regulation of enzymes...... involved in substrate utilization in the liver. Mice were subjected to 1 h of treadmill exercise, and livers were removed immediately, 4 or 10 h after exercise. Glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxylase (PEPCK) mRNA contents in the liver increased immediately after exercise, while...... immediately after exercise and at 10 h of recovery, respectively. These findings suggest that acute changes in PEPCK and G6Pase protein contents do not contribute to the regulation of gluconeogenic enzyme activity during 1 h of non-exhaustive exercise. In addition, the observation that PDH-E1α, AMPK, and ACC...

  7. The Effect of Prolonged Fasting on Total Lipid Synthesis and Enzyme Activities in the Liver of the European Eel (Anguilla anguilla)

    DEFF Research Database (Denmark)

    Abraham, S. A.; Hansen, Heinz Johs. Max; Hansen, F.N.

    1984-01-01

    The extent of fatty acid synthesis from [1-14C]acetate in liver slices was reduced 6-fold when eels were fasted for 1-7 wk and 20-fold when fasted for 39 wk, thereafter hepatic lipogenesis seemed to remain constant for up to 95 wk of fasting. After a 1-3 wk fast some hepatic enzyme activities were...... total lipid synthesis and lipogenic enzyme activity in eel liver was 30.degree. C....... reduced (acetyl-CoA carboxylase decreased 2-fold and fatty acid synthetase declined 5-fold); others remained unchanged (G-6-P dehydrogenase, 6-phosphogluconate dehydrogenase, .alpha.-glycerol phosphate dehydrogenase as well as malic enzyme and ATP-citrate lyase). The optimum temperature for measuring both...

  8. Undaria pinnatifida and Fucoxanthin Ameliorate Lipogenesis and Markers of Both Inflammation and Cardiovascular Dysfunction in an Animal Model of Diet-Induced Obesity

    Directory of Open Access Journals (Sweden)

    Ameyalli Grasa-López

    2016-08-01

    Full Text Available Brown algae and its carotenoids have been shown to have a positive influence on obesity and its comorbidities. This study evaluated the effect of Undaria pinnatifida and fucoxanthin on biochemical, physiological and inflammation markers related to obesity and on the expression of genes engaged on white adipose tissue lipid metabolism in a murine model of diet-induced obesity. The treatments improved energy expenditure, β-oxidation and adipogenesis by upregulating PPARα, PGC1α, PPARγ and UCP-1. Adipogenesis was also confirmed by image analysis of the retroperitoneal adipose tissue, by measuring cell area, perimeter and cellular density. Additionally, the treatments, ameliorated adipose tissue accumulation, insulin resistance, blood pressure, cholesterol and triglycerides concentration in serum, and reduced lipogenesis and inflammation by downregulating acetyl-CoA carboxylase (ACC gene expression, increasing serum concentration and expression of adiponectin as well as downregulating IL-6 expression. Both fucoxanthin and Undaria pinnatifida may be considered for treating obesity and other diseases related.

  9. The ACACA gene is a potential candidate gene for fat content in sheep milk.

    Science.gov (United States)

    Moioli, B; Scatà, M C; De Matteis, G; Annicchiarico, G; Catillo, G; Napolitano, F

    2013-08-01

    No major gene has yet been reported in sheep that explains the variation of milk fat content. The coding region of the acetyl-CoA carboxylase alpha (ACACA) gene, which plays an important role in de novo fatty acid synthesis, had been investigated, but no non-synonymous mutations have been reported. In this study, the genomic regions encoding the three promoters of the ACACA gene were directly sequenced in 264 sheep of three different breeds, and 10 SNPs were identified. Allele frequencies of most SNPs significantly differed (P = 0.05-0.0001) between breeds. The SNPs that potentially altered either gene regulatory elements or putative binding sites of transcription factors were made evident through in silico analysis. The association analysis with milk traits, performed for one SNP of PIII (GenBank AJ292286, g.1330G>T), showed a significant allelic substitution effect (+0.33%, P sheep milk.

  10. Role of PGC-1{alpha} in exercise and fasting induced adaptations in mouse liver

    DEFF Research Database (Denmark)

    Haase, Tobias Nørresø; Jørgensen, Stine Ringholm; Leick, Lotte

    2011-01-01

    The transcriptional coactivator peroxisome proliferator activated receptor (PPAR)-¿ coactivator (PGC)-1a plays a role in regulation of several metabolic pathways. By use of whole body PGC-1a knockout (KO) mice we investigated the role of PGC-1a in fasting, acute exercise and exercise training...... induced regulation of key proteins in gluconeogenesis and metabolism in the liver. In both wild type (WT) and PGC-1a KO mice liver, the mRNA content of the gluconeogenic proteins glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was upregulated during fasting. Pyruvate...... carboxylase (PC) remained unchanged after fasting in WT mice, but was upregulated in PGC-1a KO mice. In response to a single exercise bout G6Pase mRNA was upregulated in both genotypes, whereas no significant changes were detected in PEPCK or PC mRNA. While G6Pase and PC protein remained unchanged, liver...

  11. Ketogenic Diet: An Early Option for Epilepsy Treatment, Instead of A Last Choice Only

    Directory of Open Access Journals (Sweden)

    Huei-Shyong Wang

    2012-02-01

    Full Text Available Ketogenic diet (KD was usually tried as a last resort in the treatment of intractable epilepsy after failure of many antiepileptics and even epilepsy surgery. Glucose transporter-1 deficiency and pyruvate dehydrogenase deficiency must be treated with KD as the first choice because of inborn errors of glucose metabolism. Infantile spasms, tuberous sclerosis complex, Rett syndrome, Doose syndrome, Dravet syndrome, etc., appear to respond to KD, and it has been suggested by the international consensus statement to use KD early. We believe that all patients with epilepsy, except those with contraindicated situations such as pyruvate carboxylase deficiency, porphyria, β-oxidation defects, primary carnitine deficiency, etc., may try KD before trying other regimens.

  12. Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

    Science.gov (United States)

    Leyva, Luis A; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  13. Undaria pinnatifida and Fucoxanthin Ameliorate Lipogenesis and Markers of Both Inflammation and Cardiovascular Dysfunction in an Animal Model of Diet-Induced Obesity

    Science.gov (United States)

    Grasa-López, Ameyalli; Miliar-García, Ángel; Quevedo-Corona, Lucía; Paniagua-Castro, Norma; Escalona-Cardoso, Gerardo; Reyes-Maldonado, Elba; Jaramillo-Flores, María-Eugenia

    2016-01-01

    Brown algae and its carotenoids have been shown to have a positive influence on obesity and its comorbidities. This study evaluated the effect of Undaria pinnatifida and fucoxanthin on biochemical, physiological and inflammation markers related to obesity and on the expression of genes engaged on white adipose tissue lipid metabolism in a murine model of diet-induced obesity. The treatments improved energy expenditure, β-oxidation and adipogenesis by upregulating PPARα, PGC1α, PPARγ and UCP-1. Adipogenesis was also confirmed by image analysis of the retroperitoneal adipose tissue, by measuring cell area, perimeter and cellular density. Additionally, the treatments, ameliorated adipose tissue accumulation, insulin resistance, blood pressure, cholesterol and triglycerides concentration in serum, and reduced lipogenesis and inflammation by downregulating acetyl-CoA carboxylase (ACC) gene expression, increasing serum concentration and expression of adiponectin as well as downregulating IL-6 expression. Both fucoxanthin and Undaria pinnatifida may be considered for treating obesity and other diseases related. PMID:27527189

  14. 乙酰辅酶A羧化酶在能量平衡和运动减肥中的作用研究进展

    Institute of Scientific and Technical Information of China (English)

    谢岚; 艾华

    2010-01-01

    @@ 近年来,在肥胖的发病机制、预防和治疗的研究中,乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACC)的作用越来越受到重视.体脂过多堆积是肥胖病的基本病理特征.在脂肪合成过程中,不仅ACC是限速酶,而且其催化产物丙二酸单酰辅酶A(malonyl-CoA,MA)在一定程度上控制着脂肪酸的代谢.本文就近年来ACC在能量平衡和肥胖病方面的研究进展做一综述.

  15. Metagenomic and proteomic analyses to elucidate the mechanism of anaerobic benzene degradation

    Energy Technology Data Exchange (ETDEWEB)

    Abu Laban, Nidal [Helmholtz (Germany)

    2011-07-01

    This paper presents the mechanism of anaerobic benzene degradation using metagenomic and proteomic analyses. The objective of the study is to find out the microbes and biochemistry involved in benzene degradation. Hypotheses are proposed for the initial activation mechanism of benzene under anaerobic conditions. Two methods for degradation, molecular characterization and identification of benzene-degrading enzymes, are described. The physiological and molecular characteristics of iron-reducing enrichment culture are given and the process is detailed. Metagenome analysis of iron-reducing culture is presented using a pie chart. From the metagenome analysis of benzene-degrading culture, putative mobile element genes were identified in the aromatic-degrading configurations. Metaproteomic analysis of iron-reducing cultures and the anaerobic benzene degradation pathway are also elucidated. From the study, it can be concluded that gram-positive bacteria are involved in benzene degradation under iron-reducing conditions and that the catalysis mechanism of putative anaerobic benzene carboxylase needs further investigation.

  16. Phytotoxicities of fluoranthene and phenanthrene deposited on needle surfaces of the evergreen conifer, Japanese red pine (Pinus densiflora Sieb. et Zucc.).

    Science.gov (United States)

    Oguntimehin, Ilemobayo; Nakatani, Nobutake; Sakugawa, Hiroshi

    2008-07-01

    Polycyclic aromatic hydrocarbons (PAHs) have been widely studied with respect to their carcinogenic and mutagenic effects on animals and human cells. Phenanthrene (PHE) and fluoranthene (FLU) effects on the needle photosynthetic traits of 2-year-old Japanese red pine (Pinus densiflora Sieb. et. Zucc.) seedlings were investigated. Three months after fumigation of foliage with solutions containing these PAHs (10 microM each), FLU had negative effects on net photosynthesis at near-saturating irradiance, stomatal conductance, initial chlorophyll fluorescence, and the contents of total chlorophyll, magnesium, and ribulose 1,5-bisphosphate carboxylase (rubisco) of current-year needles. PHE had similar negative effects to FLU but in lesser magnitude. The effects of the PAHs were mitigated by the addition of an OH-radical scavenger (mannitol) into the PAH solutions. PAHs deposited on the surface of pine needles may induce the generation of reactive oxygen species in the photosynthetic apparatus, a manner closely resembling the action of the herbicide paraquat.

  17. Ozone risk assessment for plants: Central role of metabolism-dependent changes in reducing power

    Energy Technology Data Exchange (ETDEWEB)

    Dizengremel, Pierre [Faculte des Sciences et Techniques, UMR1137 Ecologie et Ecophysiologie Forestieres, Nancy-Universite, BP239, F-54506 Vandoeuvre-les-Nancy Cedex (France)], E-mail: pierre.dizengremel@scbiol.uhp-nancy.fr; Le Thiec, Didier [INRA, UMR1137 Ecologie et Ecophysiologie Forestieres, Centre INRA de Nancy, F-54280 Champenoux (France)], E-mail: le_thiec@nancy.inra.fr; Bagard, Matthieu [Faculte des Sciences et Techniques, UMR1137 Ecologie et Ecophysiologie Forestieres, Nancy-Universite, BP239, F-54506 Vandoeuvre-les-Nancy Cedex (France)], E-mail: matthieu.bagard@scbiol.uhp-nancy.fr; Jolivet, Yves [Faculte des Sciences et Techniques, UMR1137 Ecologie et Ecophysiologie Forestieres, Nancy-Universite, BP239, F-54506 Vandoeuvre-les-Nancy Cedex (France)], E-mail: yves.jolivet@scbiol.uhp-nancy.fr

    2008-11-15

    The combination of stomatal-dependent ozone flux and total ascorbate level is currently presented as a correct indicator for determining the degree of sensitivity of plants to ozone. However, the large changes in carbon metabolism could play a central role in the strategy of the foliar cells in response to chronic ozone exposure, participating in the supply of reducing power and carbon skeletons for repair and detoxification, and modifying the stomatal mode of functioning. To reinforce the accuracy of the definition of the threshold for ozone risk assessment, it is proposed to also consider the redox pool (NAD(P)H), the ratio between carboxylases and the water use efficiency as indicators of the differential ozone tolerance of plants. - We propose reducing power, Rubisco/PEPc ratio and water use efficiency as additional indicators in ozone risk assessment for plants.

  18. The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: molecular and physiological characterization of PEPC kinase mutants.

    Science.gov (United States)

    Meimoun, Patrice; Gousset-Dupont, Aurélie; Lebouteiller, Bénédicte; Ambard-Bretteville, Françoise; Besin, Evelyne; Lelarge, Caroline; Mauve, Caroline; Hodges, Michael; Vidal, Jean

    2009-05-19

    Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism.

  19. Salt Stress Perception and Plant Growth Regulators in the Halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Thomas, J. C.; Bohnert, H. J.

    1993-01-01

    We selected indicators of four different metabolic processes (Crassulacean acid metabolism [CAM], amino acid and nitrogen mobilization metabolism, osmoprotection, and plant defense mechanisms) to study the relationship between salt-stress-mediated and plant growth regulator (PGR)-induced responses in Mesembryanthemum crystallinum (ice plant). Nacl and PGRs (cytokinin and abscisic acid [ABA]) are efficient elicitors of the well-studied Nacl stress responses: induction of the CAM form of phosphoenolpyruvate carboxylase, proline pinitol accumulation, and the increase of an osmotin-like protein. NaCl and cytokinin are more effective than ABA in stimulating accumulation of proline and an osmotin-like protein before the plants are committed to flowering. The results are consistent with a plant defense-induction model, in which environmental stress and PGRs are distinct signals whose subsequent effects lead to overlapping responses, the magnitude of which depends on plant developmental status. PMID:12232022

  20. Utilization of {sup 13}C-enriched substrates for the NMR study of the channelling of Krebs cycle intermediates in glioma C6; Utilisation de substrats enrichis en {sup 13}C pour l`etude par RMN de la canalisation des intermediaires du cycle de Krebs dans le gliome C6

    Energy Technology Data Exchange (ETDEWEB)

    Merle, M.; Peron, M.; Valeins, H.; Canioni, P. [Bordeaux-2 Univ., 33 (France)

    1994-12-31

    Unequal enrichments are observed for the C2 and C3 carbons of glutamate (C2>C3) and of aspartate (C3>C2) during incubation of C6 cells with (1-{sup 13} C) glucose. In order to study if this result is the result of an entry of {sup 13}C at the oxalo-acetate level or of another phenomenon, the enrichment distribution on asparte C1 and C4 carbons of C6 cells incubated with (1-{sup 13} C) glucose and the enrichment of C2 and C3 carbons of glutamate during cell incubation with (2-{sup 13} C) acetate, i.e. cases where the entry of {sup 13}C in the cycle, via the activity of the pyruvate carboxylase, is very unlikely, are examined. 4 figs., 1 tab., 1 ref.

  1. Mechanisms of inorganic carbon acquisition in two estuarine Rhodophyceans: Bostrychia scorpioides (Hudson) ex Kützing Montagne and Catenella caespitosa (Withering) L. M. Irvine.

    Science.gov (United States)

    Ruiz-Nieto, Miriam; Fernández, José A; Niell, F Xavier; Carmona, Raquel

    2014-09-01

    Marine macroalgae possess a range of mechanisms to increase the availability of CO2 for fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase. Of these, possession of a periplasmic or external carbonic anhydrase and the ability to use bicarbonate ions is widely distributed. The mechanisms of carbon acquisition were studied in two estuarine red macroalgae Bostrychia scorpioides and Catenella caespitosa using a range of techniques. pH-drift and CO2-depletion experiments at constant pH suggested that CO2 is the main source of inorganic carbon in both species. Inhibitors indicated that internal and external carbonic anhydrase were present in both species. Inhibitors also suggested that uptake of bicarbonate is unlikely to be present (P < 0.05).

  2. Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

    DEFF Research Database (Denmark)

    Madsen, Christian Toft; Sylvestersen, Kathrine Beck; Young, Clifford

    2015-01-01

    The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin...... cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin...... deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells....

  3. Introduction of a synthetic CO₂-fixing photorespiratory bypass into a cyanobacterium.

    Science.gov (United States)

    Shih, Patrick M; Zarzycki, Jan; Niyogi, Krishna K; Kerfeld, Cheryl A

    2014-04-01

    Global photosynthetic productivity is limited by the enzymatic assimilation of CO2 into organic carbon compounds. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the carboxylating enzyme of the Calvin-Benson cycle, poorly discriminates between CO2 and O2, leading to photorespiration and the loss of fixed carbon and nitrogen. With the advent of synthetic biology, it is now feasible to design, synthesize, and introduce biochemical pathways in vivo. We engineered a synthetic photorespiratory bypass based on the 3-hydroxypropionate bi-cycle into the model cyanobacterium, Synechococcus elongatus sp. PCC 7942. The heterologously expressed cycle is designed to function as both a photorespiratory bypass and an additional CO2-fixing pathway, supplementing the Calvin-Benson cycle. We demonstrate the function of all six introduced enzymes and identify bottlenecks to be targeted in subsequent bioengineering. These results have implications for efforts to improve photosynthesis and for the "green" production of high value products of biotechnological interest.

  4. Metabolic control analysis of L-lactate synthesis pathway in Rhizopus oryzae As 3.2686.

    Science.gov (United States)

    Ke, Wei; Chang, Shu; Chen, Xiaoju; Luo, Shuizhong; Jiang, Shaotong; Yang, Peizhou; Wu, Xuefeng; Zheng, Zhi

    2015-11-01

    The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during L-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the L-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution.

  5. Organic Acid Concentrations and the Relative Enzymatic Changes During the Development of the Citrus Fruits

    Institute of Scientific and Technical Information of China (English)

    LUO An-cai; YANG Xiao-hong; DENG Ying-yi; LI Chun-fan; XIANG Ke-shu; LI Dao-gao

    2003-01-01

    Seasonal changes in enzyme activities and citrate concentration during the development of citrus fruits were investigated. The result showed that the organic acid concentrations reached a peak in the 100 - 130days after anthesis and gradually declined during later stages of fruit maturation in most varieties of citrus,but declined only slightly thereafter in lemon [Citrus lin on (L.) Burm]; there is no relation between the activity of citrate synthetase(CS) and the different acid concentration in different citrus fruits; the increase of the activity of the cytosolic aconitase in the late period of the development of citrus fruits accelerated the degradation of citric acid in citrus fruits; the higher the activity of phosphoenolpyruvate carboxylase(PEPC) and the ratio of NAD-dependent isocitrate hydrogenase(PEPC/NAD-IDH- ), the more the concentrations of organic acids in citrus fruit.

  6. Hypolipidemic and antioxidant activity of aqueous extract of fruit of Withania coagulans (Stocks) Dunal in cholesterol-fed hyperlipidemic rabbit model.

    Science.gov (United States)

    Shukla, Kirtikar; Dikshit, Piyush; Shukla, Rimi; Sharma, Sonal; Gambhir, Jasvinder K

    2014-09-01

    Withania coagulans (family: Solanaceae, English: Indian Cheese Maker, Hindi: Doda Paneer) fruit is known for its ethanopharmacological significance in health care system of India. Diet rich in high-fat is an important risk factor for diabetes, atherosclerosis and macro and microvascular complications. Treatment with aqueous extract of fruit of W. coagulans (aqWC; 250 mg/kg body weight) in cholesterol-fed animals resulted in significant decrease in the levels of total cholesterol, triacylglycerol, low density lipoprotein, tissue lipid content and acetyl CoA carboxylase activity whereas, the level of high density lipoprotein and activity of HMGCoA reductase also recovered partially. Treatment with aqWC also significantly decreased plasma lipid peroxide levels and increased reduced glutathione and superoxide dismutase activities. These results suggest that the aqueous extract of W. coagulans has potent lipid lowering and antioxidant activities.

  7. A molecular phylogeny of the heterokont algae based on analyses of choroplast-encoded rbcL sequence data

    DEFF Research Database (Denmark)

    Daugbjerg, Niels; Andersen, Robert A.

    1997-01-01

    Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae...... was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first...... of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum...

  8. Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly.

    Science.gov (United States)

    Bates, Philip D; Johnson, Sean R; Cao, Xia; Li, Jia; Nam, Jeong-Won; Jaworski, Jan G; Ohlrogge, John B; Browse, John

    2014-01-21

    Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [(14)C]acetate and [(3)H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [(14)C]acetate and [(14)C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl-CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl-CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.

  9. Observations of the uptake of carbonyl sulfide (COS by trees under elevated atmospheric carbon dioxide concentrations

    Directory of Open Access Journals (Sweden)

    L. Sandoval-Soto

    2012-08-01

    Full Text Available Global change forces ecosystems to adapt to elevated atmospheric concentrations of carbon dioxide (CO2. We understand that carbonyl sulfide (COS, a trace gas which is involved in building up the stratospheric sulfate aerosol layer, is taken up by vegetation with the same triad of the enzymes which are metabolizing CO2, i.e. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, phosphoenolpyruvate carboxylase (PEP-Co and carbonic anhydrase (CA. Therefore, we discuss a physiological/biochemical acclimation of these enzymes affecting the sink strength of vegetation for COS. We investigated the acclimation of two European tree species, Fagus sylvatica and Quercus ilex, grown inside chambers under elevated CO2, and determined the exchange characteristics and the content of CA after a 1–2 yr period of acclimation from 350 ppm to 800 ppm CO2. We demonstrate that a compensation point, by definition, does not exist. Instead, we propose to discuss a point of uptake affinity (PUA. The results indicate that such a PUA, the CA activity and the deposition velocities may change and may cause a decrease of the COS uptake by plant ecosystems, at least as long as the enzyme acclimation to CO2 is not surpassed by an increase of atmospheric COS. As a consequence, the atmospheric COS level may rise causing an increase of the radiative forcing in the troposphere. However, this increase is counterbalanced by the stronger input of this trace gas into the stratosphere causing a stronger energy reflection by the stratospheric sulfur aerosol into space (Brühl et al., 2012. These data are very preliminary but may trigger a discussion on COS uptake acclimation to foster measurements with modern analytical instruments.

  10. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress

    Institute of Scientific and Technical Information of China (English)

    Malay Kumar ADAK; Nirmalya GHOSH; Dilip Kumar DASGUPTA; Sudha GUPTA

    2011-01-01

    The detrimental effects of submergence on physiological performances of some rice varieties with special references to carbohydrate metabolisms and their allied enzymes during post-flowering stages have been documented and clarified in the present investigation.It was found that photosynthetic rate and concomitant translocation of sugars into the panicles were both related to the yield.The detrimental effects of the complete submergence were recorded in generation of sucrose,starch,sucrose phosphate synthase and phosphorylase activity in the developing panicles of the plants as compared to those under normal or control (i.e.non-submerged) condition.The accumulation of starch was significantly lower in plants under submergence and that was correlated with ADP-glucose pyrophosphorylase activity.Photosynthetic rate was most affected under submergence in varying days of post-flowering and was also related to the down regulation of Ribulose bisphosphate carboxylase activity.However,under normal or control condition,there recorded a steady maintenance of photosynthetic rate at the post-flowering stages and significantly higher values of Ribulose bisphosphate carboxylase activity.Still,photosynthetic rate of the plants under both control and submerged conditions had hardly any significant correlation with sugar accumulation and other enzymes of carbohydrate metabolism like invertase with grain yield.Finally,plants under submergence suffered significant loss of yield by poor grain filling which was related to impeded carbohydrate metabolism in the tissues.It is evident that loss of yield under submergence is attributed both by lower sink size or sink capacity (number of panicles,in this case) as well as subdued carbohydrate metabolism in plants and its subsequent partitioning into the grains.

  11. Metabolism of the seed and endocarp of cherry (Prunus avium L.) during development.

    Science.gov (United States)

    Walker, Robert P; Battistelli, Alberto; Moscatello, Stefano; Chen, Zhi-Hui; Leegood, Richard C; Famiani, Franco

    2011-08-01

    In this study some aspects of organic and amino acid metabolism in cherry endocarp and seed were investigated during their development. The abundance and location of a number of enzymes involved in these processes were investigated. These enzymes were aspartate aminotransferase (AspAT; EC:2.6.1.1), glutamine synthetase (GS; EC:6.3.1.2), phosphoenolpyruvate carboxylase (PEPC; EC:4.1.1.31), phosphoenolpyruvate carboxykinase (PEPCK; EC:4.1.1.49), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC:4.1.1.39). There was a transient and massive accumulation of vegetative storage proteins in the endocarp. These proteins were remobilised as the endocarp lignified and at the same time that proteins were accumulated in the seed. This raised the possibility that a proportion of imported amino acids were temporarily stored in the endocarp as protein, and that these were later utilised by the seed when it started to accumulate storage proteins. Rubisco was present in the embryo and integuments of the seed although no chlorophyll was present. This is the first time that Rubisco has been detected in non-green seeds. The maximum abundance of Rubisco in the seed coincided with the deposition of seed storage proteins. A possible function for Rubisco in cherry seed is discussed. PEPCK was located in the integuments and appeared when seed storage proteins were being accumulated. In the integuments and embryo AspAT, GS, PEPC and Rubisco also appeared, or greatly increased in abundance, when seed storage proteins were being deposited.

  12. Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease.

    Science.gov (United States)

    Du, Chunyang; Wu, Ming; Liu, Huan; Ren, Yunzhuo; Du, Yunxia; Wu, Haijiang; Wei, Jinying; Liu, Chuxin; Yao, Fang; Wang, Hui; Zhu, Yan; Duan, Huijun; Shi, Yonghong

    2016-10-01

    Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin

  13. Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

    Science.gov (United States)

    Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

    2015-11-01

    Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants.

  14. Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Macherel, D; Kobayashi, H; Akazawa, T

    1988-01-01

    Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

  15. Responses of two summer annuals to interactions of atmospheric carbon dioxide and soil nitrogen

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, R.B.

    1987-01-01

    The competitive relationship between Chenopodium album L. (C{sub 3}) and Amaranthus hybridus L. (C{sub 4}) was investigated in two atmospheric CO{sub 2} levels and tow soil nitrogen levels. Biomass and leaf surface area of Amaranthus plants did not respond to CO{sub 2} enrichment. Only in high nitrogen did Chenopodium plants respond to increased CO{sub 2} with greater biomass and leaf surface area. Nitrogen use efficiency (NUE) was higher in Amaranthus than in Chenopodium in all treatments except for the high-nitrogen high-CO{sub 2} treatment. Under conditions of high nitrogen and low CO{sub 2}, Chenopodium was a poor competitor, but competition favored Chenopodium in high nitrogen and high CO{sub 2}. In low nitrogen and high CO{sub 2}, competition favored Chenopodium on a dry weight basis, but favored Amaranthus on a seed weight basis, reflecting early senescence of Chenopodium. In low nitrogen and high CO{sub 2}, competition favored Amaranthus on a dry weight basis, but favored Chenopodium on a seed weight basis. Physiological aspects of the growth of Chenopodium and Amaranthus were studied. Acclimation to elevated CO{sub 2} occurred at the enzyme level in Chenopodium. Under conditions of high nitrogen and no competition, individual Chenopodium plants responded to elevated CO{sub 2} with greater biomass, leaf surface area, and maximum net photosynthetic rates. In high nitrogen, leaf nitrogen, soluble protein, and RuBP carboxylase activity of Chenopodium decreased and NUE increased when grown in elevated CO{sub 2}. In low nitrogen without competition, Chenopodium showed no significant response to CO{sub 2} enrichment. Amarantus grown in high and low nitrogen without competition showed no significant changes in leaf nitrogen, soluble protein, carboxylase activity, chlorophyll, or NUE of in response to CO{sub 2} enrichment.

  16. Saponins, especially platycodin D, from Platycodon grandiflorum modulate hepatic lipogenesis in high-fat diet-fed rats and high glucose-exposed HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Department of Pharmaceutical Engineering, International University of Korea, Jinju (Korea, Republic of); Choi, Jae Ho; Kim, Hyung Gyun; Khanal, Tilak; Song, Gye Young [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Myoung Soo [College of Agriculture and Life Sciences, Chungnam National University, Daejeon (Korea, Republic of); Lee, Hyun-Sun [Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Chung, Young Chul; Lee, Young Chun [Division of Food Science, International University of Korea, Jinju (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of)

    2013-03-01

    AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP-1c) pathway. Saponins, particularly platycodin D, from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have a variety of pharmacological properties, including antioxidant and hepatoprotective properties. The aim of this study was to investigate the effects of CKS on hepatic lipogenesis and on the expression of genes involved in lipogenesis, and the mechanisms involved. CKS attenuated fat accumulation and the induction of the lipogenic genes encoding SREBP-1c and fatty acid synthase in the livers of HFD-fed rats and in steatotic HepG2 cells. Blood biochemical analyses and histopathological examinations showed that CKS prevented liver injury. CKS and platycodin D each increased the phosphorylation of AMPK and acetyl-CoA carboxylase in HFD-fed rats and HepG2 cells. The use of specific inhibitors showed that platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells. This study demonstrates that CKS or platycodin D alone can regulate hepatic lipogenesis via an AMPK-dependent signalling pathway. - Highlights: ► CKS attenuated fat accumulation in HFD-fed rats and in steatotic HepG2 cells. ► CKS and its major component, platycodin D, inhibited the levels of SREBP-1 and FAS. ► CKS and platycodin D increased the phosphorylation of AMPK and ACC. ► Platycodin D activated AMPK via SIRT1/CaMKKβ in HepG2 cells.

  17. Thiourea, a ROS scavenger, regulates source-to-sink relationship to enhance crop yield and oil content in Brassica juncea (L..

    Directory of Open Access Journals (Sweden)

    Manish Pandey

    Full Text Available In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.

  18. Coffee polyphenols suppress diet-induced body fat accumulation by downregulating SREBP-1c and related molecules in C57BL/6J mice.

    Science.gov (United States)

    Murase, Takatoshi; Misawa, Koichi; Minegishi, Yoshihiko; Aoki, Masafumi; Ominami, Hideo; Suzuki, Yasuto; Shibuya, Yusuke; Hase, Tadashi

    2011-01-01

    The prevalence of obesity is increasing globally, and obesity is a major risk factor for type 2 diabetes and cardiovascular disease. We investigated the effects of coffee polyphenols (CPP), which are abundant in coffee and consumed worldwide, on diet-induced body fat accumulation. C57BL/6J mice were fed either a control diet, a high-fat diet, or a high-fat diet supplemented with 0.5 to 1.0% CPP for 2-15 wk. Supplementation with CPP significantly reduced body weight gain, abdominal and liver fat accumulation, and infiltration of macrophages into adipose tissues. Energy expenditure evaluated by indirect calorimetry was significantly increased in CPP-fed mice. The mRNA levels of sterol regulatory element-binding protein (SREBP)-1c, acetyl-CoA carboxylase-1 and -2, stearoyl-CoA desaturase-1, and pyruvate dehydrogenase kinase-4 in the liver were significantly lower in CPP-fed mice than in high-fat control mice. Similarly, CPP suppressed the expression of these molecules in Hepa 1-6 cells, concomitant with an increase in microRNA-122. Structure-activity relationship studies of nine quinic acid derivatives isolated from CPP in Hepa 1-6 cells suggested that mono- or di-caffeoyl quinic acids (CQA) are active substances in the beneficial effects of CPP. Furthermore, CPP and 5-CQA decreased the nuclear active form of SREBP-1, acetyl-CoA carboxylase activity, and cellular malonyl-CoA levels. These findings indicate that CPP enhances energy metabolism and reduces lipogenesis by downregulating SREBP-1c and related molecules, which leads to the suppression of body fat accumulation.

  19. Addition of fish oil to diets for dairy cows. II. Effects on milk fat and gene expression of mammary lipogenic enzymes.

    Science.gov (United States)

    Ahnadi, Charaf E; Beswick, Naomi; Delbecchi, Louis; Kennelly, John J; Lacasse, Pierre

    2002-11-01

    Sixteen Holstein cows in mid-lactation were used to determine whether alterations of mammary fatty acid metabolism are responsible for the milk fat depression associated with consumption of fish oil. Cows were given a total mixed ration with no added fish oil (control), unprotected fish oil (3.7 % of dry matter), or glutaraldehyde-protected microcapsules of fish oil (1.5% or 3.0% of dry matter) for 4 weeks. Milk samples were taken once a week and a mammary biopsy was taken from a rear quarter at the end of the treatment period. Milk fat content was lower in cows given unprotected fish oil (26.0 g/kg), 1.5% protected fish oil (24.6 g/kg) and 3% protected fish oil (20.4 g/kg) than in cows fed the control diet (36.0 g/kg). This was mainly due to a decrease in the synthesis of short-chain fatty acids. Consumption of protected fish oil decreased the abundance of lipogenic enzymes mRNA in the mammary gland. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase mRNAs for cows given 3% protected fish oil averaged only 30%, 25% and 25% of control values, respectively. Dietary addition of unprotected fish oil slightly decreased mRNA abundance of these enzymes but markedly reduced the amount of lipoprotein lipase mRNA. Milk fat content was significantly correlated with gene expression of acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase but not lipoprotein lipase. These results suggest that fish oil reduces milk fat percentage by inhibiting gene expression of mammary lipogenic enzymes.

  20. Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T/sub 3/ generation

    Energy Technology Data Exchange (ETDEWEB)

    Bianco, A.C.; Silva, J.E.

    1987-09-01

    The authors have examined the activity of three lipogenic enzymes (malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase), the activity of the mitochondrial FAD-dependent ..cap alpha..-glycerolphosphate dehydrogenase (..cap alpha..-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23/sup 0/C, but the levels of ..cap alpha..-GPD and UCP were markedly reduced. Forty-eight hours at 4/sup 0/C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and ..cap alpha..-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by ..cap alpha..-GDP. Dose-response relationship analysis of the UCP response to T/sub 3/ indicated that the normalization of the response to cold requires saturation of the nuclear T/sub 3/ receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T/sub 3/ required for the full expression of BAT thermogenic potential.

  1. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  2. Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Shioya Suteaki

    2007-06-01

    Full Text Available Abstract Background Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis, which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition. Results We performed a metabolic flux analysis (MFA with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS. The flux from phosphoenolpyruvate (PEP to oxaloacetate (Oxa catalyzed by phosphoenolpyruvate carboxylase (PEPc was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc. Conclusion It was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.

  3. Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough.

    Science.gov (United States)

    Aschenbach, Jörg R; Kristensen, Niels B; Donkin, Shawn S; Hammon, Harald M; Penner, Gregory B

    2010-12-01

    Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short-chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl-CoA carboxylase, methylmalonyl-CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6-bisphosphatase and glucose 6-phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high-producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An

  4. Hydrogen peroxide regulated photosynthesis in C4-pepc transgenic rice.

    Science.gov (United States)

    Ren, C G; Li, X; Liu, X L; Wei, X D; Dai, C C

    2014-01-01

    In this study, we investigated the photosynthetic physiological basis in 'PC' transgenic rice (Oryza sativa L.), showing high-level expression of the gene encoding C4 phosphoenolpyruvate carboxylase (pepc), by hydrogen peroxide (H2O2). The C4-PEPC gene (pepc) from maize in the transgenic rice plants was checked by PCR. Comparison of yield components and photosynthetic indices between PC and untransformed wild-type (WT) plants indicated that increased yield in PC was associated with higher net photosynthetic rate and higher activities of phosphoenolpyruvate carboxylase (PEPC). Both PC and WT plants were treated with 1 mmol L(-1) abscisic acid (ABA), 0.04% 1-butanol (BA), 2 mmol L(-1) neomycin (NS), or 2 mmol L(-1) diphenyleneiodonium chloride (DPI) to investigate the relationship between photosynthesis and levels of H2O2 and phosphatidic acid. In both PC and WT, ABA induced H2O2 generation and simultaneous decrease in stomatal conductance (g(s)). PC plants treated with BA showed decreased H2O2 content and strongly increased g(s) within 2 h of treatment. Similar results were observed in response to DPI treatment in PC. However, WT did not observe the decrease of H2O2 during the treatments of BA and DPI. The reduced H2O2 content in PC caused by BA treatment differed to that induced by DPI because BA did not inhibit NADPH oxidase activities. While BA induced a larger PEPC activity in PC, and higher catalase activity as well. These results indicated that the regulation of endogenous H2O2 metabolism of PC could be helpful for enhancing photosynthetic capability.

  5. High temperature acclimation of C4 photosynthesis is linked to changes in photosynthetic biochemistry.

    Science.gov (United States)

    Dwyer, Simon A; Ghannoum, Oula; Nicotra, Adrienne; von Caemmerer, Susanne

    2007-01-01

    With average global temperatures predicted to increase over the next century, it is important to understand the extent and mechanisms of C4 photosynthetic acclimation to modest increases in growth temperature. To this end, we compared the photosynthetic responses of two C4 grasses (Panicum coloratum and Cenchrus ciliaris) and one C4 dicot (Flaveria bidentis) to growth at moderate (25/20 degrees C, day/night) or high (35/30 degrees C, day/night) temperatures. In all three C4 species, CO2 assimilation rates (A) underwent significant thermal acclimation, such that when compared at growth temperatures, A increased less than what would be expected given the strong response of A to short-term changes in leaf temperature. Thermal photosynthetic acclimation was further manifested by an increase in the temperature optima of A, and a decrease in leaf nitrogen content and leaf mass per area in the high- relative to the moderate-temperature-grown plants. Reduced photosynthetic capacity at the higher growth temperature was underpinned by selective changes in photosynthetic components. Plants grown at the higher temperature had lower amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase and cytochrome f and activity of carbonic anhydrase. The activities of photosystem II (PSII) and phosphoenolpyruvate carboxylase were not affected by growth temperature. Chlorophyll fluorescence measurements of F. bidentis showed a corresponding decrease in the quantum yield of PSII (phi(PSII)) and an increase in non-photochemical quenching (phi(NPQ)). It is concluded that through these biochemical changes, C4 plants maintain the balance between the various photosynthetic components at each growth temperature, despite the differing temperature dependence of each process. As such, at higher temperatures photosynthetic nitrogen use efficiency increases more than A. Our results suggest C4 plants will show only modest changes in photosynthetic rates in response to changes in growth temperature

  6. POST-EXERCISE LACTATE PRODUCTION AND METABOLISM IN THREE SPECIES OF AQUATIC AND TERRESTRIAL DECAPOD CRUSTACEANS

    Science.gov (United States)

    Walsh

    1994-01-01

    Aquatic and terrestrial crustaceans are dependent on both aerobic and anaerobic metabolism for energy production during exercise. Anaerobic energy production is marked by an accumulation of lactate in both muscle tissue and haemolymph, but the metabolic fate of lactate is not clear. Lactate recycling via gluconeogenesis and the potential role of carbonic anhydrase (CA) in supplying bicarbonate for the carboxylation of pyruvate were investigated in three species of decapod crustaceans: Callinectes sapidus (aquatic), Cardisoma guanhumi (semi-terrestrial) and Gecarcinus lateralis (terrestrial). CA activity was found in mitochondria and cytoplasmic fractions of gill, hepatopancreas and muscle of all three species. Significant activities of key enzymes of gluconeogenesis (e.g. pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose bisphosphatase), however, could not be detected. Exercise to exhaustion produced a species-specific pattern of accumulation and clearance of lactate in tissue and haemolymph, indicating a differential degree of reliance on anaerobic energy production. Treatment with acetazolamide, a CA inhibitor, did not significantly alter the pattern of lactate dynamics in animals given repeated bouts of exhaustive exercise interspersed with periods of recovery. Injection of [U-14C]lactate resulted in the appearance of label in both muscle glycogen and excreted carbon dioxide, suggesting multiple metabolic fates for lactate. Lactate turnover rates for G. lateralis were similar to those reported for fish. In these animals, gluconeogenesis possibly proceeds via the reversal of pyruvate kinase, or via the typical Cori cycle but so slowly that the uncatalysed supply of bicarbonate is sufficient to keep pace with the low activities of pyruvate carboxylase and the subsequent low rates of pyruvate carboxylation.

  7. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  8. Effect of ursolic acid and Rosiglitazone combination on hepatic lipid accumulation in high fat diet-fed C57BL/6J mice.

    Science.gov (United States)

    Sundaresan, Arjunan; Radhiga, Thangaiyan; Pugalendi, Kodukkur Viswanathan

    2014-10-15

    This study investigated the combined effect of ursolic acid (UA) and Rosiglitazone (RSG) on lipid regulatory genes in high fat diet (HFD)-fed mice. Male C57BL/6J mice were fed either normal diet or HFD for 10 weeks, after which animals in each dietary group were divided into following six groups, (normal diet, normal diet plus UA and RSG, HFD alone, HFD plus UA, HFD plus RSG, and HFD plus UA and RSG), for the next 5 weeks. UA (5mg/kg BW) and RSG (4mg/kg BW) were administered as suspensions directly into the stomach using a gastric tube. At the end of the study (106th day), their liver was analyzed for lipid content. RT-PCR and western blotting methods were used to analyze lipid regulatory genes. HFD-fed mice showed increased activities of hepatic marker enzymes (aspartate aminotransferase and alanine aminotransferase) in plasma and an increased concentration of total cholesterol, triglyceride and free fatty acid in liver. These results were confirmed by upregulated mRNA expression of lipogenic genes such as sterol-regulatory-element-binding protein-1c, fatty acid synthase and acetyl-CoA carboxylase and downregulated mRNA expression of fatty acid oxidative genes such as carnitine palmitoyltransferase-1, acetyl-CoA carboxylase and peroxisome proliferator activated receptor-α in HFD-fed mice. Combined treatment (UA/RSG) significantly reduced the hepatic marker enzyme activities and decreased the lipid accumulation in liver. Furthermore, combination treatment (UA/RSG) down-regulated lipogenic genes and upregulated fatty acid oxidative genes in HFD-fed mice. This study suggests that UA in combination with RSG reduced lipid accumulation in liver.

  9. Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids.

    Science.gov (United States)

    Hamza, Neïla; Silvestre, Frédéric; Mhetli, Mohamed; Khemis, Ines Ben; Dieu, Marc; Raes, Martine; Cahu, Chantal; Kestemont, Patrick

    2010-06-01

    A comparative proteomic approach was used to assess the protein expression profile in the liver of 34days old pikeperch larvae fed from day 10 post hatching, with three isoproteic and isolipidic formulated diets varying by their phospholipid (PL) contents (% dry diet weight): 1.4% (PL1), 4.7% (PL5) and 9.5% (PL9). Using 2D-DIGE minimal labelling of liver extracts, we were able to show 56 protein spots with a differential intensity (pproteins were unambiguously identified using nanoLC-MS/MS tandem mass spectrometry. In the PL9 larvae, our results indicate that the glycolytic pathway could be down-regulated due to the under-expression of the fructose biphosphate aldolase B and the phosphoglucomutase 1. Meanwhile, propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was under-expressed. In addition, another gluconeogenic and lipogenic enzyme, pyruvate carboxylase, was identified in 3 different spots as being under-expressed in fish fed with the intermediate PL level (PL5). A high PL content increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. Moreover, several stress proteins (glutathione S-transferase M, glucose regulated protein 75 and peroxiredoxin-1) were modulated in response to the dietary PL level and fatty acid composition. In the larvae fed with the lowest dietary PL content (PL1), over-expression of both GSTM and GRP75 might indicate a cellular stress in this experimental treatment, while the under-expression of Prx1 might indicate a lower defence against oxidative stress. In conclusion, this nutriproteomic approach showed significant modifications of protein expression in the liver of pikeperch larvae fed different PL contents, highlighting the importance of these nutrients and their influence on metabolism processes and on stress response.

  10. Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica1[C][W

    Science.gov (United States)

    Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

    2011-01-01

    The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

  11. Conserved and divergent rhythms of crassulacean acid metabolism-related and core clock gene expression in the cactus Opuntia ficus-indica.

    Science.gov (United States)

    Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

    2011-08-01

    The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

  12. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Baolong [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States); Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education (China); Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States); Zempleni, Janos, E-mail: jzempleni2@unl.edu [Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE (United States)

    2011-08-19

    Highlights: {yields} Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). {yields} Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. {yields} HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  13. Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942.

    Science.gov (United States)

    Orus, M. I.; Rodriguez, M. L.; Martinez, F.; Marco, E.

    1995-04-01

    Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

  14. Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Valenzuela Jacob

    2012-06-01

    DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

  15. Insulin sensitizing effects of oligomannuronate-chromium (III complexes in C2C12 skeletal muscle cells.

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    Cui Hao

    Full Text Available BACKGROUND: It was known that the insulin resistance in skeletal muscle is a major pathogenic factor in diabetes mellitus. Therefore prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the therapy of type 2 diabetes. In the present study, we investigated the ability of marine oligosaccharides oligomannuronate and its chromium (III complexes from brown alga to enhance insulin sensitivity in C2C12 skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that oligomannuronate, especially its chromium (III complexes, enhanced insulin-stimulated glucose uptake and increased the mRNA expression of glucose transporter 4 (GLUT4 and insulin receptor (IR after their internalization into C2C12 skeletal muscle cells. Additionally, oligosaccharides treatment also significantly enhanced the phosphorylation of proteins involved in both AMP activated protein kinase (AMPK/acetyl-CoA carboxylase (ACC and phosphoinositide 3-kinase (PI3K/protein kinase B (Akt signaling pathways in C2C12 cells, indicating that the oligosaccharides activated both the insulin signal pathway and AMPK pathways as their mode of action. Moreover, oligosaccharides distributed to the mitochondria after internalization into C2C12 cells and increased the expression of transcriptional regulator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, carnitine palmitoyl transferase-1 (CPT-1, and phosphorylated acetyl-CoA carboxylase (p-ACC, which suggested that the actions of these oligosaccharides might be associated with mitochondria through increasing energy expenditure. All of these effects of marine oligosaccharides were comparable to that of the established anti-diabetic drug, metformin. In addition, the treatment with oligosaccharides showed less toxicity than that of metformin. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that oligomannuonate and its chromium (III complexes improved

  16. MR spectroscopy-based brain metabolite profiling in propionic acidaemia: metabolic changes in the basal ganglia during acute decompensation and effect of liver transplantation

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    McKiernan Patrick J

    2011-05-01

    Full Text Available Abstract Background Propionic acidaemia (PA results from deficiency of Propionyl CoA carboxylase, the commonest form presenting in the neonatal period. Despite best current management, PA is associated with severe neurological sequelae, in particular movement disorders resulting from basal ganglia infarction, although the pathogenesis remains poorly understood. The role of liver transplantation remains controversial but may confer some neuro-protection. The present study utilises quantitative magnetic resonance spectroscopy (MRS to investigate brain metabolite alterations in propionic acidaemia during metabolic stability and acute encephalopathic episodes. Methods Quantitative MRS was used to evaluate brain metabolites in eight children with neonatal onset propionic acidaemia, with six elective studies acquired during metabolic stability and five studies during acute encephalopathic episodes. MRS studies were acquired concurrently with clinically indicated MR imaging studies at 1.5 Tesla. LCModel software was used to provide metabolite quantification. Comparison was made with a dataset of MRS metabolite concentrations from a cohort of children with normal appearing MR imaging. Results MRI findings confirm the vulnerability of basal ganglia to infarction during acute encephalopathy. We identified statistically significant decreases in basal ganglia glutamate+glutamine and N-Acetylaspartate, and increase in lactate, during encephalopathic episodes. In white matter lactate was significantly elevated but other metabolites not significantly altered. Metabolite data from two children who had received liver transplantation were not significantly different from the comparator group. Conclusions The metabolite alterations seen in propionic acidaemia in the basal ganglia during acute encephalopathy reflect loss of viable neurons, and a switch to anaerobic respiration. The decrease in glutamine + glutamate supports the hypothesis that they are consumed to

  17. AB031. Spectrum of IEMs in Vietnamese patients: data from 10 years of selected screening and diagnosis

    Science.gov (United States)

    Dung, Vu Chi; Khánh, Nguyễn Ngọc; Mai, Nguyen Chi; Hương, Bùi Thị; Thao, Bui Phuong; Ngoc, Can Thi Bich; Hoan, Nguyen Thi; Hai, Le Thanh; Dung, Khu Thi Khanh; Fukao, Toshiyuki; Yamaguchi, Seiji

    2015-01-01

    Background and objective Vietnam is the easternmost country on the Indochina Peninsula in Southeast Asia. With an estimated 90 million inhabitants as of 2013, it is the world’s 13th-most-populous country, and the eighth-most-populous Asian country. Congenital anomalies accounted about 22% of causes of deaths in children under-5 [2010]. The first service for IEMs was set up at the Northern referral center of Pediatrics-National Hospital of Pediatrics, Hanoi (NHP) in 2004 officially. The NHP in Hanoi provides services to the population of North Vietnam (~30 million people). The aim of this report is to highlight disease spectrum of tandem mass spectrometry (MS/MS) target disease in Vietnam. Methods A total of 2,405 high-risk cases with IEMs were studied at NHP during 10 years [2005-2014]. Dry blood and urine samples were analyzed using MS/MS (amino acid & acylcarnitine analysis) & GC/MS (organic acid anaysis) at Shimane University, Japan from 2005. Organic acids analysis for fresh urine samples was performed at NHP using GC/MS at NHP from 2010. Amino acid analysis for plasma samples were performed using HPLC at NHP from 2012. Results Oganic acidemia (OAs), amino acid disorders (AAs), urea cycle disorders (UCDs) and fatty acid oxidation disorders (FAOD) were identified in 235/2,405 cases (9.8%). A total of 118/235 patients (50.2%) were OAs with 12 different disorders: BKT (33 cases), PPA (21 cases), 5-oxoprolinuria (19 cases), MMA (14 cases), Glutaricaciduria type II (GA II) (11 cases), 3-methylglutaconic aciduria (4 cases), isovaleric academia (3 cases), multiple carboxylase deficiency (MCD) (2 cases), 3-methylcrotonylCoA carboxylase deficiency (2 cases). A total of 42/235 patients (17.9%) were amino acid disorders including 35 cases with MSUD, 7 cases with PKU and 1 case with tyrosinemia type 1. The 36/235 patients (15.3%) were UCDs including OTC deficiency (13 cases), citrulinemia type 1 (1 case) and argininosuccinic aciduria (1 case). 39/235 patients (16

  18. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

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    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  19. Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

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    Thuy Do

    Full Text Available Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit, gki (glucokinase, lepA (GTP-binding protein, recP (transketolase, sodA (superoxide dismutase, and tyrS (tyrosyl-tRNA synthetase] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B and spaP (surface protein antigen I/II] to increase sequence type diversity. The number of alleles found varied between 20 (lepA and 37 (spaP. Overall, 121 sequence types (STs were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of

  20. Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

    Science.gov (United States)

    Do, Thuy; Gilbert, Steven C; Clark, Douglas; Ali, Farida; Fatturi Parolo, Clarissa C; Maltz, Marisa; Russell, Roy R; Holbrook, Peter; Wade, William G; Beighton, David

    2010-02-05

    Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra

  1. Improving Glutamate Yield by Adding NaHCO3 and Regulating pH in the Glutamate Fermentation%通过添加NaHCO3和调节pH提高谷氨酸得率

    Institute of Scientific and Technical Information of China (English)

    曹艳; Enock Mpofu; 丁健; 段作营; 史仲平

    2012-01-01

    Fermentation performances under the condition of NaHCO3 supplement and pH regulation during glutamate fermentation were investigated. When pH was raised to 8. 0 before or at the same time with adding NaHCO3, comparable glutamate production with control (above 75 g/L) was achieved and the consumption amount of glucose was decreased greatly. And glutamate yield was increased by 36% and 34% respectively. Enzymes metabolic analysis showed that glutamate yield could not be promoted by increase of pyruvate carboxylase activity alone. The overall fermentation performance improvement could be achieved only when pyruvate carboxylase and isocitrate dehydrogenase were stimulated, while α-ketoglutarate dehydrogenase complex and pyruvate dehydrogenase were repressed simultaneously.%研究了在谷氨酸发酵产酸期分别添加NaHCO3、调节pH及两者耦联操作条件下的发酵性能.结果表明:只有在升高pH值、添加NaHCO3同时进行或先升高pH值后添加NaHCO3的情况下,葡萄糖消耗量大幅下降,谷氨酸得率显著提高,分别比对照提高36%和34%,且谷氨酸产量可以达到正常水平(75 g/L以上).酶学代谢分析表明,仅仅提高丙酮酸羧化酶活性不能提高谷氨酸得率,只有各个关键酶相互协调配合,即适度弱化丙酮酸脱氢酶和α-酮戊二酸脱氢酶活性的同时,适度提高丙酮酸羧化酶和异柠檬酸脱氢酶活性,才能有效提高谷氨酸发酵整体性能.

  2. Structural ordering of disordered ligand-binding loops of biotin protein ligase into active conformations as a consequence of dehydration.

    Directory of Open Access Journals (Sweden)

    Vibha Gupta

    Full Text Available Mycobacterium tuberculosis (Mtb, a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC, an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA. The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of approximately 3.5 A in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In

  3. Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice.

    Science.gov (United States)

    Lin, HsiangChun; Karki, Shanta; Coe, Robert A; Bagha, Shaheen; Khoshravesh, Roxana; Balahadia, C Paolo; Ver Sagun, Julius; Tapia, Ronald; Israel, W Krystler; Montecillo, Florencia; de Luna, Albert; Danila, Florence R; Lazaro, Andrea; Realubit, Czarina M; Acoba, Michelle G; Sage, Tammy L; von Caemmerer, Susanne; Furbank, Robert T; Cousins, Asaph B; Hibberd, Julian M; Quick, W Paul; Covshoff, Sarah

    2016-05-01

    The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.

  4. Impact of Raw and Bioaugmented Olive-Mill Wastewater and Olive-Mill Solid Waste on the Content of Photosynthetic Molecules in Tobacco Plants.

    Science.gov (United States)

    Parrotta, Luigi; Campani, Tommaso; Casini, Silvia; Romi, Marco; Cai, Giampiero

    2016-08-03

    Disposal and reuse of olive-mill wastes are both an economic and environmental problem, especially in countries where the cultivation of olive trees is extensive. Microorganism-based bioaugmentation can be used to reduce the pollutant capacity of wastes. In this work, bioaugmentation was used to reduce the polyphenolic content of both liquid and solid wastes. After processing, bioaugmented wastes were tested on the root development of maize seeds and on photosynthesis-related molecules of tobacco plants. In maize, we found that bioaugmentation made olive-mill wastes harmless for seed germination. In tobacco, we analyzed the content of RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) and of the photosynthetic pigments lutein, chlorophylls, and β-carotene. Levels of RuBisCO were negatively affected by untreated wastewater but increased if plants were treated with bioaugmented wastewater. On the contrary, levels of RuBisCO increased in the case of plants treated with raw olive-mill solid waste. Pigment levels showed dissimilar behavior because their concentration increased if plants were irrigated with raw wastewater or treated with raw olive-mill solid waste. Treatment with bioaugmented wastes restored pigment content. Findings show that untreated wastes are potentially toxic at the commencement of treatment, but plants can eventually adapt after an initial stress period. Bioaugmented wastes do not induce immediate damages, and plants rapidly recover optimal levels of photosynthetic molecules.

  5. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    Science.gov (United States)

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  6. Dietary fiber prevents obesity-related liver lipotoxicity by modulating sterol-regulatory element binding protein pathway in C57BL/6J mice fed a high-fat/cholesterol diet.

    Science.gov (United States)

    Han, Shufen; Jiao, Jun; Zhang, Wei; Xu, Jiaying; Wan, Zhongxiao; Zhang, Weiguo; Gao, Xiaoran; Qin, Liqiang

    2015-10-29

    Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/cholesterol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibited lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles.

  7. Flux analysis of central metabolic pathways in the Fe(III)-reducing organism Geobacter metallireducens via 13C isotopiclabeling

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Chakraborty, Romy; Martin, Hector Garcia; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-08-13

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The model indicated that over 90 percent of the acetate wascompletely oxidized to CO2 via a complete tricarboxylic acid (TCA) cyclewhile reducing iron. Pyruvate carboxylase and phosphoenolpyruvatecarboxykinase were present under these conditions, but enzymes in theglyoxylate shunt and malic enzyme were absent. Gluconeogenesis and thepentose phosphate pathway were mainly employed for biosynthesis andaccounted for less than 3 percent of total carbon consumption. The modelalso indicated surprisingly high reversibility in the reaction betweenoxoglutarate and succinate. This step operates close to the thermodynamicequilibrium possibly because succinate is synthesized via a transferasereaction, and its product, acetyl-CoA, inhibits the conversion ofoxoglutarate to succinate. These findings enable a better understandingof the relationship between genome annotation and extant metabolicpathways in G. metallireducens.

  8. Metabolic responses of primary and transformed cells to intracellular Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Nadine Gillmaier

    Full Text Available The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using (13C-labelled glucose or glutamine as carbon tracers. The (13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.

  9. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Chian-Jiun Liou

    2015-01-01

    Full Text Available Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK, resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C, ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway.

  10. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    Energy Technology Data Exchange (ETDEWEB)

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.; (GSKNC); (Duke); (UNC)

    2009-06-15

    Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined -helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  11. Acclimation of Arabidopsis thaliana to long-term CO{sub 2} enrichment and nitrogen supply is basically a matter of growth rate adjustment

    Energy Technology Data Exchange (ETDEWEB)

    Tocquin, P.; Ormenese, S.; Pieltain, A.; Detry, N.; Bernier, G.; Perilleux, C. [Univ. of Liege, Dept. of Life Sciences, Lab. of Plant Physiology, Liege (Belgium)

    2006-12-15

    The long-term response of Arabidopsis thaliana to increasing CO{sub 2} was evaluated in plants grown in 800 {mu}l l{sup -1} CO{sub 2} from sowing and maintained, in hydroponics, on three nitrogen supplies: 'low', 'medium' and 'high'. The global response to high CO{sub 2} and N-supply was evaluated by measuring growth parameters in parallel with photosynthetic activity, leaf carbohydrates, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) messenger RNA and protein, stomatal conductance (g-s) and density. CO{sub 2} enrichment was found to stimulate biomass production, whatever the N-supply. This stimulation was transient on low N-supply and persisted throughout the whole vegetative growth only in high N-supply. Acclimation on low N-high C0{sub 2} was not associated with carbohydrate accumulation or with a strong reduction in Rubisco amount or activity. At high N-supply, growth stimulation by high CO{sub 2} was mainly because of the acceleration of leaf production and expansion while other parameters such as specific leaf area, root/shoot ratio and g{sub s} appeared to be correlated with total leaf area. Our results thus suggest that, in strictly controlled and stable growing conditions, acclimation of A. thaliana to long-term CO{sub 2} enrichment is mostly controlled by growth rate adjustment. (au)

  12. Overexpression of G6PDH does not affect the behavior of HEK-293 clones stably expressing interferon-α2b

    Directory of Open Access Journals (Sweden)

    Iness Hammami

    2016-07-01

    Full Text Available HEK293 cells are gaining in interest for the production of recombinant proteins. However, further understanding and engineering of cell metabolism are still needed to improve protein titers. The importance of G6PDH has already been studied regarding redox balance, and a correlation has recently been established between the oxidative pentose phosphate pathway (PPP and antibody peak production. In this work, HEK293 cells stably expressing interferon-α2b, a parental clone, and a further engineered clone expressing the cytosolic yeast pyruvate-carboxylase (PYC were transiently transfected to overexpress glucose-6-phosphate dehydrogenase (G6PDH and increase fluxes through the PPP. The aim of the study was thus to evaluate the effect of overexpressing G6PDH on the “pull” effect brought by the PYC phenotype. Results indicate that the cell metabolism is, however, highly robust, showing a highly regulated PPP damping the potential effects of overexpressing G6PDH. A metabolomic study also clearly demonstrates, by metabolites profiling, that the PYC clone has a highly robust and more efficient metabolic efficiency, compared to its parental clone.

  13. Induction of fatty liver by Coleus forskohlii extract through enhancement of de novo triglyceride synthesis in mice

    Directory of Open Access Journals (Sweden)

    Keizo Umegaki

    2014-01-01

    Full Text Available Coleus forskohlii extract (CFE, an herbal ingredient, is used for weight-loss products. CFE's alleged efficacy is attributed to forskolin. However, CFE has been shown to induce fatty liver in mice, with components other than forskolin playing a part in this effect. The present study addressed the underlying mechanism of CFE-induced fatty liver by analyzing changes in CFE-treated mice of lipid concentrations and of the levels of mRNAs encoding enzymes and transcription factors known to be related to fatty liver. Mice were fed a diet containing 0, 0.3 and 1% CFE for 2 weeks. CFE at 1% clearly induced fatty liver, as demonstrated by histological examination and confirmed by increases in triglyceride concentrations in liver. However, treated mice did not exhibit elevation in plasma levels of non-esterified fatty acids. Comprehensive analysis of liver mRNA levels revealed accumulation of multiple transcripts, including mRNAs encoding enzymes acetyl-CoA carboxylase and long-chain elongase; transcription factor peroxisome proliferator-activated receptor gamma (PPARγ; and lipid-droplet-associated fat-specific protein 27 (Fsp27. These findings suggest that the de novo synthesis and accumulation of triglyceride in the liver, through the enhanced expression of specific lipogenic mRNAs, is a major underlying mechanism of fatty liver induction by CFE.

  14. DNA analysis of natural fiber rope.

    Science.gov (United States)

    Dunbar, Mignon; Murphy, Terence M

    2009-01-01

    When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis Née (abaca, "Manila hemp"), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.

  15. Molecular cloning of C4-specific Ppc gene of sorghum and its high level expression in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang; CHI Wei; WANG Qiang; ZHANG Qide; WU Naihu

    2003-01-01

    In order to improve the carbon-assimilation ability of C3 plants, we isolated a C4-specific photosynthetic enzyme gene, Ppc (encode phosphoenolpyruvate carboxylase, PEPCase) from the genome of the C4 plant, sorghum, and transformed rice with it. As shown by sequence analysis, the gene is composed of 10 exons and 9 introns, and the full-length transcript is 5989 bp long. A recombinant expression vector, p1301PEPC, was constructed by inserting the gene into a plasmid vector, pCAMBIA1301, which was then transformed into two japonica rice varieties, Nongken 58 and Zhonghua 10, using an Agrobacterium-mediated transformation system. PCR analysis, activity measurement of PEPCase, and protein-, RNA- and DNA-based hybridization all confirmed the successful integration of the C4-specific Ppc gene into the nuclear genome of rice and its high level expression. Physiological studies revealed the photosynthetic features characterizing C4 plants such as marked lowering of CO2 compensation point and photorespiration rate, and improved carboxylation efficiency. This study provides useful experimental materialsand opens up new avenues for further studies on improving photosynthetic efficiency of elite varieties of rice.

  16. An Excel tool for deriving key photosynthetic parameters from combined gas exchange and chlorophyll fluorescence: theory and practice.

    Science.gov (United States)

    Bellasio, Chandra; Beerling, David J; Griffiths, Howard

    2016-06-01

    Combined photosynthetic gas exchange and modulated fluorometres are widely used to evaluate physiological characteristics associated with phenotypic and genotypic variation, whether in response to genetic manipulation or resource limitation in natural vegetation or crops. After describing relatively simple experimental procedures, we present the theoretical background to the derivation of photosynthetic parameters, and provide a freely available Excel-based fitting tool (EFT) that will be of use to specialists and non-specialists alike. We use data acquired in concurrent variable fluorescence-gas exchange experiments, where A/Ci and light-response curves have been measured under ambient and low oxygen. From these data, the EFT derives light respiration, initial PSII (photosystem II) photochemical yield, initial quantum yield for CO2 fixation, fraction of incident light harvested by PSII, initial quantum yield for electron transport, electron transport rate, rate of photorespiration, stomatal limitation, Rubisco (ribulose 1·5-bisphosphate carboxylase/oxygenase) rate of carboxylation and oxygenation, Rubisco specificity factor, mesophyll conductance to CO2 diffusion, light and CO2 compensation point, Rubisco apparent Michaelis-Menten constant, and Rubisco CO2 -saturated carboxylation rate. As an example, a complete analysis of gas exchange data on tobacco plants is provided. We also discuss potential measurement problems and pitfalls, and suggest how such empirical data could subsequently be used to parameterize predictive photosynthetic models.

  17. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) promote adipogenesis in 3T3-L1 adipocyte cell culture.

    Science.gov (United States)

    Kim, Jonggun; Sun, Quancai; Yue, Yiren; Yoon, Kyong Sup; Whang, Kwang-Youn; Marshall Clark, J; Park, Yeonhwa

    2016-07-01

    4,4'-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4'-dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 adipocytes. Treatment of DDT or DDE resulted in increased lipid accumulation accompanied by increased expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome-proliferator activated receptor-γ (PPARγ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase, and leptin. Moreover, treatment of DDT or DDE increased protein levels of C/EBPα, PPARγ, AMP-activated protein kinase-α (AMPKα), and ACC, while significant decrease of phosphorylated forms of AMPKα and ACC were observed. These finding suggest that increased lipid accumulation caused by DDT and DDE may mediate AMPKα pathway in 3T3-L1 adipocytes.

  18. Carbon and nitrogen metabolism in barley plants exposed to UV-B radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ghisi, R.; Trentin, A.R.; Masi, A.; Ferretti, M. [Dipartimento di Biotecnologie Agrarie, Legnaro, Padova (Italy)

    2002-10-01

    The effect of UV-B radiation on FW, leaf and stem length, photosynthetic O{sub 2} evolution, levels of carbohydrates and nitrates, and extractable activities of some of the enzymes involved in C and N metabolism was evaluated in barley (Hordeum vulgare L. cv. Express) seedlings during the 9 days following transfer to an UV-B enriched environment. The results show that under our experimental conditions UV-B radiation scarcely affects the photosynthetic competence of barley leaves, expressed as RuBP carboxylase (EC 4.1.1.39) activity, O{sub 2} evolution rate and chlorophyll content. Nevertheless, this treatment induced significant alterations of the enzyme activity of nitrate reductase (EC 1.6.6.1) and glutamine synthetase (EC 6.3.1.2), although only after a few days of treatment. The effects were not confined to the exposed tissue, but were detectable also at the root level. In fact, nitrate reductase decreased in response to UV-B in both leaf and root tissue, whereas glutamine synthetase was affected only in the root. In contrast, nitrate content was not influenced by the treatment, neither in root nor in leaf tissue, whilst leaf sucrose diminished in exposed plants only on the last day of treatment. (au)

  19. The role of glutamine synthetase in energy production and glutamine metabolism during oxidative stress.

    Science.gov (United States)

    Aldarini, Nohaiah; Alhasawi, Azhar A; Thomas, Sean C; Appanna, Vasu D

    2017-01-17

    Oxidative stress is known to severely impede aerobic adenosine triphosphate (ATP) synthesis. However, the metabolically-versatile Pseudomonas fluorescens survives this challenge by invoking alternative ATP-generating networks. When grown in a medium with glutamine as the sole organic nutrient in the presence of H2O2, the microbe utilizes glutamine synthetase (GS) to modulate its energy budget. The activity of this enzyme that mediates the release of energy stored in glutamine was sharply increased in the stressed cells compared to the controls. The enhanced activities of such enzymes as acetate kinase, adenylate kinase and nucleotide diphosphate kinase ensured the efficacy of this ATP producing-machine by transferring the high energy phosphate. The elevated amounts of phosphoenol pyruvate carboxylase and pyruvate orthophosphate dikinase recorded in the H2O2 exposed cells provided another route to ATP independent of the reduction of O2. This is the first demonstration of a metabolic pathway involving GS dedicated to ATP synthesis. The phospho-transfer network that is pivotal to the survival of the microorganism under oxidative stress may reveal therapeutic targets against infectious microbes reliant on glutamine for their proliferation.

  20. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.

    2000-10-04

    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  1. Protamine sulfate precipitation method depletes abundant plant seed-storage proteins: A case study on legume plants.

    Science.gov (United States)

    Kim, Yu Ji; Wang, Yiming; Gupta, Ravi; Kim, So Wun; Min, Chul Woo; Kim, Yong Chul; Park, Ki Hun; Agrawal, Ganesh Kumar; Rakwal, Randeep; Choung, Myoung-Gun; Kang, Kyu Young; Kim, Sun Tae

    2015-05-01

    Depletion of abundant proteins is one of the effective ways to improve detection and identification of low-abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable for their in-depth proteome investigation. In this study, we provide evidence that the PSP method can also be effectively used for depletion of abundant seed-storage proteins (SSPs) from the total seed proteins of diverse legume plants including soybean, broad bean, pea, wild soybean, and peanut. The 0.05% protamine sulfate (PS) was sufficient to deplete major SSPs from all legumes tested except for peanut where 0.1% PS was required. SDS-PAGE, Western blotting and 2DE analyses of PS-treated soybean and peanut seed proteins showed enriched spots in PS-supernatant than total proteins. Coefficient of variation percentage (%CV) and principal component analysis of 2DE spots support the reproducibility, suitability, and efficacy of the PSP method for quantitative and comparative seed proteome analysis. MALDI-TOF-TOF successfully identified some protein spots from soybean and peanut. Hence, this simple, reproducible, economical PSP method has a broader application in depleting plant abundant proteins including SSPs in addition to RuBisCO, allowing discussion for comprehensive proteome establishment and parallel comparative studies in plants.

  2. Effects of Glucose Feeding on Respiration and Photosynthesis in Photoautotrophic Dianthus caryophyllus Cells: Mass Spectrometric Determination of Gas Exchange.

    Science.gov (United States)

    Avelange, M H; Sarrey, F; Rébillé, F

    1990-11-01

    When glucose (20 millimolar) was added to photoautotrophic cell suspension cultures of Dianthus caryophyllus, there was during the first 10 hours an accumulation of carbohydrates and phosphorylated compounds. These biochemical changes were accompanied by a progressive decrease of net photosynthesis and a twofold increase of the dark respiratory rate. The rise of respiration was associated with a rise of fumarase and cytochrome c oxidase activities, two mitochondrial markers. Gas exchange of illuminated cells were performed with a mass spectrometry technique and clearly established that during the first hours of glucose feeding, the decrease of net photosynthesis was essentially due to an increase of respiration in light, whereas the photosynthetic processes (gross O(2) evolution and gross CO(2) fixation) were almost not affected. However, after 24 hours of experiment, O(2) evolution and CO(2) fixation started to decline in turn. While ribulose-1,5-bisphosphate carboxylase activity was little affected during the first 48 hours of the experiment, the maximal light-induced phosphoribulokinase activity dramatically decreased with time and represented after 48 hours only 30% of its initial activity. It is postulated that the decrease in phosphoribulokinase activity was at least partially responsible for the decrease of CO(2) fixation and the metabolic events involved in this regulation are discussed.

  3. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    Science.gov (United States)

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).

  4. Chronic exposure to paclobutrazol causes hepatic steatosis in male rockfish Sebastiscus marmoratus and the mechanism involved

    Energy Technology Data Exchange (ETDEWEB)

    Sun Lingbin [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); Li Jinshou [Department of Biological Engineering, Ningde Normal University, Ningde City, Fujian (China); Zuo Zhenghong [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); Chen Meng [State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen (China); Wang Chonggang, E-mail: cgwang@xmu.edu.cn [State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen (China); State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen (China)

    2013-01-15

    Paclobutrazol (PBZ) is a triazole-containing fungicide which is widely used in agriculture. Acute toxicity can follow its extensive use but it is generally weaker than traditional pesticides such as organochlorine and organophosphorus. However, its adverse effects on aquatic organisms need to be investigated. This study was conducted to investigate the effect of PBZ exposure on the hepatic lipid metabolism of Sebastiscus marmoratus. After PBZ exposure for 50 days, hepatic lipid droplets were enlarged and the hepatic total lipid, triglyceride, total cholesterol and free fatty acid content had increased in a dose dependent manner compared to the control. The mRNA expression of lipid metabolism associated genes such as peroxisome proliferator-activated receptors (PPARs), androgen receptor, acetyl-CoA carboxylase 1, fatty acid synthesis, fatty acid bing protein 4, liver X receptor {alpha} (LXR{alpha}) and stearoyl-CoA desaturase were up-regulated by PBZ exposure. These results indicated that triazole-containing fungicides might affect the metabolism and health of fish via the multi-signal pathways of nuclear receptors such as PPARs and LXR.

  5. Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways.

    Science.gov (United States)

    Erdem, Cemal; Nagle, Alison M; Casa, Angelo J; Litzenburger, Beate C; Wang, Yu-Fen; Taylor, D Lansing; Lee, Adrian V; Lezon, Timothy R

    2016-09-01

    Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro.

  6. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    Energy Technology Data Exchange (ETDEWEB)

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  7. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    Science.gov (United States)

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  8. Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production.

    Science.gov (United States)

    Chen, Xiulai; Wang, Yuancai; Dong, Xiaoxiang; Hu, Guipeng; Liu, Liming

    2017-02-22

    L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L(-1), 0.30 g g(-1), and 0.32 g L(-1) h(-1) in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L(-1), 0.31 g g(-1), and 0.32 g L(-1) h(-1), respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.

  9. [Effects of drought stress on photosynthesis capability of Spiraea fritschiana and Spiraea bunmalba 'Goldmound'].

    Science.gov (United States)

    Liu, Hui-Min; Che, Yan-Shuang; Che, Dai-Di; Yan, Yong-Qing; Wu, Feng-Zhi

    2010-08-01

    In this paper, Spiraea fritschiana and Spiraea bunmalba 'Goldmound' were treated with mild, moderate, and severe drought to study the dynamic changes of their photosynthesis capability, and two-dimensional electrophoresis and mass spectrometry were adopted to analyze and identify the differences in the protein expression of the two species before and after the treatments, and the physiological mechanisms inducing the changes of the photosynthesis capability. Drought treatments had significant effects on the photosynthesis capability of the two species. Under drought stress, the maximum photosynthetic rate, light compensation point, and light saturation point decreased gradually, suggesting that the responses of the two species to drought stress were progressive. The two species presented stronger recovery capability after the mild and moderate stresses, but weaker recovery capability after severe stress. After the inducement of drought stress, the weaker drought-resistant S. bunmalba 'Goldmound' had six protein spots lost, eleven new protein spots appeared, thirteen protein spots up-regulation expression, and four protein spots down-regulation expression. All of the proteins were low molecular weight acidic proteins, of which, there were three kinds of different proteins that had been induced expression by drought and were the oxygen-enhanced protein factor 1 and 2 and the degradation fragments of large subunit 1,5-ribulose bisphosphate carboxylase/oxygenase. The drought- resistant difference of the two Spiraea species was related to the changes of their photosynthesis capability during drought stress.

  10. Effects of Natural Products on Fructose-Induced Nonalcoholic Fatty Liver Disease (NAFLD

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    Qian Chen

    2017-01-01

    Full Text Available As a sugar additive, fructose is widely used in processed foods and beverages. Excessive fructose consumption can cause hepatic steatosis and dyslipidemia, leading to the development of metabolic syndrome. Recent research revealed that fructose-induced nonalcoholic fatty liver disease (NAFLD is related to several pathological processes, including: (1 augmenting lipogenesis; (2 leading to mitochondrial dysfunction; (3 stimulating the activation of inflammatory pathways; and (4 causing insulin resistance. Cellular signaling research indicated that partial factors play significant roles in fructose-induced NAFLD, involving liver X receptor (LXRα, sterol regulatory element binding protein (SREBP-1/1c, acetyl-CoA carboxylase (ACC, fatty acid synthase (FAS, stearoyl-CoA desaturase (SCD, peroxisome proliferator–activated receptor α (PPARα, leptin nuclear factor-erythroid 2-related factor 2 (Nrf2, nuclear factor kappa B (NF-κB, tumor necrosis factor α (TNF-α, c-Jun amino terminal kinase (JNK, phosphatidylinositol 3-kinase (PI3K and adenosine 5′-monophosphate (AMP-activated protein kinase (AMPK. Until now, a series of natural products have been reported as regulators of NAFLD in vivo and in vitro. This paper reviews the natural products (e.g., curcumin, resveratrol, and (−-epicatechin and their mechanisms of ameliorating fructose-induced NAFLD over the past years. Although, as lead compounds, natural products usually have fewer activities compared with synthesized compounds, it will shed light on studies aiming to discover new drugs for NAFLD.

  11. Reduced AMPK-ACC and mTOR signaling in muscle from older men, and effect of resistance exercise

    Science.gov (United States)

    Li, Mengyao; Verdijk, Lex B.; Sakamoto, Kei; Ely, Brian; van Loon, Luc J.C.; Musi, Nicolas

    2012-01-01

    AMP-activated protein kinase (AMPK) is a key energy-sensitive enzyme that controls numerous metabolic and cellular processes. Mammalian target of rapamycin (mTOR) is another energy/nutrient-sensitive kinase that controls protein synthesis and cell growth. In this study we determined whether older versus younger men have alterations in the AMPK and mTOR pathways in skeletal muscle, and examined the effect of a long term resistance type exercise training program on these signaling intermediaries. Older men had decreased AMPKα2 activity and lower phosphorylation of AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). mTOR phosphylation also was reduced in muscle from older men. Exercise training increased AMPKα1 activity in older men, however, AMPKα2 activity, and the phosphorylation of AMPK, ACC and mTOR, were not affected. In conclusion, older men have alterations in the AMPK-ACC and mTOR pathways in muscle. In addition, prolonged resistance type exercise training induces an isoform-selective up regulation of AMPK activity. PMID:23000302

  12. Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice.

    Science.gov (United States)

    Taniguchi, Yojiro; Ohkawa, Hiroshi; Masumoto, Chisato; Fukuda, Takuya; Tamai, Tesshu; Lee, Kwanghong; Sudoh, Sizue; Tsuchida, Hiroko; Sasaki, Haruto; Fukayama, Hiroshi; Miyao, Mitsue

    2008-01-01

    Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.

  13. Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

    Science.gov (United States)

    Francki, Michael G; Whitaker, Peta; Smith, Penelope M; Atkins, Craig A

    2002-11-01

    Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.

  14. Biotin deprivation impairs mitochondrial structure and function and has implications for inherited metabolic disorders.

    Science.gov (United States)

    Ochoa-Ruiz, Estefanía; Díaz-Ruiz, Rodrigo; Hernández-Vázquez, Alaín de J; Ibarra-González, Isabel; Ortiz-Plata, Alma; Rembao, Daniel; Ortega-Cuéllar, Daniel; Viollet, Benoit; Uribe-Carvajal, Salvador; Corella, José Ahmed; Velázquez-Arellano, Antonio

    2015-11-01

    Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect,whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases.Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.

  15. Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p.

    Science.gov (United States)

    Madsen, Christian T; Sylvestersen, Kathrine B; Young, Clifford; Larsen, Sara C; Poulsen, Jon W; Andersen, Marianne A; Palmqvist, Eva A; Hey-Mogensen, Martin; Jensen, Per B; Treebak, Jonas T; Lisby, Michael; Nielsen, Michael L

    2015-07-09

    The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.

  16. Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia

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    Mahnaz Darvish-Damavandi

    2016-09-01

    Full Text Available Propionic acidemia (PA is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC. This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA, derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs and mitochondria targeting sequences (MTS to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA.

  17. [Effects of diets on growth, serum biochemical indices and lipid metabolism in Coilia nasus].

    Science.gov (United States)

    Wei, Guang-Lian; Xu, Gang-Chun; Gu, Ruo-Bo; Xu, Pao

    2013-12-01

    Effects of diets on growth, serum biochemical indices, and enzyme activities related to lipid metabolism in fingerlings Coilia nasus at age of 6 months were investigated during 60-day experiment in this study. Fingerlings with similar body length and mass were fed with one of 3 types of diets (diet 1: soft pellet; diet 2: soft pellet mixed with fish oil; diet 3: slow-sinking hard pellet). Fish fed with diets 2 or 3 had significantly higher total body mass, rate of mass gain, specific growth rate, and fullness coefficient than those fed with diet 1. Fish fed with diet 3 exhibited the lower food coefficient compared to those fed with diets 1 or 2. Growth traits (length, length to mass ratio, length to width ratio, hepatopancreas somatic indices and viscera somatic index) and serum biochemical indices (total protein, albumin, blood glucose, cholesterol and triglycerides) in all three treatments were not significantly different. Fish fed with diet 1 exhibited significantly higher carnitine palmitoyltransferase-I than those fed with diets 2 or 3, while fish fed with diet 2 exhibited significantly lower carnitine palmitoyltransferase-II. However, amylase, pepsin, lipase activity, lipoprotein lipase and acetyl-coa carboxylase had no significant difference in fish body among all groups. Results suggested that fish oil as a diet supplement highly facilitated fish growing. The slow-sinking pellet had the highest utilization efficiency and was suitable to feed fish fingerlings of C. nasus.

  18. Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

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    Fabien Coze

    Full Text Available Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2 strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

  19. Metabolic traits of an uncultured archaeal lineage -MSBL1- from brine pools of the Red Sea

    KAUST Repository

    Mwirichia, Romano

    2016-01-13

    The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden–Meyerhof–Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide a unique narrow niche) when glucose and other fermentable sugars are not available.

  20. GGCX-Associated Phenotypes: An Overview in Search of Genotype-Phenotype Correlations

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    Eva Y. G. De Vilder

    2017-01-01

    Full Text Available Gamma-carboxylation, performed by gamma-glutamyl carboxylase (GGCX, is an enzymatic process essential for activating vitamin K-dependent proteins (VKDP with important functions in various biological processes. Mutations in the encoding GGCX gene are associated with multiple phenotypes, amongst which vitamin K-dependent coagulation factor deficiency (VKCFD1 is best known. Other patients have skin, eye, heart or bone manifestations. As genotype–phenotype correlations were never described, literature was systematically reviewed in search of patients with at least one GGCX mutation with a phenotypic description, resulting in a case series of 47 patients. Though this number was too low for statistically valid correlations—a frequent problem in orphan diseases—we demonstrate the crucial role of the horizontally transferred transmembrane domain in developing cardiac and bone manifestations. Moreover, natural history suggests ageing as the principal determinant to develop skin and eye symptoms. VKCFD1 symptoms seemed more severe in patients with both mutations in the same protein domain, though this could not be linked to a more perturbed coagulation factor function. Finally, distinct GGCX functional domains might be dedicated to carboxylation of very specific VKDP. In conclusion, this systematic review suggests that there indeed may be genotype–phenotype correlations for GGCX-related phenotypes, which can guide patient counseling and management.

  1. The Effect and Mechanism of Tamoxifen-Induced Hepatocyte Steatosis in Vitro

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    Fei Zhao

    2014-03-01

    Full Text Available The aim of this study was to determine the effect and mechanism of tamoxifen (TAM-induced steatosis in vitro. HepG 2 (Human hepatocellular liver carcinoma cell line cells were treated with different concentrations of TAM for 72 h. Steatosis of hepatocytes was determined after Oil Red O staining and measurement of triglyceride (TG concentration. The expressions of genes in the TG homeostasis pathway, including sterol regulatory element-binding protein-1c (SREBP-1c, peroxisome proliferator-activated receptor γ (PPARγ, CCAAT/enhancer-binding protein α (C/EBPα, fatty acid synthase (FAS, acetyl-CoA carboxylase (ACC, stearoyl-CoA desaturase (SCD, carnitine palmitoyltransferase 1 (CPT1 and microsomal triglyceride transfer protein (MTP, were examined using quantitative real-time PCR and Western blot analysis. Cell proliferation was examined using the cell counting kit-8 (CCK-8 assay. We found that hepatocytes treated with TAM had: (1 induced hepatocyte steatosis and increased hepatocyte TG; (2 upregulation of SREBP-1c, FAS, ACC, SCD and MTP mRNA expressions (300%, 600%, 70%, 130% and 160%, respectively; (3 corresponding upregulation of protein expression; and (4 no difference in HepG 2 cell proliferation. Our results suggest that TAM can induce hepatocyte steatosis in vitro and that the enhancement of fatty acid synthesis through the upregulations of SREBP-1c and its downstream target genes (FAS, ACC and SCD may be the key mechanism of TAM-induced hepatocyte steatosis.

  2. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

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    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  3. Emodin, a Naturally Occurring Anthraquinone Derivative, Ameliorates Dyslipidemia by Activating AMP-Activated Protein Kinase in High-Fat-Diet-Fed Rats

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    Thing-Fong Tzeng

    2012-01-01

    Full Text Available The aim of this study was to investigate the antiobesity and antihyperlipidaemic effects of emodin on high-fat diet (HFD-induced obese rats, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. After being fed HFD for two weeks, Wistar rats were dosed orally with emodin (40 and 80 mg kg−1 or pioglitazone (20 mg kg−1, once daily for eight weeks. Emodin (80 mg kg−1 per day displayed similar characteristics to pioglitazone (20 mg kg−1 per day in reducing body weight gain, plasma lipid levels as well as coronary artery risk index and atherogenic index of HFD-fed rats. Emodin also caused dose related reductions in the hepatic triglyceride and cholesterol contents and lowered hepatic lipid droplets accumulation in HFD-fed rats. Emodin and pioglitazone enhanced the phosphorylation of AMP-activated protein kinase (AMPK and its primary downstream targeting enzyme, acetyl-CoA carboxylase, up-regulated gene expression of carnitine palmitoyl transferase 1, and down-regulated sterol regulatory element binding protein 1 and fatty acid synthase protein levels in hepatocytes of HFD-fed rats. Our findings suggest emodin could attenuate lipid accumulation by decreasing lipogenesis and increasing mitochondrial fatty acid β-oxidation mediated by activation of the AMPK signaling pathway.

  4. Short-term oleoyl-estrone treatment affects capacity to manage lipids in rat adipose tissue

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    Remesar Xavier

    2007-08-01

    Full Text Available Abstract Background Short-term OE (oleoyl-estrone treatment causes significant decreases in rat weight mainly due to adipose tissue loss. The aim of this work was to determine if OE treatment affects the expression of genes that regulate lipid metabolism in white adipose tissue. Results Gene expression in adipose tissue from female treated rats (48 hours was analysed by hybridization to cDNA arrays and levels of specific mRNAs were determined by real-time PCR. Treatment with OE decreased the expression of 232 genes and up-regulated 75 other genes in mesenteric white adipose tissue. The use of real-time PCR validate that, in mesenteric white adipose tissue, mRNA levels for Lipoprotein Lipase (LPL were decreased by 52%, those of Fatty Acid Synthase (FAS by 95%, those of Hormone Sensible Lipase (HSL by 32%, those of Acetyl CoA Carboxylase (ACC by 92%, those of Carnitine Palmitoyltransferase 1b (CPT1b by 45%, and those of Fatty Acid Transport Protein 1 (FATP1 and Adipocyte Fatty Acid Binding Protein (FABP4 by 52% and 49%, respectively. Conversely, Tumour Necrosis Factor (TNFα values showed overexpression (198%. Conclusion Short-term treatment with OE affects adipose tissue capacity to extract fatty acids from lipoproteins and to deal with fatty acid transport and metabolism.

  5. Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

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    Guo Honghui

    2012-01-01

    Full Text Available Abstract Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC and carnitine palmitoyl transferase 1 (CPT-1 pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g, a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease.

  6. The Compound of Mangiferin-Berberine Salt Has Potent Activities in Modulating Lipid and Glucose Metabolisms in HepG2 Cells

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    Can Wang

    2016-01-01

    Full Text Available The mangiferin-berberine (MB salt was synthesized by ionic bonding of mangiferin (M and berberine (B at an equal molecular ratio. This study aimed to investigate the activities of MB salt in modulating lipid and glucose metabolisms in HepG2 cells. After 24 h treatment of the studying compounds, cellular AMP-activated protein kinase α (AMPKα/acetyl-CoA carboxylase (ACC protein levels and carnitine palmitoyltransferase (CPT 1 activities, intracellular lipid contents, mRNA expression levels of target genes, glucose consumption, and glucose production amounts were determined. Compound C (CC was used in the blocking experiments. Our results showed that MB salt increased p-AMPKα (Thr172/p-ACC (Ser79 levels and CPT1 activity and suppressed oleic acid- (OA- induced lipid accumulation and upregulation of lipogenic genes potently in HepG2 cells. The above activities of MB salt were AMPK dependent and were superior to those of M or B when administered at an equal molar concentration. MB salt enhanced basal and insulin-stimulated glucose consumption and suppressed gluconeogenesis more potently than M or B alone. The inhibiting activity of MB salt on cellular gluconeogenesis was AMPK dependent. Our results may support MB salt as a new kind of agent for the development of novel lipid or glucose-lowering drugs in the future.

  7. The Roots of Atractylodes macrocephala Koidzumi Enhanced Glucose and Lipid Metabolism in C2C12 Myotubes via Mitochondrial Regulation

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    Mi Young Song

    2015-01-01

    Full Text Available The root of Atractylodes macrocephala Koidzumi (Atractylodis Rhizoma Alba, ARA is a Traditional Korean Medicine and has been commonly used for weight control. Mitochondrial dysfunction appears to be a key contributor to insulin resistance, and therefore mitochondrial targeting drugs represent an important potential strategy for the treatment of insulin resistance and obesity. In this study, the authors investigated the regulatory effects of ARA on mitochondrial function with respect to the stimulation of glucose and lipid metabolism in C2C12 myotubes. After differentiating C2C12 myotubes, cells were treated with or without different concentrations (0.2, 0.5, and 1.0 mg/mL of ARA extract. ARA extract significantly increased the expression of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC1α and the downregulations of its targets, nuclear respiratory factor-1 (NRF-1, transcription factor A (TFAM, and total ATP content in C2C12 myotubes. ARA extract also increased the expressions of PGC1α activator and of the metabolic sensors, AMP-activated protein kinase (AMPK, and acetyl-CoA carboxylase and sirtuin (SIRT 1. Furthermore, it significantly increased glucose uptake by enhancing glucose consumption and subsequently decreased FFA contents and increased carnitine palmitoyltransferase (CPT 1b expression. Our study indicates that ARA has a potential for stimulating mitochondrial function and energy metabolism in muscle.

  8. Treatment of Nonalcoholic Fatty Liver Disease with Total Alkaloids in Rubus aleaefolius Poir through Regulation of Fat Metabolism

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    Ying Li

    2014-01-01

    Full Text Available Total alkaloids in Rubus aleaefolius Poir (TARAP is a folk medicinal herb that has been used clinically in China to treat nonalcoholic fatty liver disease (NAFLD for many years. However, the mechanism of its anti-NAFLD effect is largely unknown. In this study, we developed a NAFLD rat model by supplying a modified high-fat diet (mHFD ad libitum for 8 weeks and evaluated the therapeutic effect of TARAP in NAFLD rats as well as the underlying molecular mechanism. We found that TARAP could reduce the serum triglycerides (TG, total cholesterol (TC, and low-density lipoprotein (LDL-C levels and increase the serum high-density lipoprotein (HDL-C level in NAFLD rats. In addition, TARAP treatment reduced expression of fatty acid synthetase (FAS, and acetyl-CoA carboxylase (ACC and upregulated the expression of carnitine palmitoyltransferase (CPT. Our results suggest that regulation of lipid metabolism may be a mechanism by which TARAP treats NAFLD.

  9. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism

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    Anna M. Giudetti

    2016-05-01

    Full Text Available The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs. The citrate carrier (CiC and the carnitine/acylcarnitine transporter (CACT are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism.

  10. A Combination of Flaxseed Oil and Astaxanthin Improves Hepatic Lipid Accumulation and Reduces Oxidative Stress in High Fat-Diet Fed Rats

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    Jiqu Xu

    2017-03-01

    Full Text Available Hepatic lipid accumulation and oxidative stress are crucial pathophysiological mechanisms for non-alcoholic fatty liver disease (NAFLD. Thus, we examined the effect of a combination of flaxseed oil (FO and astaxanthin (ASX on hepatic lipid accumulation and oxidative stress in rats fed a high-fat diet. ASX was dissolved in flaxseed oil (1 g/kg; FO + ASX. Animals were fed diets containing 20% fat, where the source was lard, or 75% lard and 25% FO + ASX, or 50% lard and 50% FO + ASX, or FO + ASX, for 10 weeks. Substitution of lard with FO + ASX reduced steatosis and reduced hepatic triacylglycerol and cholesterol. The combination of FO and ASX significantly decreased hepatic sterol regulatory element-binding transcription factor 1 and 3-hydroxy-3-methylglutaryl-CoA reductase but increased peroxisome proliferator activated receptor expression. FO + ASX significantly suppressed fatty acid synthase and acetyl CoA carboxylase but induced carnitine palmitoyl transferase-1 and acyl CoA oxidase expression. FO + ASX also significantly elevated hepatic SOD, CAT and GPx activity and GSH, and markedly reduced hepatic lipid peroxidation. Thus, FO and ASX may reduce NAFLD by reversing hepatic steatosis and reducing lipid accumulation and oxidative stress.

  11. Acetylated and propionated derivatives of swertiamarin have anti-adipogenic effects

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    Hitesh B Vaidya

    2014-01-01

    Full Text Available Objective: To investigate whether the acetylated and propionated derivatives (LMP-09-1 and -2 of swertiamarin have anti-adipogenic effects. Materials and Methods: 3T3-L1 pre-adipocytes were grown in Dulbecco′s Modified Eagle′s Medium (DMEM containing 10% calf serum; fully confluent cells were differentiated with insulin, dexamethasone, and 3-isobutylmethylxanthine in the presence and absence of LMP-09-1 and -2 (100 μg/mL for 10 days. Control cells received same amount of dimethylsulfoxide (DMSO. On day ten, cells were analyzed for triglycerides accumulation and the expression of genes involved in adipogenesis, lipogenesis, and lipolysis. In another set of experiment, effects of LMP-09-1 and 2 were studied for isoproterenol induced lipolysis using fully mature adipocytes. Results: LMP-09-1 and -2 caused a significant (P < 0.001 reduction in intracellular triglycerides accumulation. Both LMP-09-1 and -2 significantly (P < 0.001 decreased the mRNA expression of peroxisome proliferator activated receptor-γ and acetyl-CoA carboxylase-1, and increased isoproterenol induced lipolysis in adipocytes. LMP-09-1 induced lipolysis even in the absence of isoproterenol, and also showed a significant up-regulation of carnitine palmitoyl transferase-1α and hormone sensitive lipase (HSL gene expression. Conclusions: These findings show that swertiamarin derivatives, LMP-09-1 and -2 have a potent anti-adipogenic effect.

  12. Uteroplacental insufficiency down regulates insulin receptor and affects expression of key enzymes of long-chain fatty acid (LCFA metabolism in skeletal muscle at birth

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    Puglianiello Antonella

    2008-05-01

    Full Text Available Abstract Background Epidemiological studies have revealed a relationship between early growth restriction and the subsequent development of insulin resistance and type 2 diabetes. Ligation of the uterine arteries in rats mimics uteroplacental insufficiency and serves as a model of intrauterine growth restriction (IUGR and subsequent developmental programming of impaired glucose tolerance, hyperinsulinemia and adiposity in the offspring. The objective of this study was to investigate the effects of uterine artery ligation on the skeletal muscle expression of insulin receptor and key enzymes of LCFA metabolism. Methods Bilateral uterine artery ligation was performed on day 19 of gestation in Sprague-Dawley pregnant rats. Muscle of the posterior limb was dissected at birth and processed by real-time RT-PCR to analyze the expression of insulin receptor, ACCα, ACCβ (acetyl-CoA carboxylase alpha and beta subunits, ACS (acyl-CoA synthase, AMPK (AMP-activated protein kinase, alpha2 catalytic subunit, CPT1B (carnitine palmitoyltransferase-1 beta subunit, MCD (malonyl-CoA decarboxylase in 14 sham and 8 IUGR pups. Muscle tissue was treated with lysis buffer and Western immunoblotting was performed to assay the protein content of insulin receptor and ACC. Results A significant down regulation of insulin receptor protein (p Conclusion Our data suggest that uteroplacental insufficiency may affect skeletal muscle metabolism down regulating insulin receptor and reducing the expression of key enzymes involved in LCFA formation and oxidation.

  13. Des-γ-Carboxy Prothrombin (DCP as a Potential Autologous Growth Factor for the Development of Hepatocellular Carcinoma

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    Yu-Sheng Zhang

    2014-08-01

    Full Text Available Des-γ-carboxy prothrombin (DCP is a prothrombin precursor produced in hepatocellular carcinoma (HCC. Because of deficiency of vitamin K or γ-glutamyl carboxylase in HCC cells, the 10 glutamic acid (Glu residues in prothrombin precursor did not completely carboxylate to γ-carboxylated glutamic acid (Gla residues, leaving some Glu residues remained in N-terminal domain. These prothrombin precursors with Glu residues are called DCPs. DCP displays insufficient coagulation activity. Since Liebman reported an elevated plasma DCP in patients with HCC, DCP has been used in the diagnosis of HCC. Recently, its biological malignant potential has been specified to describe DCP as an autologous growth factor to stimulate HCC growth and a paracrine factor to integrate HCC with vascular endothelial cells. DCP was found to stimulate HCC growth through activation of the DCP-Met-JAK1-STAT3 signaling pathway. DCP might increase HCC invasion and metastasis through activation of matrix metalloproteinase (MMPs and the ERK1/2 MAPK signaling pathway. DCP has also been found to play a crucial role in the formation of angiogenesis. DCP could increase the angiogenic factors released from HCC and vascular endothelial cells. These effects of DCP in angiogenesis might be related to activation of the DCP-KDR-PLC-γ-MAPK signaling pathway. In this article, we summarized recent studies on DCP in biological roles related to cancer progression and angiogenesis in HCC.

  14. Can exploiting natural genetic variation in leaf photosynthesis contribute to increasing rice productivity? A simulation analysis.

    Science.gov (United States)

    Gu, Junfei; Yin, Xinyou; Stomph, Tjeerd-Jan; Struik, Paul C

    2014-01-01

    Rice productivity can be limited by available photosynthetic assimilates from leaves. However, the lack of significant correlation between crop yield and leaf photosynthetic rate (A) is noted frequently. Engineering for improved leaf photosynthesis has been argued to yield little increase in crop productivity because of complicated constraints and feedback mechanisms when moving up from leaf to crop level. Here we examined the extent to which natural genetic variation in A can contribute to increasing rice productivity. Using the mechanistic model GECROS, we analysed the impact of genetic variation in A on crop biomass production, based on the quantitative trait loci for various photosynthetic components within a rice introgression line population. We showed that genetic variation in A of 25% can be scaled up equally to crop level, resulting in an increase in biomass of 22-29% across different locations and years. This was probably because the genetic variation in A resulted not only from Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase)-limited photosynthesis but also from electron transport-limited photosynthesis; as a result, photosynthetic rates could be improved for both light-saturated and light-limited leaves in the canopy. Rice productivity could be significantly improved by mining the natural variation in existing germ-plasm, especially the variation in parameters determining light-limited photosynthesis.

  15. Inhibitory effects of Rubi Fructus extracts on hepatic steatosis development in high-fat diet-induced obese mice.

    Science.gov (United States)

    Nam, Mi Kyung; Choi, Hye Ran; Cho, Jin Sook; Cho, Soo Min; Ha, Ki Chan; Kim, Tae-Hyeon; Ryu, Hee-Young; Lee, Young-Ik

    2014-10-01

    The present study was performed to investigate the potential effects of the unripened dried fruit of Rubus coreanus Miq., Rubi Fructus (RF), on hepatic steatosis and lipid metabolism in mice fed with a high-fat diet (HFD) known to induce obesity and hyperlipidaemia. Rubi Fructus extract (RFex) fed mice demonstrated a reduced body weight and adipose tissue weight. RFex fed mice also demonstrated decreased aminotransferase levels, lipid contents [triglyceride (TG), total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C)], leptin content and increased high‑density lipoprotein-cholesterol (HDL‑C) contents in the plasma. These effects were accompanied by a decreased expression of lipogenic genes, including sterol regulatory element binding protein-1c, liver X receptor, fatty acid synthase (FAS), acetyl‑CoA carboxylase, cluster of differentiation 36, lipoprotein lipase and decreased lipogenic enzyme FAS and 3-hydroxy-3 methylglutamyl coenzyme reductase enzyme activities, while elevating carnitine palmitoyltrasferase-1 activity. Based on these results, the present study hypothesized that the inhibitory effect on hepatic steatosis of RFex is the result of the suppression of lipid synthesis in mice fed with HFD, suggesting that RFex may be beneficial in preventing hepatic steatosis and liver lipotoxicity.

  16. Mathematical modelling of the citric acid cycle for the analysis of glutamine isotopomers from cerebellar astrocytes incubated with [1(-13)C]glucose.

    Science.gov (United States)

    Merle, M; Martin, M; Villégier, A; Canioni, P

    1996-08-01

    A mathematical model of the citric acid cycle devoted to the analysis of 13C-NMR data was developed for determining the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. Different variants of the model were considered depending on the reversibility of the conversion of fumarate into malate and oxaloacetate. The model also included the possibility of orientation-conserved transfer of the four-carbon citric acid cycle intermediates, leading to conversion of succinyl-CoA C1 into either malate C1 or C4. It was used to analyse NMR data from glutamine isotopomers produced by cerebellar astrocytes incubated with [1-13C]glucose. Partial cycling (39%) between oxaloacetate and fumarate was evident from the analysis. Application of the model to glutamate isotopomers from granule cells incubated with [1-13C]glucose [Martin, M.. Portais, J.C.. Labouesse. J., Canioni. P, & Merle, M. (1993) Eur. J. Biochem. 217, 617-625] indicated that total cycling of oxaloacetate into fumarate was, in this case, required to get the best fit. The results emphasized some important differences in carbon metabolism between cerebellar astrocytes and granule cells concerning the sources of carbon fuelling the citric acid cycle and the carbon fluxes on different pathways.

  17. Dietary conjugated linoleic acid modify gene expression in liver, muscles, and fat tissues of finishing pigs.

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    Tous, N; Theil, P K; Lauridsen, C; Lizardo, R; Vilà, B; Esteve-Garcia, E

    2012-12-01

    The aim of this study was to investigate underlying mechanisms of dietary conjugated linoleic acid (CLA) on lipid metabolism in various tissues of pigs. Sixteen gilts (73 ± 3 kg) were fed a control (containing sunflower oil) or an experimental diet in which 4% of sunflower oil was replaced by CLA, and slaughtered at an average BW of 117 ± 4.9 kg. Transcription of peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), sterol regulatory element binding protein (SREBP1), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), delta-6-desaturase (D6D), and stearoyl CoA desaturase (SCD) were determined by real-time PCR in longissimus thoracis (LT) and semimembranosus (SM) muscles, LT subcutaneous and SM intermuscular fat, and in the liver. Fatty acid (FA) composition was analyzed using gas chromatography in these tissues, except for SM intermuscular fat. Dietary CLA increased PPARγ in LT muscle (P tissues studied (P fat. Transcription of genes related to FA synthesis was reduced by CLA in SM muscle and liver (SREBP1, both P fat. Saturated FA were increased in all studied tissues (P tissue-specific way by CLA. It was concluded that dietary CLA affected transcription of genes and fat metabolism in a tissue-specific manner.

  18. Effects of dietary fat saturation on fatty acid composition and gene transcription in different tissues of Iberian pigs.

    Science.gov (United States)

    Benítez, R; Núñez, Y; Fernández, A; Isabel, B; Fernández, A I; Rodríguez, C; Barragán, C; Martín-Palomino, P; López-Bote, C; Silió, L; Óvilo, C

    2015-04-01

    The effect of two diets, respectively enriched with SFA (S) and PUFA (P), on FA tissue composition and gene expression was studied in fattened Iberian pigs. The FA composition of adipose, muscular and liver tissues was affected by dietary treatment. S group showed higher MUFA and MUFA/SFA ratio and lower PUFA and n-6/n-3 ratio than P group in all analyzed tissues. In muscle and liver the extracted lipids were separated into neutral lipids and polar lipid fractions which showed significantly different responses to the dietary treatment, especially in liver where no significant effect of diet was observed in NL fraction. The expression of six candidate genes related to lipogenesis and FA oxidation was analyzed by qPCR. In liver, stearoyl CoA desaturase (SCD), acetyl CoA carboxylase alpha (ACACA) and malic enzyme 1 (ME1) genes showed higher expression in S group. SCD, ACACA, ME1, and fatty acid synthase (FASN) gene expression levels showed a wide variation across the tested tissues, with much higher expression levels observed in adipose tissue than other tissues. Tissue FA profile and gene expression results support the deposition of dietary FA, the lipogenic effect of dietary saturated fat in liver and the employment of saturated dietary fat for endogenous synthesis of MUFA in all the analyzed tissues.

  19. Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China.

    Science.gov (United States)

    Bi, Yaling; Liu, Weitang; Guo, Wenlei; Li, Lingxu; Yuan, Guohui; Du, Long; Wang, Jinxin

    2016-01-01

    Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose-response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

  20. Calorie Restriction Prevents Metabolic Aging Caused by Abnormal SIRT1 Function in Adipose Tissues.

    Science.gov (United States)

    Xu, Cheng; Cai, Yu; Fan, Pengcheng; Bai, Bo; Chen, Jie; Deng, Han-Bing; Che, Chi-Ming; Xu, Aimin; Vanhoutte, Paul M; Wang, Yu

    2015-05-01

    Adipose tissue is a pivotal organ determining longevity, due largely to its role in maintaining whole-body energy homeostasis and insulin sensitivity. SIRT1 is a NAD-dependent protein deacetylase possessing antiaging activities in a wide range of organisms. The current study demonstrates that mice with adipose tissue-selective overexpression of hSIRT1(H363Y), a dominant-negative mutant that disrupts endogenous SIRT1 activity, show accelerated development of metabolic aging. These mice, referred to as Adipo-H363Y, exhibit hyperglycemia, dyslipidemia, ectopic lipid deposition, insulin resistance, and glucose intolerance at a much younger age than their wild-type littermates. The metabolic defects of Adipo-H363Y are associated with abnormal epigenetic modifications and chromatin remodeling in their adipose tissues, as a result of excess accumulation of biotin, which inhibits endogenous SIRT1 activity, leading to increased inflammation, cellularity, and collagen deposition. The enzyme acetyl-CoA carboxylase 2 plays an important role in biotin accumulation within adipose tissues of Adipo-H363Y. Calorie restriction prevents biotin accumulation, abolishes abnormal histone biotinylation, and completely restores the metabolic and adipose functions of Adipo-H363Y. The effects are mimicked by short-term restriction of biotin intake, an approach potentially translatable to humans for maintaining the epigenetic and chromatin remodeling capacity of adipose tissues and preventing aging-associated metabolic disorders.

  1. Biotinidase Deficiency in Newborns as Respiratory Distress and Tachypnea: A Case Report

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    Shahin KOOHMANAEE

    2015-06-01

    Full Text Available How to Cite This Article: Kohmanaee Sh, Zarkesh M, Tabrizi M, Hassanzadeh Rad A, Divshali S, Dalili S. Biotinidase Deficiency in Newborns as Respiratory Distress and Tachypnea: A Case Report. Iran J Child Neurol. Spring 2015; 9(2:58-60.AbstractObjectiveBiotin is a coenzyme composed of four carboxylases. It presents in amino acid catabolism, fatty acid synthesis, and gluconeogenesis. Biotinidase recycles the vitamin biotin. A biotinidase deficiency is a neurocutaneous disorder with autosomal recessive inheritance. The symptoms can be successfully treatedor prevented by administering pharmacological doses of biotin. Although, according to neonatal prenatal medicine (2011, a biotinidase deficiency does not manifest during the neonatal period. In this study, we report on a case of biotinidase deficiency in the first week of birth.Case ReportA 3100 g term boy was born via cesarean section. After 3 days, he was referred to the 17th Shahrivar Hospital with the chief complaint of tachypnea and grunting.Laboratory results revealed that liver and renal function tests, serum electrolytes, and blood indexes except ammonia were all normal. Within few days after the administration of oral biotin, the patient showed dramatic improvement and was discharged. However, within 4 months he was admitted two other times with the complaints of diarrhea and pneumonia. Unfortunately, he expired after 4 months.ConclusionAccording to our results, it seems that clinicians should accurately assess suspicious patients and even assess infants for biotinidase deficiency.

  2. Glutamate metabolism in the brain focusing on astrocytes

    DEFF Research Database (Denmark)

    Schousboe, Arne; Scafidi, Susanna; Bak, Lasse Kristoffer

    2014-01-01

    Metabolism of glutamate, the main excitatory neurotransmitter and precursor of GABA, is exceedingly complex and highly compartmentalized in brain. Maintenance of these neurotransmitter pools is strictly dependent on the de novo synthesis of glutamine in astrocytes which requires both the anaplero......Metabolism of glutamate, the main excitatory neurotransmitter and precursor of GABA, is exceedingly complex and highly compartmentalized in brain. Maintenance of these neurotransmitter pools is strictly dependent on the de novo synthesis of glutamine in astrocytes which requires both...... the anaplerotic enzyme pyruvate carboxylase and glutamine synthetase. Glutamate is formed directly from glutamine by deamidation via phosphate activated glutaminase a reaction that also yields ammonia. Glutamate plays key roles linking carbohydrate and amino acid metabolism via the tricarboxylic acid (TCA) cycle......, as well as in nitrogen trafficking and ammonia homeostasis in brain. The anatomical specialization of astrocytic endfeet enables these cells to rapidly and efficiently remove neurotransmitters from the synaptic cleft to maintain homeostasis, and to provide glutamine to replenish neurotransmitter pools...

  3. Piceatannol Exerts Anti-Obesity Effects in C57BL/6 Mice through Modulating Adipogenic Proteins and Gut Microbiota

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    Yen-Chen Tung

    2016-10-01

    Full Text Available Obesity is a global health concern. Piceatannol (Pic, an analog of resveratrol (Res, has many reported biological activities. In this study, we investigated the anti-obesity effect of Pic in a high-fat diet (HFD-induced obese animal model. The results showed that Pic significantly reduced mouse body weight in a dose-dependent manner without affecting food intake. Serum total cholesterol (TC, low-density lipoprotein (LDL, high-density lipoprotein (HDL levels, and blood glucose (GLU were significantly lowered in Pic-treated groups. Pic significantly decreased the weight of liver, spleen, perigonadal and retroperitoneal fat compared with the HFD group. Pic significantly reduced the adipocyte cell size of perigonadal fat and decreased the weight of liver. Pic-treated mice showed higher phosphorylated adenosine 5′-monophosphate-activated protein kinase (pAMPK and phosphorylated acetyl-CoA carboxylase (pACC protein levels and decreased protein levels of CCAAT/enhancer-binding protein C/EBPα, peroxisome proliferator-activated receptor PPARγ and fatty acid synthase (FAS, resulting in decreased lipid accumulation in adipocytes and the liver. Pic altered the composition of the gut microbiota by increasing Firmicutes and Lactobacillus and decreasing Bacteroidetes compared with the HFD group. Collectively, these results suggest that Pic may be a candidate for obesity treatment.

  4. NanoESI Mass Spectrometry of Rubisco and Rubisco Activase Structures and Their Interactions with Nucleotides and Sugar Phosphates

    Science.gov (United States)

    Blayney, Michelle J.; Whitney, Spencer M.; Beck, Jennifer L.

    2011-09-01

    Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.

  5. High survival frequencies at low herbicide use rates in populations of Lolium rigidum result in rapid evolution of herbicide resistance.

    Science.gov (United States)

    Neve, P; Powles, S

    2005-12-01

    The frequency of phenotypic resistance to herbicides in previously untreated weed populations and the herbicide dose applied to these populations are key determinants of the dynamics of selection for resistance. In total, 31 Lolium rigidum populations were collected from sites with no previous history of exposure to herbicides and where there was little probability of gene flow from adjacent resistant populations. The mean survival frequency across all 31 populations following two applications of commercial rates (375 g ha(-1)) of the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicide, diclofop-methyl was 0.43%. Survivors from five of these populations were grown to maturity and seed was collected. Dose-response experiments compared population level resistance to diclofop-methyl in these selected lines with their original parent populations. A single cycle of herbicide selection significantly increased resistance in all populations (LD(50) R:S ratios ranged from 2.8 to 23.2), confirming the inheritance and genetic basis of phenotypic resistance. In vitro assays of ACCase inhibition by diclofop acid indicated that resistance was due to a non-target-site mechanism. Following selection with diclofop-methyl, the five L. rigidum populations exhibited diverse patterns of cross-resistance to ACCase and ALS-inhibiting herbicides, suggesting that different genes or gene combinations were responsible for resistance. The relevance of these results to the management of herbicide resistance are discussed.

  6. Fitness cost due to herbicide resistance may trigger genetic background evolution.

    Science.gov (United States)

    Darmency, Henri; Menchari, Yosra; Le Corre, Valérie; Délye, Christophe

    2015-01-01

    This article investigates the possible existence of mechanisms counterbalancing the negative pleiotropic effects on development and reproduction that are conferred by alleles responsible for herbicide resistance in the weed Alopecurus myosuroides. We considered three herbicide-resistant, mutant acetyl-coenzyme A carboxylase (ACCase) alleles, Leu1781, Asn2041, and Gly2078, found in eight resistant populations. Of these, Gly2078 is the only allele with a known fitness cost. We compared plants homozygous for wild-type ACCase alleles that were siblings of plants carrying a given mutant resistant ACCase allele with plants from three populations where resistance did not evolve. In each of two series of experiments, we measured germination dynamics, seedling vigor, plant height, vegetative biomass, and seed production. The wild-type siblings of plants carrying Gly2078 performed better in the field, on average, than wild-type plants that were sibling of plants carrying other mutant ACCase alleles, and particularly those carrying Leu1781. We propose that rapid evolution of the genetic background of plants from the populations where the Gly2078 allele originally arose could partially counterbalance Gly2078 fitness cost, enhancing the spread of the resistant genotypes.

  7. DNA analysis of herbarium Specimens of the grass weed Alopecurus myosuroides reveals herbicide resistance pre-dated herbicides.

    Science.gov (United States)

    Délye, Christophe; Deulvot, Chrystel; Chauvel, Bruno

    2013-01-01

    Acetyl-CoA carboxylase (ACCase) alleles carrying one point mutation that confers resistance to herbicides have been identified in arable grass weed populations where resistance has evolved under the selective pressure of herbicides. In an effort to determine whether herbicide resistance evolves from newly arisen mutations or from standing genetic variation in weed populations, we used herbarium specimens of the grass weed Alopecurus myosuroides to seek mutant ACCase alleles carrying an isoleucine-to-leucine substitution at codon 1781 that endows herbicide resistance. These specimens had been collected between 1788 and 1975, i.e., prior to the commercial release of herbicides inhibiting ACCase. Among the 734 specimens investigated, 685 yielded DNA suitable for PCR. Genotyping the ACCase locus using the derived Cleaved Amplified Polymorphic Sequence (dCAPS) technique identified one heterozygous mutant specimen that had been collected in 1888. Occurrence of a mutant codon encoding a leucine residue at codon 1781 at the heterozygous state was confirmed in this specimen by sequencing, clearly demonstrating that resistance to herbicides can pre-date herbicides in weeds. We conclude that point mutations endowing resistance to herbicides without having associated deleterious pleiotropic effects can be present in weed populations as part of their standing genetic variation, in frequencies higher than the mutation frequency, thereby facilitating their subsequent selection by herbicide applications.

  8. Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides.

    Science.gov (United States)

    Menchari, Yosra; Camilleri, Christine; Michel, Séverine; Brunel, Dominique; Dessaint, Fabrice; Le Corre, Valérie; Délye, Christophe

    2006-01-01

    Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion. In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase (ACCase) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of Côte d'Or) by genotyping 13 151 plants originating from 243 fields. Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated. These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides.

  9. DNA analysis of herbarium Specimens of the grass weed Alopecurus myosuroides reveals herbicide resistance pre-dated herbicides.

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    Christophe Délye

    Full Text Available Acetyl-CoA carboxylase (ACCase alleles carrying one point mutation that confers resistance to herbicides have been identified in arable grass weed populations where resistance has evolved under the selective pressure of herbicides. In an effort to determine whether herbicide resistance evolves from newly arisen mutations or from standing genetic variation in weed populations, we used herbarium specimens of the grass weed Alopecurus myosuroides to seek mutant ACCase alleles carrying an isoleucine-to-leucine substitution at codon 1781 that endows herbicide resistance. These specimens had been collected between 1788 and 1975, i.e., prior to the commercial release of herbicides inhibiting ACCase. Among the 734 specimens investigated, 685 yielded DNA suitable for PCR. Genotyping the ACCase locus using the derived Cleaved Amplified Polymorphic Sequence (dCAPS technique identified one heterozygous mutant specimen that had been collected in 1888. Occurrence of a mutant codon encoding a leucine residue at codon 1781 at the heterozygous state was confirmed in this specimen by sequencing, clearly demonstrating that resistance to herbicides can pre-date herbicides in weeds. We conclude that point mutations endowing resistance to herbicides without having associated deleterious pleiotropic effects can be present in weed populations as part of their standing genetic variation, in frequencies higher than the mutation frequency, thereby facilitating their subsequent selection by herbicide applications.

  10. Clozapine promotes glycolysis and myelin lipid synthesis in cultured oligodendrocytes

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    Johann eSteiner

    2014-11-01

    Full Text Available Clozapine has stronger systemic metabolic side effects than haloperidol and it was hypothesized that therapeutic antipsychotic and adverse metabolic effects might be related. Considering that cerebral disconnectivity through oligodendrocyte dysfunction has been implicated in schizophrenia, it is important to determine the effect of these drugs on oligodendrocyte energy metabolism and myelin lipid production.Effects of clozapine and haloperidol on glucose and myelin lipid metabolism were evaluated and compared in cultured OLN-93 oligodendrocytes. First, glycolytic activity was assessed by measurement of extra- and intracellular glucose and lactate levels. Next, the expression of glucose (GLUT and monocarboxylate (MCT transporters was determined after 6h and 24h. And finally mitochondrial respiration, acetyl-CoA carboxylase, free fatty acids, and expression of the myelin lipid galactocerebroside were analyzed.Both drugs altered oligodendrocyte glucose metabolism, but in opposite directions. Clozapine improved the glucose uptake, production and release of lactate, without altering GLUT and MCT. In contrast, haloperidol led to higher extracellular levels of glucose and lower levels of lactate, suggesting reduced glycolysis. Antipsychotics did not alter significantly the number of functionally intact mitochondria, but clozapine enhanced the efficacy of oxidative phosphorylation and expression of galactocerebroside.Our findings support the superior impact of clozapine on white matter integrity in schizophrenia as previously observed, suggesting that this drug improves the energy supply and myelin lipid synthesis in oligodendrocytes. Characterizing the underlying signal transduction pathways may pave the way for novel oligodendrocyte-directed schizophrenia therapies.

  11. Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

    Science.gov (United States)

    Raghavan, V.

    1992-01-01

    Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

  12. Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

    Science.gov (United States)

    Neuner, Andreas; Wagner, Ines; Sieker, Tim; Ulber, Roland; Schneider, Konstantin; Peifer, Susanne; Heinzle, Elmar

    2013-01-20

    Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

  13. The economic importance of acaricides in the control of phytophagous mites and an update on recent acaricide mode of action research.

    Science.gov (United States)

    Van Leeuwen, Thomas; Tirry, Luc; Yamamoto, Atsushi; Nauen, Ralf; Dermauw, Wannes

    2015-06-01

    Acaricides are one of the cornerstones of an efficient control program for phytophagous mites. An analysis of the global acaricide market reveals that spider mites such as Tetranychus urticae, Panonychus citri and Panonychus ulmi are by far the most economically important species, representing more than 80% of the market. Other relevant mite groups are false spider mites (mainly Brevipalpus), rust and gall mites and tarsonemid mites. Acaricides are most frequently used in vegetables and fruits (74% of the market), including grape vines and citrus. However, their use is increasing in major crops where spider mites are becoming more important, such as soybean, cotton and corn. As revealed by a detailed case study of the Japanese market, major shifts in acaricide use are partially driven by resistance development and the commercial availability of compounds with novel mode of action. The importance of the latter cannot be underestimated, although some compounds are successfully used for more than 30 years. A review of recent developments in mode of action research is presented, as such knowledge is important for devising resistance management programs. This includes spirocyclic keto-enols as inhibitors of acetyl-CoA carboxylase, the carbazate bifenazate as a mitochondrial complex III inhibitor, a novel class of complex II inhibitors, and the mite growth inhibitors hexythiazox, clofentezine and etoxazole that interact with chitin synthase I.

  14. Glucose and Intermediary Metabolism and Astrocyte-Neuron Interactions Following Neonatal Hypoxia-Ischemia in Rat.

    Science.gov (United States)

    Brekke, Eva; Berger, Hester Rijkje; Widerøe, Marius; Sonnewald, Ursula; Morken, Tora Sund

    2017-01-01

    Neonatal hypoxia-ischemia (HI) and the delayed injury cascade that follows involve excitotoxicity, oxidative stress and mitochondrial failure. The susceptibility to excitotoxicity of the neonatal brain may be related to the capacity of astrocytes for glutamate uptake. Furthermore, the neonatal brain is vulnerable to oxidative stress, and the pentose phosphate pathway (PPP) may be of particular importance for limiting this kind of injury. Also, in the neonatal brain, neurons depend upon de novo synthesis of neurotransmitters via pyruvate carboxylase in astrocytes to increase neurotransmitter pools during normal brain development. Several recent publications describing intermediary brain metabolism following neonatal HI have yielded interesting results: (1) Following HI there is a prolonged depression of mitochondrial metabolism in agreement with emerging evidence of mitochondria as vulnerable targets in the delayed injury cascade. (2) Astrocytes, like neurons, are metabolically impaired following HI, and the degree of astrocytic malfunction may be an indicator of the outcome following hypoxic and hypoxic-ischemic brain injury. (3) Glutamate transfer from neurons to astrocytes is not increased following neonatal HI, which may imply that astrocytes fail to upregulate glutamate uptake in response to the massive glutamate release during HI, thus contributing to excitotoxicity. (4) In the neonatal brain, the activity of the PPP is reduced following HI, which may add to the susceptibility of the neonatal brain to oxidative stress. The present review aims to discuss the metabolic temporal alterations observed in the neonatal brain following HI.

  15. FGF1 and FGF19 reverse diabetes by suppression of the hypothalamic-pituitary-adrenal axis.

    Science.gov (United States)

    Perry, Rachel J; Lee, Sangwon; Ma, Lie; Zhang, Dongyan; Schlessinger, Joseph; Shulman, Gerald I

    2015-04-28

    Fibroblast growth factor-1 (FGF1) and FGF19 have been shown to improve glucose metabolism in diabetic rodents, but how this occurs is unknown. Here to investigate the mechanism of action of these growth factors, we perform intracerebroventricular (i.c.v.) injections of recombinant FGF1 or FGF19 in an awake rat model of type 1 diabetes (T1D) and measure rates of whole-body lipolysis, hepatic acetyl CoA content, pyruvate carboxylase activity and hepatic glucose production. We show that i.c.v. injection of FGF19 or FGF1 leads to a ∼60% reduction in hepatic glucose production, hepatic acetyl CoA content and whole-body lipolysis, which results from decreases in plasma ACTH and corticosterone concentrations. These effects are abrogated by an intra-arterial infusion of corticosterone. Taken together these studies identify suppression of the HPA axis and ensuing reductions in hepatic acetyl CoA content as a common mechanism responsible for mediating the acute, insulin-independent, glucose-lowering effects of FGF1 and FGF19 in rodents with poorly controlled T1D.

  16. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  17. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    Science.gov (United States)

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  18. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

    Science.gov (United States)

    Runguphan, Weerawat; Keasling, Jay D

    2014-01-01

    As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals.

  19. A molecular analysis of ground sloth diet through the last glaciation.

    Science.gov (United States)

    Hofreiter, M; Poinar, H N; Spaulding, W G; Bauer, K; Martin, P S; Possnert, G; Pääbo, S

    2000-12-01

    DNA was extracted from five coprolites, excavated in Gypsum Cave, Nevada and radiocarbon dated to approximately 11 000, 20 000 and 28 500 years BP. All coprolites contained mitochondrial DNA sequences identical to a DNA sequence determined from a bone of the extinct ground sloth Nothrotheriops shastensis. A 157-bp fragment of the chloroplast gene for the large subunit of the ribulosebisphosphate carboxylase (rbcL) was amplified from the boluses and several hundred clones were sequenced. In addition, the same DNA fragment was sequenced from 99 plant species that occur in the vicinity of Gypsum Cave today. When these were compared to the DNA sequences in GenBank, 69 were correctly (two incorrectly) assigned to taxonomic orders. The plant sequences from the five coprolites as well as from one previously studied coprolite were compared to rbcL sequences in GenBank and the contemporary plant species. Thirteen families or orders of plants that formed part of the diet of the Shasta ground sloth could be identified, showing that the ground sloth was feeding on trees as well as herbs and grasses. The plants in the boluses further indicate that the climate 11 000 years BP was dryer than 20 000 and 28 500 years BP. However, the sloths seem to have visited water sources more frequently at 11 000 BP than at earlier times.

  20. Feeding a High Concentrate Diet Down-Regulates Expression of ACACA, LPL and SCD and Modifies Milk Composition in Lactating Goats.

    Directory of Open Access Journals (Sweden)

    Hui Tao

    Full Text Available High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD group and low concentrate diet (LCD group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS in plasma and decreased milk fat content, vaccenic acid (VA and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c, lipoprotein lipase (LPL, fatty acid synthetase (FASN and acetyl-CoA carboxylase α (ACACA, ACCα involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.

  1. Rosiglitazone, a PPAR-γ agonist, fails to attenuate CLA-induced milk fat depression and hepatic lipid accumulation in lactating mice.

    Science.gov (United States)

    Vyas, Diwakar; Teter, Beverly B; Delmonte, Pierluigi; Erdman, Richard A

    2014-07-01

    Our objective was to investigate the combination of rosiglitazone (ROSI) and conjugated linoleic acid (CLA) on mammary and hepatic lipogenesis in lactating C57Bl/6 J mice. Twenty-four lactating mice were randomly assigned to one of four treatments applied from postpartum day 6 to day 10. Treatments included: (1) control diet, (2) control plus 1.5 % dietary CLA (CLA) substituted for soybean oil, (3) control plus daily intra-peritoneal (IP) rosiglitazone injections (10 mg/kg body weight) (ROSI), and (4) CLA plus ROSI (CLA-ROSI). Dam food intake and milk fat concentration were depressed with CLA. However, no effects were observed with ROSI. The CLA-induced milk fat depression was due to reduced expression for mammary lipogenic genes involved in de-novo fatty acid (FA) synthesis, FA uptake and desaturation, and triacyglycerol synthesis. Liver weight (g/100 g body weight) was increased by CLA due to an increase in lipid accumulation triggering a compensatory reduction in mRNA abundance of hepatic lipogenic enzymes, including acetyl-CoA carboxylase I and stearoyl-CoA desaturase I. On the contrary, no effects were observed with ROSI on hepatic and mammary lipogenic gene and enzyme expression. Overall, feeding CLA to lactating mice induced milk fat depression and increased hepatic lipid accumulation, probably due to the presence of trans-10, cis-12 CLA isomer, while ROSI failed to significantly attenuate both hepatic steatosis and reduction in milk fat content.

  2. Trans-10,cis-12-CLA dysregulate lipid and glucose metabolism and induce hepatic NR4A receptors.

    Science.gov (United States)

    Navarro, Maria A; Badimon, Lina; Rodriguez, Cristina; Arnal, Carmen; Noone, Enda J; Roche, Helen M; Osada, Jesus; Martinez-Gonzalez, Jose

    2010-01-01

    Our aim was to assess the effect of two isomers of conjugated linoleic acids (CLA), cis-9,trans-11-CLA (c9,t11-CLA) and trans-10,cis-12-CLA (t10,c12-CLA), on glucose metabolism and hepatic expression of NR4A receptors, key transcription factors regulating gluconeogenesis. ApoE-deficient mice were fed isocaloric, isonitrogenous westernized diets enriched with c9,t11-CLA, t10,c12-CLA or linoleic acid (control diet). Plasma glucose, NEFA, triglyceride and cholesterol concentrations were significantly higher in the t10,c12-CLA group compared with c9,t11-CLA or control group. Plasma insulin concentrations were lowered by c9,t11-CLA compared with either control or t10,c12-CLA group. Hepatic expression of NR4A receptors (Nur77, Nurr1 and NOR-1) was induced by t10,c12-CLA while c9,t11-CLA had not effect. Consistently t10,c12-CLA up-regulated key genes involved in gluconeogenesis including glucose-6-phosphatase, enolase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Hepatic expression of NR4A receptors correlated with plasma NEFA, with the expression of their target gene fatty acid transporter (FAT)/CD36 and with the accumulation of fat in the liver. These results suggest that t10,c12-CLA promote dysregulation of lipid and glucose metabolism, at least in part, by an isomer-specific modulation of hepatic expression of NR4A receptors.

  3. The effect of long term under- and over-feeding on the expression of genes related to lipid metabolism in mammary tissue of sheep.

    Science.gov (United States)

    Tsiplakou, Eleni; Flemetakis, Emmanouil; Kouri, Evangelia-Diamanto; Sotirakoglou, Kyriaki; Zervas, George

    2015-02-01

    Milk fatty acid (FA) synthesis by the mammary gland involves expression of a large number of genes whose nutritional regulation remains poorly defined. In this study, we examined the effect of long-term under- and over-feeding on the expression of genes (acetyl Co A carboxylase, ACC; fatty acid synthetase, FAS; lipoprotein lipase, LPL; stearoyl Co A desaturase, SCD; peroxisome proliferator activated receptor γ2, PPARγ2; sterol regulatory element binding protein-1, SREBP-1c; and hormone sensitive lipase, HSL) related to FA metabolism in sheep mammary tissue (MT). Twenty-four lactating sheep were divided into three homogenous sub-groups and fed the same ration in quantities covering 70% (underfeeding), 100% (control) and 130% (overfeeding) of their energy and crude protein requirements. The results showed a significant reduction of mRNA of ACC, FAS, LPL and SCD in the MT of underfed sheep, and a significant increase on the mRNA of LPL and SREBP-1c in the MT of overfed compared with the control respectively. In conclusion, the negative, compared to positive, energy balance in sheep down-regulates ACC, FAS, LPL, SCD, SREBP-1c and PPARγ2 expression in their MT which indicates that the decrease in nutrient availability may lead to lower rates of lipid synthesis.

  4. The response of Cupriavidus metallidurans CH34 to spaceflight in the international space station.

    Science.gov (United States)

    Leys, Natalie; Baatout, Sarah; Rosier, Caroline; Dams, Annik; s'Heeren, Catherine; Wattiez, Ruddy; Mergeay, Max

    2009-08-01

    The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the 'International Space Station' (ISS), strain CH34 was grown for 10-12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.

  5. Molecular Pathogenesis of Liver Steatosis Induced by Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    Jun; Cheng; Min; Li; Ping; Gao; Jin-ling; Dong; Qi; Wang

    2012-01-01

    Liver steatosis is a pathological hallmark in patients with chronic hepatitis C(CHC).Increased lipid uptake,decreased lipid secretion,increased lipid synthesis and decreased lipid degradation are all involved in pathogenesis of steatosis induced by hepatitic C virus(HCV) infection.Level of low density lipoprotein receptor(LDL-R) and activity of peroxisome proliferator-activated receptor(PPAR) α is related to liver uptake of lipid from circulation,and affected by HCV.Secretion via microsomal triglyceride transfer protein(MTTP),and formation of very low density lipoprotein(VLDL) have been hampered by HCV infection.Up-regulation of lipid synthesis related genes,such as sterol regulatory element-binding protein(SREBP)-1,SREBP-2,SREBP-1c,fatty acid synthase(FASN),HMG CoA reductase(HMGCR),liver X receptor(LXR),acetyl-CoA carboxylase 1(ACC1),hepatic CB(1) receptors,retinoid X receptor(RXR) α,were the main stay of liver steatosis pathogenesis.Degradation of lipid in liver is decreased in patients with CHC.There is strong evidence that heterogeneity of HCV core genes of different genotypes affect their effects of liver steatosis induction.A mechanism in which steatosis is involved in HCV life cycle is emerging.

  6. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Qin Feng

    2013-01-01

    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  7. Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Sandeep Dave

    Full Text Available The phytotherapeutic protein stem bromelain (SBM is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2, fatty acid synthase (FAS, lipoprotein lipase (LPL, CD36, and acetyl-CoA carboxylase (ACC were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B, and GTP binding protein G(iα(1, as well as sustained expression of hormone sensitive lipase (HSL. These data indicate that SBM, together with all-trans retinoic-acid (atRA, may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.

  8. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  9. DNA barcoding for species identification in the Palmae family.

    Science.gov (United States)

    Naeem, A; Khan, A A; Cheema, H M N; Khan, I A; Buerkert, A

    2014-12-04

    DNA barcoding is a promising tool for species identification at the molecular level. The barcoding system is well established for species differentiation in animals, while it is less common in plants. We evaluated 2 barcoding regions, maturase K (matK) and ribulose bisphosphate carboxylase (rbcL), to compare species of Palmae according to amplification success, discrimination power, and inter- and intra-specific divergence. Both regions appear to have potential to discriminate most species of Palmae, but 2 species, Phoenix dactylifera and Phoenix sylvestris, did not show variation in the nucleotides of the barcode genes. P. sylvestris is said to be the sister species of P. dactilyfera according to its morphological and genetic proximity to the cultivated date palm. Thus, the status of these 2 species needs to be re-evaluated considering more genes as barcodes. Furthermore, rbcL has a higher discrimination power (90%) than matK (66.6%) and can thus be potentially used as a standard barcode to discriminate the species of Palmae.

  10. Time-course investigation of Phytophthora infestans infection of potato leaf from three cultivars by quantitative proteomics

    Directory of Open Access Journals (Sweden)

    Mia Kruse Guldstrand Larsen

    2016-03-01

    We used label-free quantitative proteomics to investigate the infection with P. infestans in a time-course study over 258 h. Several key issues limits proteome analysis of potato leaf tissue [5–7]. Firstly, the immense complexity of the plant proteome, which is further complicated by the presence of highly abundant proteins, such as ribulose bisphosphate carboxylase/oxygenase (RuBisCO. Secondly, plant leaf and potato, in particular, contain abundant levels amounts of phenols and polyphenols, which hinder or completely prevent a successful protein extraction. Hitherto, protein profiling of potato leaf tissues have been limited to few proteome studies and only 1484 proteins have been extracted and comprehensively described [5,8,9]. We here present the detailed methods and raw data by optimized gel-enhanced label free quantitative approach. The methodology enabled us to detect and quantify between 3248 and 3529 unique proteins from each cultivar, and up to 758 P. infestans derived proteins. The complete dataset is available via ProteomeXchange, with the identifier PXD002767.

  11. β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells

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    Min Zhang

    2015-11-01

    Full Text Available Background/Aims: In dairy cows, β-hydroxybutyrate (BHBA is utilized as precursors of de novo synthesized fatty acids in mammary gland. Ketotic cows are characterized by excessive negative energy balance (NEB, which can further increase the blood BHBA concentration. Sterol regulatory element-binding protein1 (SREBP1 and cell death-inducing DNA fragmentation factor-alpha-like effector α (Cidea play crucial roles in lipid synthesis. Therefore, we hypothesized that BHBA could stimulate SREBP1/Cidea pathway to increase milk fat synthesis in bovine mammary epithelial cells. Methods: Bovine mammary epithelial cells were treated with different concentrations of BHBA and transfected with adenovirus to silence SREBP1 expression. The effects of BHBA on the lipid synthesis in bovine mammary epithelial cells were investigated. Results: The results showed that BHBA could significantly increase the expression of SREBP1, fatty acid synthase (FAS, acetyl-CoA carboxylase α (ACC-α, Cidea and diacylglycerol transferase-1 (DGAT-1, as well as the triglycerides (TG content in bovine mammary epithelial cells. BHBA treatment also increased the transfer of mature SREBP1 to nucleus compared with control group. However, SREBP1 silencing could significantly down-regulate the overexpression of FAS, ACC-α, Cidea and DGAT-1, as well as TG content induced by BHBA. Conclusion: The present data indicate that BHBA can significantly increase TG secretion mediated by SREBP1 in bovine mammary epithelial cells.

  12. Metabolic adaptation in transplastomic plants massively accumulating recombinant proteins.

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    Julia Bally

    Full Text Available BACKGROUND: Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we used proteomics to characterize tobacco (Nicotiana tabacum plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD or a green fluorescent protein (GFP. While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO(2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. CONCLUSIONS/SIGNIFICANCE: The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation.

  13. Bioinformatic analysis of the distribution of inorganic carbon transporters and prospective targets for bioengineering to increase Ci uptake by cyanobacteria.

    Science.gov (United States)

    Gaudana, Sandeep B; Zarzycki, Jan; Moparthi, Vamsi K; Kerfeld, Cheryl A

    2015-10-01

    Cyanobacteria have evolved a carbon-concentrating mechanism (CCM) which has enabled them to inhabit diverse environments encompassing a range of inorganic carbon (Ci: [Formula: see text] and CO2) concentrations. Several uptake systems facilitate inorganic carbon accumulation in the cell, which can in turn be fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase. Here we survey the distribution of genes encoding known Ci uptake systems in cyanobacterial genomes and, using a pfam- and gene context-based approach, identify in the marine (alpha) cyanobacteria a heretofore unrecognized number of putative counterparts to the well-known Ci transporters of beta cyanobacteria. In addition, our analysis shows that there is a huge repertoire of transport systems in cyanobacteria of unknown function, many with homology to characterized Ci transporters. These can be viewed as prospective targets for conversion into ancillary Ci transporters through bioengineering. Increasing intracellular Ci concentration coupled with efforts to increase carbon fixation will be beneficial for the downstream conversion of fixed carbon into value-added products including biofuels. In addition to CCM transporter homologs, we also survey the occurrence of rhodopsin homologs in cyanobacteria, including bacteriorhodopsin, a class of retinal-binding, light-activated proton pumps. Because they are light driven and because of the apparent ease of altering their ion selectivity, we use this as an example of re-purposing an endogenous transporter for the augmentation of Ci uptake by cyanobacteria and potentially chloroplasts.

  14. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

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    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  15. Cyanophycin mediates the accumulation and storage of fixed carbon in non-heterocystous filamentous cyanobacteria from coniform mats.

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    Biqing Liang

    Full Text Available Thin, filamentous, non-heterocystous, benthic cyanobacteria (Subsection III from some marine, lacustrine and thermal environments aggregate into macroscopic cones and conical stromatolites. We investigate the uptake and storage of inorganic carbon by cone-forming cyanobacteria from Yellowstone National Park using high-resolution stable isotope mapping of labeled carbon (H(13CO3 (- and immunoassays. Observations and incubation experiments in actively photosynthesizing enrichment cultures and field samples reveal the presence of abundant cyanophycin granules in the active growth layer of cones. These ultrastructurally heterogeneous granules rapidly accumulate newly fixed carbon and store 18% of the total particulate labeled carbon after 120 mins of incubation. The intracellular distribution of labeled carbon during the incubation experiment demonstrates an unexpectedly large contribution of PEP carboxylase to carbon fixation, and a large flow of carbon and nitrogen toward cyanophycin in thin filamentous, non-heterocystous cyanobacteria. This pattern does not occur in obvious response to a changing N or C status. Instead, it may suggest an unusual interplay between the regulation of carbon concentration mechanisms and accumulation of photorespiratory products that facilitates uptake of inorganic C and reduces photorespiration in the dense, surface-attached communities of cyanobacteria from Subsection III.

  16. Krebs cycle enzymes from livers of old mice are differentially regulated by caloric restriction.

    Science.gov (United States)

    Hagopian, Kevork; Ramsey, Jon J; Weindruch, Richard

    2004-08-01

    Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and alpha-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The alpha-ketoglutarate/glutamate and malate/alpha-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain.

  17. Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens.

    Science.gov (United States)

    Takeuchi, Kasumi; Kiefer, Patrick; Reimmann, Cornelia; Keel, Christoph; Dubuis, Christophe; Rolli, Joëlle; Vorholt, Julia A; Haas, Dieter

    2009-12-11

    Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

  18. Effects of 60 Hz sinusoidal magnetic field on in vitro establishment, multiplication, and acclimatization phases of Coffea arabica seedlings.

    Science.gov (United States)

    Isaac Alemán, Elizabeth; Oliveira Moreira, Rafael; Almeida Lima, Andre; Chaves Silva, Samuel; González-Olmedo, Justo Lorenzo; Chalfun-Junior, Antonio

    2014-09-01

    The influence of extremely low frequency electromagnetic fields on net photosynthesis, transpiration, photosynthetic pigment concentration, and gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS1), during in vitro establishment, in vitro multiplication and acclimatization phases of coffee seedlings were investigated. Untreated coffee plants were considered as control, whereas treated plants were exposed to a 60 Hz sinusoidal magnetic field of 2 mT of magnetic induction during 3 min. This magnetic field was generated by an electromagnet, connected to a wave generator. The results revealed that magnetically treated plants showed a significant increase in net photosynthesis (85.4% and 117.9%, in multiplication and acclimatization phases, respectively), and in photosynthetic pigment concentration (66.6% for establishment phase, 79.9% for multiplication phase, and 43.8% for acclimatization phase). They also showed a differential RBCS1 gene expression (approximately twofold) and a decrease of transpiration rates in regard to their control plants. In conclusion, the findings suggest that the application of 60 Hz magnetic field to in vitro coffee plants may improve the seedlings quality by modifying some photosynthetic physiological and molecular processes, increasing their vigor, and ensuring better plant development in later stages.

  19. Rosmarinus officinalis polyphenols produce anti-depressant like effect through monoaminergic and cholinergic functions modulation.

    Science.gov (United States)

    Sasaki, Kazunori; El Omri, Abdelfatteh; Kondo, Shinji; Han, Junkyu; Isoda, Hiroko

    2013-02-01

    Rosmarinus officinalis (R. officinalis), a culinary aromatic and medicinal plant, is very rich in polyphenols and flavonoids with high antioxidant properties. This plant was reported to exert multiple benefits for neuronal system and alleviate mood disorder. In our previous study, we demonstrated that R. officinalis and its active compounds, luteolin (Lut), carnosic acid (CA), and rosmarinic acid (RA), exhibited neurotrophic effects and improved cholinergic functions in PC12 cells in correlation with mitogen-activated protein kinase (MAPK), ERK1/2 signaling pathway. The current study was conducted to evaluate and understand the anti-depressant effect of R. officinalis using tail suspension test (TST) in ICR mice and PC12 cells as in vitro neuronal model. Proteomics analysis of PC12 cells treated with R. officinalis polyphenols (ROP) Lut, CA, and RA revealed a significant upregulation of tyrosine hydroxylase (TH) and pyruvate carboxylase (PC) two major genes involved in dopaminergic, serotonergic and GABAergic pathway regulations. Moreover, ROP were demonstrated to protect neuronal cells against corticosterone-induced toxicity. These results were concordant with decreasing immobility time in TST and regulation of several neurotransmitters (dopamine, norepinephrine, serotonin and acetylcholine) and gene expression in mice brain like TH, PC and MAPK phosphatase (MKP-1). To the best of our knowledge this is the first evidence to contribute to the understanding of molecular mechanism behind the anti-depressant effect of R. officinalis and its major active compounds.

  20. RAP-PCR fingerprinting reveals time-dependent expression of development-related genes following differentiation process of Bacillus thuringiensis.

    Science.gov (United States)

    Huang, Tianpei; Yu, Xiaomin; Gelbič, Ivan; Guan, Xiong

    2015-09-01

    Gene expression profiles are important data to reveal the functions of genes putatively involved in crucial biological processes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and specifically primed reverse transcription polymerase chain reaction (RT-PCR) were combined to screen differentially expressed genes following development of a commercial Bacillus thuringiensis subsp. kurstaki strain 8010 (serotype 3a3b). Six differentially expressed transcripts (RAP1 to RAP6) were obtained. RAP1 encoded a putative triple helix repeat-containing collagen or an exosporium protein H related to spore pathogenicity. RAP2 was homologous to a ClpX protease and an ATP-dependent protease La (LonB), which likely acted as virulence factors. RAP3 was homologous to a beta subunit of propionyl-CoA carboxylase required for the development of Myxococcus xanthus. RAP4 had homology to a quinone oxidoreductase involved in electron transport and ATP formation. RAP5 showed significant homology to a uridine kinase that mediates phosphorylation of uridine and azauridine. RAP6 shared high sequence identity with 3-methyl-2-oxobutanoate-hydroxymethyltransferase (also known as ketopantoate hydroxymethyltransferase or PanB) involved in the operation of the tricarboxylic acid cycle. The findings described here would help to elucidate the molecular mechanisms underlying the differentiation process of B. thuringiensis and unravel novel pathogenic genes.

  1. Metagenomic insights into the dominant Fe(II oxidizing Zetaproteobacteria from an iron mat at Lōi’hi, Hawai’I

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    Esther eSinger

    2013-03-01

    Full Text Available Zetaproteobacteria are among the most prevalent Fe(II oxidizing bacteria at deep-sea hydrothermal vents; however, knowledge about their environmental significance is limited. We provide metagenomic insights into an iron mat at the Lōʻihi Seamount, Hawai’I, revealing novel genomic information of locally dominant Zetaproteobacteria lineages. These lineages were previously estimated to account for ~13% of all local Zetaproteobacteria based on 16S clone library data. Biogeochemically relevant genes include nitrite reductases, which were previously not identified in Zetaproteobacteria, sulfide:quinone oxidases, and ribulose-1,5-bisphosphate carboxylase (RuBisCo. Genes assumed to be involved in Fe(II oxidation correlate in synteny and share 70% AASim with those previously identified in the related Zetaproteobacterium Mariprofundus ferrooxydans PV-1. Overall, Zetaproteobacteria genes appear to originate primarily from within the Proteobacteria and the Fe(II oxidizing Leptospirillum spp. and are predicted to facilitate adaptation to a deep-sea hydrothermal vent environment in addition to microaerophilic Fe(II and H2S oxidation. This dataset represents the first metagenomic study of Fe(II oxidizing bacteria (FeOB from an iron oxide mat at a deep-sea hydrothermal habitat.

  2. Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

    Science.gov (United States)

    Osborn, Hannah L; Alonso-Cantabrana, Hugo; Sharwood, Robert E; Covshoff, Sarah; Evans, John R; Furbank, Robert T; von Caemmerer, Susanne

    2017-01-01

    In C4 species, the major β-carbonic anhydrase (β-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3(-), which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in β-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C(18)O(16)O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

  3. Phylogeny of a genomically diverse group of elymus (poaceae allopolyploids reveals multiple levels of reticulation.

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    Roberta J Mason-Gamer

    Full Text Available The grass tribe Triticeae (=Hordeeae comprises only about 300 species, but it is well known for the economically important crop plants wheat, barley, and rye. The group is also recognized as a fascinating example of evolutionary complexity, with a history shaped by numerous events of auto- and allopolyploidy and apparent introgression involving diploids and polyploids. The genus Elymus comprises a heterogeneous collection of allopolyploid genome combinations, all of which include at least one set of homoeologs, designated St, derived from Pseudoroegneria. The current analysis includes a geographically and genomically diverse collection of 21 tetraploid Elymus species, and a single hexaploid species. Diploid and polyploid relationships were estimated using four molecular data sets, including one that combines two regions of the chloroplast genome, and three from unlinked nuclear genes: phosphoenolpyruvate carboxylase, β-amylase, and granule-bound starch synthase I. Four gene trees were generated using maximum likelihood, and the phylogenetic placement of the polyploid sequences reveals extensive reticulation beyond allopolyploidy alone. The trees were interpreted with reference to numerous phenomena known to complicate allopolyploid phylogenies, and introgression was identified as a major factor in their history. The work illustrates the interpretation of complicated phylogenetic results through the sequential consideration of numerous possible explanations, and the results highlight the value of careful inspection of multiple independent molecular phylogenetic estimates, with particular focus on the differences among them.

  4. Phylogeny of a genomically diverse group of elymus (poaceae) allopolyploids reveals multiple levels of reticulation.

    Science.gov (United States)

    Mason-Gamer, Roberta J

    2013-01-01

    The grass tribe Triticeae (=Hordeeae) comprises only about 300 species, but it is well known for the economically important crop plants wheat, barley, and rye. The group is also recognized as a fascinating example of evolutionary complexity, with a history shaped by numerous events of auto- and allopolyploidy and apparent introgression involving diploids and polyploids. The genus Elymus comprises a heterogeneous collection of allopolyploid genome combinations, all of which include at least one set of homoeologs, designated St, derived from Pseudoroegneria. The current analysis includes a geographically and genomically diverse collection of 21 tetraploid Elymus species, and a single hexaploid species. Diploid and polyploid relationships were estimated using four molecular data sets, including one that combines two regions of the chloroplast genome, and three from unlinked nuclear genes: phosphoenolpyruvate carboxylase, β-amylase, and granule-bound starch synthase I. Four gene trees were generated using maximum likelihood, and the phylogenetic placement of the polyploid sequences reveals extensive reticulation beyond allopolyploidy alone. The trees were interpreted with reference to numerous phenomena known to complicate allopolyploid phylogenies, and introgression was identified as a major factor in their history. The work illustrates the interpretation of complicated phylogenetic results through the sequential consideration of numerous possible explanations, and the results highlight the value of careful inspection of multiple independent molecular phylogenetic estimates, with particular focus on the differences among them.

  5. In vitro anti-diabetic effect and chemical component analysis of 29 essential oils products

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    Hsiu-Fang Yen

    2015-03-01

    Full Text Available Twenty-nine commercial essential oil (EO products that were purchased from the Taiwan market, including three different company-made Melissa officinalis essential oils, were assayed on their glucose consumption activity and lipid accumulation activity on 3T3-L1 adipocytes. The EOs of M. officinalis were significantly active in both model assays. By contrast, EOs of peppermint, lavender, bergamot, cypress, niaouli nerolidol, geranium-rose, and revensara did not increase glucose consumption activity from media, but displayed inhibited lipid accumulation activity (65–90% of lipid accumulation vs. the control 100%. Because of the promising activity of M. officinalis EOs, three different products were collected and compared for their gas chromatography chemical profiles and bioactivity. The Western blot data suggest that the key factors of the adenosine monophosphate-activated protein kinase/acetyl-CoA carboxylase pathway can be mediated by M. officinalis EOs. Together with biodata, gas chromatography–mass spectrometry profiles suggested mixtures of citrals and minor compounds of M. officinalis EOs may play an important role on effect of antidiabetes.

  6. Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hui Jia; Peng-Peng Zhang; Ding-Ji Shi; Hua-Ling Mi; Jia-Cheng Zhu; Xi-Wen Huang; Pei-Min He

    2015-01-01

    Since pepc gene encoding phosphoenolpyruvate carboxylase (PEPCase) has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated (forward) or downregulated (reverse) pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction (RT‐qPCR), Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I (PSI) activity was inhibited, as reflected by Y(I), and Y(NA) was elevated, and dark reduction of P700þ was stimulated, indicating enhanced cyclic electron flow (CEF) around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

  7. Connection of Nicotine to Diet-Induced Obesity and Non-Alcoholic Fatty Liver Disease: Cellular and Mechanistic Insights

    Science.gov (United States)

    Sinha-Hikim, Amiya P.; Sinha-Hikim, Indrani; Friedman, Theodore C.

    2017-01-01

    Non-alcoholic fatty liver disease (NAFLD) poses a serious health hazard affecting 20–40% of adults in the general population in the USA and over 70% of the obese and extremely obese people. In addition to obesity, nicotine is recognized as a risk factor for NAFLD, and it has been reported that nicotine can exaggerate obesity-induced hepatic steatosis. The development of NAFLD has serious clinical complications because of its potential progression from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. Multiple mechanisms can be involved in nicotine plus high-fat diet-induced (HFD) hepatic steatosis. Emerging evidence now suggests that nicotine exacerbates hepatic steatosis triggered by HFD, through increased oxidative stress and hepatocellular apoptosis, decreased phosphorylation (inactivation) of adenosine-5-monophosphate-activated protein kinase and, in turn, up-regulation of sterol response-element binding protein 1-c, fatty acid synthase, and activation of acetyl-coenzyme A-carboxylase, leading to increased hepatic lipogenesis. There is also growing evidence that chronic endoplasmic reticulum stress through regulation of several pathways leading to oxidative stress, inflammation, perturbed hepatic lipid homeostasis, apoptosis, and autophagy can induce hepatic steatosis and its progression to NASH. Evidence also suggests a central role of the gut microbiota in obesity and its related disorders, including NAFLD. This review explores the contribution of nicotine and obesity to the development of NAFLD and its molecular underpinning. PMID:28239368

  8. Immunostimulatory in vitro and in vivo effects of a water-soluble extract from kale.

    Science.gov (United States)

    Nishi, Kosuke; Kondo, Ai; Okamoto, Takeaki; Nakano, Hiroyuki; Daifuku, Miho; Nishimoto, Sogo; Ochi, Kenji; Takaoka, Terumi; Sugahara, Takuya

    2011-01-01

    The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer's patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.

  9. Combinatorial biosynthesis of flavones and flavonols in Escherichia coli.

    Science.gov (United States)

    Miyahisa, Ikuo; Funa, Nobutaka; Ohnishi, Yasuo; Martens, Stefan; Moriguchi, Takaya; Horinouchi, Sueharu

    2006-06-01

    (2S)-Flavanones (naringenin and pinocembrin) are key intermediates in the flavonoid biosynthetic pathway in plants. Recombinant Escherichia coli cells containing four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:CoA ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-CoA carboxylase, have been established for efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. Further introduction of the flavone synthase I gene from Petroselinum crispum under the control of the T7 promoter and the synthetic ribosome-binding sequence in pACYCDuet-1 caused the E. coli cells to produce flavones: apigenin (13 mg/l) from tyrosine and chrysin (9.4 mg/l) from phenylalanine. Introduction into the E. coli cells of the flavanone 3beta-hydroxylase and flavonol synthase genes from the plant Citrus species led to production of flavonols: kaempferol (15.1 mg/l) from tyrosine and galangin (1.1 mg/l) from phenylalanine. The combinatorial biosynthesis of the flavones and flavonols in E. coli is promising for the construction of a library of various flavonoid compounds and un-natural flavonoids in bacteria.

  10. Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins.

    Science.gov (United States)

    Strzalka, K; Ngernprasirtsiri, J; Watanabe, A; Akazawa, T

    1987-12-16

    Amyloplasts isolated from white-wild suspension-cultured cells of sycamore (Acer pseudoplatanus L.) are found to import and process the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach, but they lack the ability to form its holoenzyme due to the absence of both the large subunit and its binding-protein. They also import the precursor of the 33-kDa extrinsic protein (p33-kDa) of the O2-evolving complex of Photosystem II from spinach, but process is only to an intermediate form (i33-kDa). Chloroplasts from green-mutant cells of sycamore process p33-kDa to its mature form in this heterologous system. These results suggest that the thylakoid-associated protease responsible for the second processing step of p33-kDa is missing in amyloplasts, possibly due to the absence of thylakoid-membranes. In contrast, the apparent import of the precursor of the light-harvesting chlorophyll a/b-binding apoprotein (pLHCP) from spinach was not detected. Sycamore amyloplasts may lack the ability to import this particular thylakoid-protein, or rapidly degrade the imported molecules in the absence of thylakoid-membranes for their proper insertion.

  11. Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells

    Science.gov (United States)

    Wang, Yuan Yuan; Attané, Camille; Milhas, Delphine; Dirat, Béatrice; Dauvillier, Stéphanie; Guerard, Adrien; Gilhodes, Julia; Lazar, Ikrame; Alet, Nathalie; Laurent, Victor; Le Gonidec, Sophie; Hervé, Caroline; Bost, Frédéric; Ren, Guo Sheng; Bono, Françoise; Escourrou, Ghislaine; Prentki, Marc; Nieto, Laurence; Valet, Philippe

    2017-01-01

    In breast cancer, a key feature of peritumoral adipocytes is their loss of lipid content observed both in vitro and in human tumors. The free fatty acids (FFAs), released by adipocytes after lipolysis induced by tumor secretions, are transferred and stored in tumor cells as triglycerides in lipid droplets. In tumor cell lines, we demonstrate that FFAs can be released over time from lipid droplets through an adipose triglyceride lipase–dependent (ATGL-dependent) lipolytic pathway. In vivo, ATGL is expressed in human tumors where its expression correlates with tumor aggressiveness and is upregulated by contact with adipocytes. The released FFAs are then used for fatty acid β-oxidation (FAO), an active process in cancer but not normal breast epithelial cells, and regulated by coculture with adipocytes. However, in cocultivated cells, FAO is uncoupled from ATP production, leading to AMPK/acetyl-CoA carboxylase activation, a circle that maintains this state of metabolic remodeling. The increased invasive capacities of tumor cells induced by coculture are completely abrogated by inhibition of the coupled ATGL-dependent lipolysis/FAO pathways. These results show a complex metabolic symbiosis between tumor-surrounding adipocytes and cancer cells that stimulate their invasiveness, highlighting ATGL as a potential therapeutic target to impede breast cancer progression. PMID:28239646

  12. Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

    Science.gov (United States)

    Noah, Alexandre Mboene; Niemenak, Nicolas; Sunderhaus, Stephanie; Haase, Christin; Omokolo, Denis Ndoumou; Winkelmann, Traud; Braun, Hans-Peter

    2013-01-14

    Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed.

  13. Ozone-induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves.

    Science.gov (United States)

    Bagard, Matthieu; Le Thiec, Didier; Delacote, Emilien; Hasenfratz-Sauder, Marie-Paule; Banvoy, Jacques; Gérard, Joëlle; Dizengremel, Pierre; Jolivet, Yves

    2008-12-01

    Young poplar trees (Populus tremula Michx. x Populus alba L. clone INRA 717-1B4) were subjected to 120 ppb of ozone for 35 days in phytotronic chambers. Treated trees displayed precocious leaf senescence and visible symptoms of injury (dark brown/black upper surface stippling) exclusively observed on fully expanded leaves. In these leaves, ozone reduced parameters related to photochemistry (Chl content and maximum rate of photosynthetic electron transport) and photosynthetic CO(2) fixation [net CO(2) assimilation, Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity and maximum velocity of Rubisco for carboxylation]. In fully expanded leaves, the rate of photorespiration as estimated from Chl fluorescence was markedly impaired by the ozone treatment together with the activity of photorespiratory enzymes (Rubisco and glycolate oxidase). Immunoblot analysis revealed a decrease in the content of serine hydroxymethyltransferase in treated mature leaves, while the content of the H subunit of the glycine decarboxylase complex was not modified. Leaves in the early period of expansion were exempt from visible symptoms of injury and remained unaffected as regards all measured parameters. Leaves reaching full expansion under ozone exposure showed potential responses of protection (stimulation of mitochondrial respiration and transitory stomatal closure). Our data underline the major role of leaf phenology in ozone sensitivity of photosynthetic processes and reveal a marked ozone-induced inhibition of photorespiration.

  14. Protein phosphorylation and photorespiration.

    Science.gov (United States)

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  15. The Costs of Photorespiration to Food Production Now and in the Future.

    Science.gov (United States)

    Walker, Berkley J; VanLoocke, Andy; Bernacchi, Carl J; Ort, Donald R

    2016-04-29

    Photorespiration is essential for C3 plants but operates at the massive expense of fixed carbon dioxide and energy. Photorespiration is initiated when the initial enzyme of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), reacts with oxygen instead of carbon dioxide and produces a toxic compound that is then recycled by photorespiration. Photorespiration can be modeled at the canopy and regional scales to determine its cost under current and future atmospheres. A regional-scale model reveals that photorespiration currently decreases US soybean and wheat yields by 36% and 20%, respectively, and a 5% decrease in the losses due to photorespiration would be worth approximately $500 million annually in the United States. Furthermore, photorespiration will continue to impact yield under future climates despite increases in carbon dioxide, with models suggesting a 12-55% improvement in gross photosynthesis in the absence of photorespiration, even under climate change scenarios predicting the largest increases in atmospheric carbon dioxide concentration. Although photorespiration is tied to other important metabolic functions, the benefit of improving its efficiency appears to outweigh any potential secondary disadvantages.

  16. The regulatory interplay between photorespiration and photosynthesis.

    Science.gov (United States)

    Timm, Stefan; Florian, Alexandra; Fernie, Alisdair R; Bauwe, Hermann

    2016-05-01

    The Calvin-Benson cycle and the photorespiratory pathway form the photosynthetic-photorespiratory supercycle that is responsible for nearly all biological CO2 fixation on Earth. In essence, supplementation with the photorespiratory pathway is necessary because the CO2-fixing enzyme of the Calvin-Benson cycle, ribulose 1,5-bisphosphate carboxylase (Rubisco), catalyses several side reactions including the oxygenation of ribulose 1,5-bisphosphate, which produces the noxious metabolite phosphoglycolate. The photorespiratory pathway recycles the phosphoglycolate to 3-phosphoglycerate and in this way allows the Calvin-Benson cycle to operate in the presence of molecular oxygen generated by oxygenic photosynthesis. While the carbon flow through the individual and combined subprocesses is well known, information on their regulatory interaction is very limited. Regulatory feedback from the photorespiratory pathway to the Calvin-Benson cycle can be presumed from numerous inhibitor experiments and was demonstrated in recent studies with transgenic plants. This complexity illustrates that we are not yet ready to rationally engineer photosynthesis by altering photorespiration since despite massive understanding of the core photorespiratory pathway our understanding of its interaction with other pathways and processes remains fragmentary.

  17. Photorespiration in C4 grasses remains slow under drought conditions.

    Science.gov (United States)

    Carmo-Silva, Ana E; Powers, Stephen J; Keys, Alfred J; Arrabaça, Maria Celeste; Parry, Martin A J

    2008-07-01

    The CO(2)-concentrating mechanism present in C(4) plants decreases the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and, consequently, photorespiratory rates in air. Under drought conditions, the intercellular CO(2) concentration may decrease and cause photorespiration to increase. The C(4) grasses Paspalum dilatatum Poiret, Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel were grown in soil and drought was imposed by ceasing to provide water. Net CO(2) assimilation (A) and stomatal conductance to water vapour decreased with leaf dehydration. Decreased carbon and increased oxygen isotope composition were also observed under drought. The response of A to CO(2) suggested that the compensation point was zero in all species irrespective of the extent of drought stress. A slight decrease of A as O(2) concentration increased above 10% provided evidence for slow photorespiratory gas exchanges. Analysis of amino acids contained in the leaves, particularly the decrease of glycine after 30 s in darkness, supported the presence of slow photorespiration rates, but these were slightly faster in Cynodon dactylon than in Paspalum dilatatum and Zoysia japonica. Although the contents of glycine and serine increased with dehydration and mechanistic modelling of C(4) photosynthesis suggested slightly increased photorespiration rates in proportion to photosynthesis, the results provide evidence that photorespiration remained slow under drought conditions.

  18. A high throughput gas exchange screen for determining rates of photorespiration or regulation of C4 activity.

    Science.gov (United States)

    Bellasio, Chandra; Burgess, Steven J; Griffiths, Howard; Hibberd, Julian M

    2014-07-01

    Large-scale research programmes seeking to characterize the C4 pathway have a requirement for a simple, high throughput screen that quantifies photorespiratory activity in C3 and C4 model systems. At present, approaches rely on model-fitting to assimilatory responses (A/C i curves, PSII quantum yield) or real-time carbon isotope discrimination, which are complicated and time-consuming. Here we present a method, and the associated theory, to determine the effectiveness of the C4 carboxylation, carbon concentration mechanism (CCM) by assessing the responsiveness of V O/V C, the ratio of RuBisCO oxygenase to carboxylase activity, upon transfer to low O2. This determination compares concurrent gas exchange and pulse-modulated chlorophyll fluorescence under ambient and low O2, using widely available equipment. Run time for the procedure can take as little as 6 minutes if plants are pre-adapted. The responsiveness of V O/V C is derived for typical C3 (tobacco, rice, wheat) and C4 (maize, Miscanthus, cleome) plants, and compared with full C3 and C4 model systems. We also undertake sensitivity analyses to determine the impact of R LIGHT (respiration in the light) and the effectiveness of the light saturating pulse used by fluorescence systems. The results show that the method can readily resolve variations in photorespiratory activity between C3 and C4 plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies.

  19. Combining Phytate/Ca2+Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

    Institute of Scientific and Technical Information of China (English)

    Muhammad A R F Sultan; LIU Hui; CHENG Yu-Feng; ZHANG Pei-pei; ZHAO Hui-xian

    2013-01-01

    Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.

  20. Molecular phylogenies support taxonomic revision of three species of Laurencia (Rhodomelaceae, Rhodophyta, with the description of a new genus

    Directory of Open Access Journals (Sweden)

    Florence Rousseau

    2017-02-01

    Full Text Available The systematics of the Laurencia complex was investigated using a taxon-rich data set including the chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL gene only and a character-rich data set combining mitochondrial cytochrome oxidase 1 (COI-5P, the rbcL marker, and the nuclear large subunit of the ribosomal operon (LSU. Bayesian and ML analyses of these data sets showed that three species hitherto placed in the genus Laurencia J.V.Lamour. were not closely related to Laurencia s. str. Laurencia caspica Zinova & Zaberzhinskaya was the sister group of the remaining Osmundea Stackh. species, L. crustiformans McDermid joined Palisada and L. flexilis Setch. consisted of an independent lineage. In light of these results a new genus, Ohelopapa F.Rousseau, Martin-Lescanne, Payri & L.Le Gall gen. nov., is proposed to accommodate L. flexilis. This new genus is morphologically characterized by four pericentral cells in each vegetative axial segment; however, it lacks ‘corps en cerise’ in cortical cells and secondary pit connections between cortical cells, which are characteristic of Laurencia. Three novel combinations are proposed to render the classification closer to a natural system: Ohelopapa flexilis (Setch. F.Rousseau, Martin-Lescanne, Payri & L.Le Gall comb. nov., Osmundea caspica (Zinova & Zaberzhinskaya Maggs & L.M.McIvor comb. nov. and Palisada crustiformans (McDermid A.R.Sherwood, A.Kurihara & K.W.Nam comb. nov.