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Sample records for branching enzyme gene

  1. Circadian oscillation of starch branching enzyme gene expression in the sorghum endosperm

    Energy Technology Data Exchange (ETDEWEB)

    Mutisya, J.; Sun, C.; Jansson, C.

    2009-08-31

    Expression of the three SBE genes, encoding starch branching enzymes, in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle. Remarkably, the oscillation in SBE expression was maintained in cultured spikes after a 48-h dark treatment, also when fed a continuous solution of sucrose or abscisic acid. Our findings suggest that the rhythmicity in SBE expression in the endosperm is independent of cues from the photosynthetic source and that the oscillator resides within the endosperm itself.

  2. Modification of potato starch composition by introduction and expression of bacterial branching enzyme genes.

    OpenAIRE

    Kortstee, A.J.

    1997-01-01

    Starch consists of two major components; amylose and amylopectin. Amylose is synthesized by the enzyme Granule-Bound Starch Syntase (GBSS) and consists of essentially linear chains of α-1,4 linked glucose residues. Amylopectin is synthesized by the combined activity of the enzymes Soluble Starch Synthase (SSS) and Branching enzyme (BE) and consists of linear α-1,4 linked glucosidic chains with α-1,6 linked branchpoints. The amount and fine structure of each of the components determine the sta...

  3. Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme

    DEFF Research Database (Denmark)

    Madsen, Søren M; Beck, Hans Christian; Ravn, Peter; Vrang, Astrid; Hansen, Anne Maria; Israelsen, Hans

    2002-01-01

    Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The...... first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the...... corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the...

  4. Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules

    DEFF Research Database (Denmark)

    Carciofi, Massimiliano; Blennow, Per Gunnar Andreas; Jensen, Susanne Langgård;

    2012-01-01

    Background Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to...... glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is...... preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. Results In...

  5. Metabolic gene clusters encoding the enzymes of two branches of the 3-oxoadipate pathway in the pathogenic yeast Candida albicans.

    Science.gov (United States)

    Gérecová, Gabriela; Neboháčová, Martina; Zeman, Igor; Pryszcz, Leszek P; Tomáška, Ľubomír; Gabaldón, Toni; Nosek, Jozef

    2015-05-01

    The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway. PMID:25743787

  6. Modification of potato starch composition by introduction and expression of bacterial branching enzyme genes.

    NARCIS (Netherlands)

    Kortstee, A.J.

    1997-01-01

    Starch consists of two major components; amylose and amylopectin. Amylose is synthesized by the enzyme Granule-Bound Starch Syntase (GBSS) and consists of essentially linear chains of α-1,4 linked glucose residues. Amylopectin is synthesized by the combined activity of the enzymes Soluble Starch Syn

  7. Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

    Institute of Scientific and Technical Information of China (English)

    WEI Ke-su; CHENG Fang-min; ZHANG Qi-fang; LIU Kui-gang

    2009-01-01

    An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures (32oC for high temperature and 22oC for optimum temperature) at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes (SBEI, SBEIII and SBEIV) encoding starch branching enzyme (SBE) in the endosperms were studied by the real-time fluorescence quantitative PCR (FQ-PCR) method. Effects of high temperature on the SBE expression in developing rice endosperms were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

  8. Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules

    DEFF Research Database (Denmark)

    Carciofi, Massimiliano; Blennow, Andreas; Jensen, Susanne L;

    2012-01-01

    endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For...

  9. Properties of the glucan branching enzyme of the hyperthermophilic bacterium Aquifex aeolicus

    OpenAIRE

    van der Maarel, MJEC; de Vos, A.; Sanders, P.; Dijkhuizen, L.

    2003-01-01

    Glucan branching enzymes are responsible for the synthesis of alpha(1-->6) glycosidic bonds in glycogen and amylopectin. The glucan branching enzyme of the hyperthermophile Aquifex aeolicus is the most thermoactive and thermostable glucan branching enzyme described. The gene encoding this glucan branching enzyme was overexpressed in E. coli and purified using gamma-cyclodextrin affinity chromatography. Subsequently, the enzyme was subjected to a biochemical characterization. The optimum tempe...

  10. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    Directory of Open Access Journals (Sweden)

    Margaret Ann Carpenter

    2015-03-01

    Full Text Available Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p<0.001 were found with SNPs in the glucan water dikinase (GWD, starch branching enzyme I (SBEI and the starch synthase III (SSIII genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato.

  11. A putative gene sbe3-rs for resistant starch mutated from SBE3 for starch branching enzyme in rice (Oryza sativa L..

    Directory of Open Access Journals (Sweden)

    Ruifang Yang

    Full Text Available Foods high in resistant starch (RS are beneficial to prevent various diseases including diabetes, colon cancers, diarrhea and chronic renal or hepatic diseases. Elevated RS in rice is important for public health since rice is a staple food for half of the world population. A japonica mutant 'Jiangtangdao 1' (RS = 11.67% was crossed with an indica cultivar 'Miyang 23' (RS = 0.41%. The mutant sbe3-rs that explained 60.4% of RS variation was mapped between RM6611 and RM13366 on chromosome 2 (LOD = 36 using 178 F(2 plants genotyped with 106 genome-wide polymorphic SSR markers. Using 656 plants from four F(3:4 families, sbe3-rs was fine mapped to a 573.3 Kb region between InDel 2 and InDel 6 using one STS, five SSRs and seven InDel markers. SBE3 which codes for starch branching enzyme was identified as a candidate gene within the putative region. Nine pairs of primers covering 22 exons were designed to sequence genomic DNA of the wild type for SBE3 and the mutant for sbe3-rs comparatively. Sequence analysis identified a missense mutation site where Leu-599 of the wild was changed to Pro-599 of the mutant in the SBE3 coding region. Because the point mutation resulted in the loss of a restriction enzyme site, sbe3-rs was not digested by a CAPS marker for SpeI site while SBE3 was. Co-segregation of the digestion pattern with RS content among 178 F(2 plants further supported sbe3-rs responsible for RS in rice. As a result, the CAPS marker could be used in marker-assisted breeding to develop rice cultivars with elevated RS which is otherwise difficult to accurately assess in crops. Transgenic technology should be employed for a definitive conclusion of the sbe3-rs.

  12. Structure Function Relationships of ADP-Glucose Pyrophosphorylase and Branching Enzyme: Manipulation of Their Genes for Alteration of Starch Quanlity and Quantity

    Energy Technology Data Exchange (ETDEWEB)

    Jack Preiss

    2006-02-16

    Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit can be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is

  13. Highly branched dextrin prepared from high-amylose maize starch using waxy rice branching enzyme (WRBE).

    Science.gov (United States)

    Tian, Yaoqi; Chen, Huangli; Zhang, Xiwen; Zhan, Jinling; Jin, Zhengyu; Wang, Jinpeng

    2016-07-15

    Branching enzyme (BE, EC 2.4.1.18) was isolated from the developing waxy rice endosperm and used to prepare a highly branched dextrin based on high-amylose maize starch (HAMS) as a substrate. The molecular mass of the starch initially degraded quickly from 2.5×10(7) to 4.1×10(5)Da, and then stabilized, with a minimal increase during the BE treatment. The resultant branched dextrin had a narrow size distribution, with a mean molecular weight of 5.1×10(5)Da and a polydispersity index (PI) of 1.567. The results of high-performance anion exchange chromatography indicated that the degree of polymerization (DP) of the branched chains ranged from 3 to 27; approximately 75.26% of these chains were short (DP<10). These findings suggest that the isolated BE can cleave long chains into oligosaccharides, subsequently transferring oligosaccharides into highly branched dextrins with a narrow size distribution and short side chains. PMID:26948647

  14. LOCALIZATION OF BRANCHING ENZYME IN POTATO-TUBER CELLS WITH THE USE OF IMMUNOELECTRON MICROSCOPY

    NARCIS (Netherlands)

    KRAM, AM; OOSTERGETEL, GT; VANBRUGGEN, EFJ

    1993-01-01

    Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma a

  15. Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

    Institute of Scientific and Technical Information of China (English)

    QIN Hua; ZHOU Shuang; ZHANG Yi-zheng

    2013-01-01

    Spatial and temporal expression patterns of Sbe1 and Sbe2 that encode starch branching enzyme (SBE) I and II, respectively, in sweet potato (Ipomoea batatas L.) were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbe1 mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for IbSbe2. The expression pattern of IbSbe1, closely resembles that of AGPase S, a gene coding for one of the subunits of ADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid (ABA) was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

  16. Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans.

    Science.gov (United States)

    Boyer, Laura; Roussel, Xavier; Courseaux, Adeline; Ndjindji, Ofilia M; Lancelon-Pin, Christine; Putaux, Jean-Luc; Tetlow, Ian J; Emes, Michael J; Pontoire, Bruno; D' Hulst, Christophe; Wattebled, Fabrice

    2016-07-01

    Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan. PMID:26715025

  17. Heterologous expression and characterization of glycogen branching enzyme from Synechocystis sp. PCC6803.

    Science.gov (United States)

    Lee, Byung-Hoo; Yoo, Young-Hee; Ryu, Je-Hoon; Kim, Tae-Jip; Yoo, Sang-Ho

    2008-08-01

    A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and 30 degrees in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an alpha-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed alpha-glucosyl transferring activity by cleaving the alpha-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new alpha-(1,6)- branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; Mp approximately = 8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP (DPw and DPn) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size (Mp, peak Mw approximately = 2.45-2.75 x 10(5)) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties. PMID:18756098

  18. Studies on starch structure and the differential properties of starch branching enzymes

    OpenAIRE

    Andersson, Lena

    2001-01-01

    Starch is a staple food in human and animal diets, but also a raw material widely used for industrial purposes. By genetical modification of starch-synthesising enzymes in crop plants, starch yields could be increased and novel starches with particular qualities could be produced for industrial use. However, the process of starch biosynthesis and its regulation is still not completely understood. One of the major groups of enzymes in starch biosynthesis is the starch branching enzymes (SBEs),...

  19. 马铃薯遗传转化体系的优化及玉米淀粉分支酶基因SBEⅡb的导入%Optimization of genetic transformation system of potato and introduction of maize starch branching enzyme gene SBEⅡb into potatoFAN

    Institute of Scientific and Technical Information of China (English)

    范亚丽; 阮颖; 刘春林

    2012-01-01

    The highly effective potato transformation systems were established and optimized using tube potato plant-lets as transformation materials in this study. The maize starch branching enzyme gene(SBE Ⅱ b)was transformed into potato using Agrobacterium tumefaciens-mediated transformation method. 762 stem sections were infec ted by Agrobaterium-mediated tumefacien method. 4 transgenic plants were obtained by PCR measurement, implying that SEE Ⅱ b gene had been integrated into potato genome. It was also found that GUS gene could normal ly be expressed in stem sections and potato microtubers of the trasgenetic potato plants.%以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系.通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株.经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达.

  20. Synergistic amylomaltase and branching enzyme catalysis to suppress cassava starch digestibility.

    Science.gov (United States)

    Sorndech, Waraporn; Meier, Sebastian; Jansson, Anita M; Sagnelli, Domenico; Hindsgaul, Ole; Tongta, Sunanta; Blennow, Andreas

    2015-11-01

    Starch provides our main dietary caloric intake and over-consumption of starch-containing foods results in escalating life-style disease including diabetes. By increasing the content of α-1,6 branch points in starch, digestibility by human amylolytic enzymes is expected to be retarded. Aiming at generating a soluble and slowly digestible starch by increasing the content and changing the relative positioning of the branch points in the starch molecules, we treated cassava starch with amylomaltase (AM) and branching enzyme (BE). We performed a detailed molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility. Step-by-step enzyme catalysis was the most efficient treatment, and it generated branch structures even more extreme than those of glycogen. All AM- and BE-treated samples showed increased resistance to degradation by porcine pancreatic α-amylase and glucoamylase as compared to cassava starch. The amylolytic products showed chain lengths and branching patterns similar to the products obtained from glycogen. Our data demonstrate that combinatorial enzyme catalysis provides a strategy to generate potential novel soluble α-glucan ingredients with low dietary digestibility assets. PMID:26256365

  1. Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Solomon, Kevin V.; Haitjema, Charles; Henske, John K.; Gilmore, Sean P.; Borges-Rivera, Diego; Lipzen, Anna; Brewer, Heather M.; Purvine, Samuel O.; Wright, Aaron T.; Theodorou, Michael K.; Grigoriev, Igor V.; Regev, Aviv; Thompson, Dawn; O' Malley, Michelle A.

    2016-03-11

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed, and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.

  2. The characterization of modified starch branching enzymes: toward the control of starch chain-length distributions.

    Directory of Open Access Journals (Sweden)

    Cheng Li

    Full Text Available Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE. Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality.

  3. The branching gene RAMOSUS1 mediates interactions among two novel signals and auxin in pea.

    Science.gov (United States)

    Foo, Eloise; Bullier, Erika; Goussot, Magali; Foucher, Fabrice; Rameau, Catherine; Beveridge, Christine Anne

    2005-02-01

    In Pisum sativum, the RAMOSUS genes RMS1, RMS2, and RMS5 regulate shoot branching via physiologically defined mobile signals. RMS1 is most likely a carotenoid cleavage enzyme and acts with RMS5 to control levels of an as yet unidentified mobile branching inhibitor required for auxin inhibition of branching. Our work provides molecular, genetic, and physiological evidence that RMS1 plays a central role in a shoot-to-root-to-shoot feedback system that regulates shoot branching in pea. Indole-3-acetic acid (IAA) positively regulates RMS1 transcript level, a potentially important mechanism for regulation of shoot branching by IAA. In addition, RMS1 transcript levels are dramatically elevated in rms3, rms4, and rms5 plants, which do not contain elevated IAA levels. This degree of upregulation of RMS1 expression cannot be achieved in wild-type plants by exogenous IAA application. Grafting studies indicate that an IAA-independent mobile feedback signal contributes to the elevated RMS1 transcript levels in rms4 plants. Therefore, the long-distance signaling network controlling branching in pea involves IAA, the RMS1 inhibitor, and an IAA-independent feedback signal. Consistent with physiological studies that predict an interaction between RMS2 and RMS1, rms2 mutations appear to disrupt this IAA-independent regulation of RMS1 expression. PMID:15659639

  4. Enzyme-instructed self-assembly of taxol promotes axonal branching

    Science.gov (United States)

    Mei, Bin; Miao, Qingqing; Tang, Anming; Liang, Gaolin

    2015-09-01

    Axonal branching is important for vertebrate neuron signaling. Taxol has a biphasic effect on axonal branching (i.e., high concentration inhibits axonal growth but low concentration restores it). To the best of our knowledge, low concentration of taxol to promote axonal branching has not been reported yet. Herein, we rationally designed a taxol derivative Fmoc-Phe-Phe-Lys(taxol)-Tyr(H2PO4)-OH (1) which could be subjected to alkaline phosphatase (ALP)-catalyzed self-assembly to form taxol nanofibers. We found that, at 10 μM, 1 has a microtubule (MT) condensation effect similar to that of taxol on mammalian cells but with more chronic toxicity than taxol on the cells. At a low concentration of 10 nM, 1 not only promoted neurite elongation as taxol did but also promoted axonal branching which was not achieved by using taxol. We propose that self-assembly of 1 along the MTs prohibited their lateral contacts and thus promoted axonal branching. Our strategy of enzyme-instructed self-assembly (EISA) of a taxol derivative provides a new tool for scientists to study the morphology of neurons, as well as their behaviours.Axonal branching is important for vertebrate neuron signaling. Taxol has a biphasic effect on axonal branching (i.e., high concentration inhibits axonal growth but low concentration restores it). To the best of our knowledge, low concentration of taxol to promote axonal branching has not been reported yet. Herein, we rationally designed a taxol derivative Fmoc-Phe-Phe-Lys(taxol)-Tyr(H2PO4)-OH (1) which could be subjected to alkaline phosphatase (ALP)-catalyzed self-assembly to form taxol nanofibers. We found that, at 10 μM, 1 has a microtubule (MT) condensation effect similar to that of taxol on mammalian cells but with more chronic toxicity than taxol on the cells. At a low concentration of 10 nM, 1 not only promoted neurite elongation as taxol did but also promoted axonal branching which was not achieved by using taxol. We propose that self-assembly of 1

  5. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs. PMID:19746353

  6. Enzyme-instructed self-assembly of taxol promotes axonal branching.

    Science.gov (United States)

    Mei, Bin; Miao, Qingqing; Tang, Anming; Liang, Gaolin

    2015-10-14

    Axonal branching is important for vertebrate neuron signaling. Taxol has a biphasic effect on axonal branching (i.e., high concentration inhibits axonal growth but low concentration restores it). To the best of our knowledge, low concentration of taxol to promote axonal branching has not been reported yet. Herein, we rationally designed a taxol derivative Fmoc-Phe-Phe-Lys(taxol)-Tyr(H2PO4)-OH (1) which could be subjected to alkaline phosphatase (ALP)-catalyzed self-assembly to form taxol nanofibers. We found that, at 10 μM, 1 has a microtubule (MT) condensation effect similar to that of taxol on mammalian cells but with more chronic toxicity than taxol on the cells. At a low concentration of 10 nM, 1 not only promoted neurite elongation as taxol did but also promoted axonal branching which was not achieved by using taxol. We propose that self-assembly of 1 along the MTs prohibited their lateral contacts and thus promoted axonal branching. Our strategy of enzyme-instructed self-assembly (EISA) of a taxol derivative provides a new tool for scientists to study the morphology of neurons, as well as their behaviours. PMID:26359218

  7. Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

    Institute of Scientific and Technical Information of China (English)

    SHEN Xihui; LIU Shuangjiang

    2005-01-01

    Although the protocatechuate branch of the β-ketoadipate pathway in Gram bacteria has been well studied, this branch is less understood in Gram+ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C.glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.

  8. Phosphorylated alpha(1 leads to 4) glucans as substrate for potato starch-branching enzyme I

    International Nuclear Information System (INIS)

    The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated alpha(1 leads to 4) glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of alpha(1 leads to 6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated alpha(1 leads to 4) glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO4(3-) and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated alpha(1 leads to 4) glucan chains

  9. Improved yields of cyclic nigerosylnigerose from starch by pretreatment with a thermostable branching enzyme.

    Science.gov (United States)

    Aga, Hajime; Okamoto, Iwao; Taniguchi, Mituki; Kawashima, Akira; Abe, Hiroko; Chaen, Hiroto; Fukuda, Shigeharu

    2010-04-01

    Cyclic nigerosylnigerose (CNN) is produced enzymatically from starch by the combined action of 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase. In our previous study, alpha-1,6-branching chains found in the structure of amylopectin and glycogen were shown to be favorable for CNN formation by the two enzymes. Therefore, we examined whether the introduction of alpha-1,6-branch points into starch using the action of branching enzyme (BE) could improve the yield of CNN from starch. Thermostable BE from Geobacillus stearothermophilus TC-91 was prepared as a purified recombinant protein. Pretreatment of amylose with BE considerably increased the CNN yield from 5% to 38%. When BE acted on tapioca starch, the CNN yield was elevated from 47% to 60%. Conversely, BE treatment of waxy corn starch containing very little amylose resulted in a negligible increase in CNN yield. In addition, BE exerted a beneficial effect when starch with a lower degree of hydrolysis was used as a substrate. The present results indicate that the addition of alpha-1,6-glucosidic linkages to starch using BE is an effective strategy to improve the yield of CNN from starch. PMID:20226381

  10. Single-enzyme kinetics with branched pathways: exact theory and series expansion

    CERN Document Server

    Garai, Ashok

    2014-01-01

    The progress of the successive rounds of catalytic conversion of substrates into product(s) by a single enzyme is characterized by the distribution of turnover times. Establishing the most general form of dependence of this distribution on the substrate concentration [S] is one of the fundamental challenges in single molecule enzymology. The distribution of the times of dwell of a molecular motor at the successive positions on its track is an analogous quantity. We derive approximate series expansions for the [ATP]-dependence of the first two moments of the dwell time distributions of motors that catalyze hydrolysis of ATP to draw input energy. Comparison between our results for motors with branched pathways and the corresponding expressions reported earlier for linear enzymatic pathways provides deep insight into the effects of the branches. Such insight is likely to help in discovering the most general form of [S]-dependence of these fundamental distributions.

  11. Starch branching enzymes and their genes in Sorghum

    OpenAIRE

    Mutisya, Joel

    2004-01-01

    Starch is an important raw material both for food and non-food purposes. It is synthesized and stored in source and sink tissues in plants. The starch deposited in amyloplasts of storage tissues possesses several physico-chemical properties, which makes it desirable for diverse applications. For industrial applications, starches high in either amylose or amylopectin are preferred in order to minimize chemical modification. The main purpose in our research is to understand starch synthesis in ...

  12. Direct evidence for the inactivation of branched-chain oxo-acid dehydrogenase by enzyme phosphorylation

    International Nuclear Information System (INIS)

    The branched-chain 2-oxo-acid dehydrogenase (BCOAD) from mitochondria of several different rat tissues is inactivated by ATP and can be reactivated by incubation in Mg2+-containing buffers. Work carried out on the system from skeletal muscle mitochondria has shown that inactivation requires the cleavage of the γ-phosphate group of ATP and that modification is covalent. The non-metabolized ATP analog, p[NH]ppA, can block the inhibitory effect of ATP when added prior to ATP addition, but cannot reverse the inhibition of the inactivated dehydrogenase. These and other data raise the possibility that BCOAD may be regulated by enzyme phosphorylation. This hypothesis is supported by the finding that various procedures which separate the enzyme from its mitochondrial environment (e.g. detergent treatment, ammonium sulfate precipitation and freeze-thawing) do not alter the degree of inhibition induced by ATP in the mitochondrial preincubation. These experiments suggested the feasibility of labelling the enzyme with 32P and purifying it. (Auth.)

  13. Characterization of the reactions of starch branching enzymes from rice endosperm.

    Science.gov (United States)

    Nakamura, Yasunori; Utsumi, Yoshinori; Sawada, Takayuki; Aihara, Satomi; Utsumi, Chikako; Yoshida, Mayumi; Kitamura, Shinichi

    2010-05-01

    To our knowledge the present paper shows for the first time the kinetic parameters of all the three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice with both amylopectin and synthetic amylose as glucan substrate. The activities of these BE isozymes with a linear glucan amylose decreased with a decrease in the molar size of amylose, and no activities of BEIIa and BEIIb were found when the degree of polymerization (DP) of amylose was lower than at least 80, whereas BEI had an activity with amylose of a DP higher than approximately 50. Detailed analyses of debranched products from BE reactions revealed the distinct chain length preferences of the individual BE isozymes. BEIIb almost exclusively transferred chains of DP7 and DP6 while BEIIa formed a wide range of short chains of DP6 to around DP15 from outer chains of amylopectin and amylose. On the other hand, BEI formed a variety of short chains and intermediate chains of a DP branched glucan while BEIIa or BEIIb could only scarcely or could not attack inner chains, respectively. The comprehensive in vitro studies revealed different enzymatic characteristics of the three BE isozymes and give a new insight into the distinct roles of individual BE isozymes in amylopectin biosynthesis in the endosperm. Based on these results, the functional distinction and interaction of BE isozymes during amylopectin biosynthesis in cereal endosperm is discussed. PMID:20305271

  14. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism

    OpenAIRE

    Shaik, Shahnoor S.; Obata, Toshihiro; Hebelstrup, Kim H.; Schwahn, Kevin; Fernie, Alisdair R.; Mateiu, Ramona Valentina; Blennow, Andreas

    2016-01-01

    Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or ...

  15. Identification of candidate genes for dissecting complex branch number trait in chickpea.

    Science.gov (United States)

    Bajaj, Deepak; Upadhyaya, Hari D; Das, Shouvik; Kumar, Vinod; Gowda, C L L; Sharma, Shivali; Tyagi, Akhilesh K; Parida, Swarup K

    2016-04-01

    The present study exploited integrated genomics-assisted breeding strategy for genetic dissection of complex branch number quantitative trait in chickpea. Candidate gene-based association analysis in a branch number association panel was performed by utilizing the genotyping data of 401 SNP allelic variants mined from 27 known cloned branch number gene orthologs of chickpea. The genome-wide association study (GWAS) integrating both genome-wide GBS- (4556 SNPs) and candidate gene-based genotyping information of 4957 SNPs in a structured population of 60 sequenced desi and kabuli accessions (with 350-400kb LD decay), detected 11 significant genomic loci (genes) associated (41% combined PVE) with branch number in chickpea. Of these, seven branch number-associated genes were further validated successfully in two inter (ICC 4958×ICC 17160)- and intra (ICC 12299×ICC 8261)-specific mapping populations. The axillary meristem and shoot apical meristem-specific expression, including differential up- and down-regulation (4-5 fold) of the validated seven branch number-associated genes especially in high branch number as compared to the low branch number-containing parental accessions and homozygous individuals of two aforesaid mapping populations was apparent. Collectively, this combinatorial genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in seven potential known/candidate genes [PIN1 (PIN-FORMED protein 1), TB1 (teosinte branched 1), BA1/LAX1 (BARREN STALK1/LIKE AUXIN1), GRAS8 (gibberellic acid insensitive/GAI, Repressor of ga13/RGA and Scarecrow8/SCR8), ERF (ethylene-responsive element-binding factor), MAX2 (more axillary growth 2) and lipase] governing chickpea branch number. The useful information generated from this study have potential to expedite marker-assisted genetic enhancement by developing high-yielding cultivars with more number of productive (pods and seeds) branches in chickpea. PMID:26940492

  16. Retrogradation behavior of corn starch treated with 1,4-α-glucan branching enzyme.

    Science.gov (United States)

    Li, Wenwen; Li, Caiming; Gu, Zhengbiao; Qiu, Yijing; Cheng, Li; Hong, Yan; Li, Zhaofeng

    2016-07-15

    The retrogradation behavior of corn starch treated with 1,4-α-glucan branching enzyme (GBE) was investigated using rheometry, pulsed nuclear magnetic resonance (PNMR), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). Dynamic time sweep analysis confirmed that the storage modulus (G') of corn starch stored at 4 °C decreased with increasing GBE treatment time. PNMR analysis demonstrated that the transverse relaxation times (T2) of corn starches treated with GBE were higher than that of control during the storage at 4 °C. DSC results demonstrated that the retrogradation enthalpy (ΔHr) of corn starch was reduced by 22.3% after GBE treatment for 10h. Avrami equation analysis showed that GBE treatment reduced the rate of starch retrogradation. FTIR analysis revealed that GBE treatment led to a decrease in hydrogen bonds within the starch. Overall, these results demonstrate that both short- and long-term retrogradation of corn starch were retarded by GBE treatment. PMID:26948619

  17. Enzyme- and pH-Sensitive Branched Polymer-Doxorubicin Conjugate-Based Nanoscale Drug Delivery System for Cancer Therapy.

    Science.gov (United States)

    Wei, Xiaoli; Luo, Qiang; Sun, Ling; Li, Xue; Zhu, Hongyan; Guan, Pujun; Wu, Min; Luo, Kui; Gong, Qiyong

    2016-05-11

    Owing to their dendritic architectural features, branched copolymers have been investigated as drug delivery systems. In this paper, an enzyme- and pH-sensitive branched poly[N-(2-hydroxypropyl)methacrylamide] (polyHPMA) copolymer-doxorubicin (DOX) conjugate possessing a molecular weight (MW) of 165 kDa was designed and prepared via a one-pot reaction and drug conjugation. This conjugate's potential as a smart, nanoscale drug delivery system (NDDS) is also investigated. The branched conjugate was capable of forming nanoparticles with a negative surface charge. The self-assembled nanoparticles were 102 nm in diameter as measured by dynamic light scattering (DLS) and 95 nm in diameter via scanning electron microscopy, respectively. The nanoparticles were degraded to low-MW products (23∼25 kDa) in the presence of papain or cathepsin B, and the degradation was monitored via DLS and size-exclusion chromatography. The nanoparticles demonstrated pH-sensitive drug release, as the DOX was attached to the branched copolymer via a hydrazone bond. In comparison to free DOX, the conjugate-based nanoparticles exhibited greater accumulation in breast tumors, resulting in enhanced antitumor therapeutic indexes. Furthermore, widespread dissemination of the conjugate among breast tumor cells was confirmed by immunohistochemical assay. Finally, no obvious systemic toxicities were observed in vivo in normal mice. Thus, the branched HPMA copolymer-DOX conjugate may be employed as a safe and efficient pH- and enzyme-responsive NDDS for cancer therapy. PMID:27102364

  18. Evaluation on the nutritional value of protein in genetically modified rice with double antisense starch-branching enzyme gene%转双反义淀粉分支酶基因大米中蛋白质的营养价值评价

    Institute of Scientific and Technical Information of China (English)

    李敏; 胡贻椿; 朴建华; 杨晓光

    2011-01-01

    目的 对转双反义淀粉分支酶基因大米和亲本大米蛋白质的营养价值进行评价.方法 采用通用的非生物学评价方法,即化学评分、氨基酸评分、蛋白质消化率校正后的氨基酸评分、必需氨基酸指数、营养指数、氨基酸比值系数和氨基酸比值系数分,评价大米蛋白质的营养价值.结果 转基因大米和亲本大米的化学评分分别为62,59;氨基酸评分分别为75,62;蛋白质消化率校正后的氨基酸评分分别为65,56;必需氨基酸指数分别为79,80;营养指数分别为11,8;氨基酸比值系数分分别为76.7,67.0;氨基酸比值系数都是l.1.结论 转双反义淀粉分支氨基因大来中抗性淀粉含童显著提高,但并未影响到其自身蛋白质的营养价值.该转基因大米蛋白质的营养价值与亲本大米具有"实质等同性".%Objective To assess the nutritional value of protein in the genetically modified rice with double antisense starch-branching enzyme (SBE) gene and its parental rice.Methods The adopted nutritional value assessment methods, including chemical score (CS), amino acid score (AAS), protein digestibility-corrected amino acid score (PDCAAS), essential amino acid index (EAAI), nutritional index (NI), ratio coefficient of amino acid (RCAA) and score of RCAA ( SRCAA ) was applied to assess the protein nutritional value of the rice.Results The CS value of genetically modified rice and parental rice were 62 and 59, AAS value were 75 and 62, PDCAAS value were 65 and 56,EAAI were 79 and 80, NI were 11 and 8, SRCAA were 76.7 and 67.0, respectively.RCAA were the same.Conclusion The nutritional value of protein in the genetically modified rice with double antisense SBE gene were not greatly changed by the increase of resistant starch content in the rice.So, the nutritional value of protein in the genetically modified rice and its parental rice was substantial equivalent.

  19. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism

    DEFF Research Database (Denmark)

    Shaik, Shahnoor S.; Obata, Toshihiro; Hebelstrup, Kim H;

    2016-01-01

    -structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper...... high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences...

  20. Synthesis of Hyperbranched Glycoconjugates by the Combined Action of Potato Phosphorylase and Glycogen Branching Enzyme from Deinococcus geothermalis

    Directory of Open Access Journals (Sweden)

    Katja Loos

    2012-02-01

    Full Text Available Potato phosphorylase is able to synthesize linear polyglucans from maltoheptaose primers. By coupling maltoheptaose to butane diamine, tris(2-aminoethylamine and amine functionalized amine functionalized poly ethyleneglycol (PEG, new primer molecules became available. The resulting di-, tri- and macro-primers were incubated with potato phosphorylase and glycogen branching enzyme from Deinococcus geothermalis. Due to the action of both enzymes, hyperbranched polyglucan arms were grown from the maltoheptaose derivatives with a maximum degree of branching of 11%. The size of the synthesized hyperbranched polyglucans could be controlled by the ratio monomer over primer. About 60%–80% of the monomers were incorporated in the glycoconjugates. The resulting hyperbranched glycoconjugates were subjected to Dynamic Light Scattering (DLS measurements in order to determine the hydrodynamic radius and it became obvious that the structures formed agglomerates in the range of 14–32 nm.

  1. Microdiversity of extracellular enzyme genes among sequenced prokaryotic genomes

    OpenAIRE

    Zimmerman, Amy E; Martiny, Adam C.; Allison, Steven D.

    2013-01-01

    Understanding the relationship between prokaryotic traits and phylogeny is important for predicting and modeling ecological processes. Microbial extracellular enzymes have a pivotal role in nutrient cycling and the decomposition of organic matter, yet little is known about the phylogenetic distribution of genes encoding these enzymes. In this study, we analyzed 3058 annotated prokaryotic genomes to determine which taxa have the genetic potential to produce alkaline phosphatase, chitinase and ...

  2. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer

    Science.gov (United States)

    Kizuka, Yasuhiko; Taniguchi, Naoyuki

    2016-01-01

    N-glycan, a fundamental and versatile protein modification in mammals, plays critical roles in various physiological and pathological events including cancer progression. The formation of N-glycan branches catalyzed by specific N-acetylglucosaminyltransferases [GnT-III, GnT-IVs, GnT-V, GnT-IX (Vb)] and a fucosyltransferase, Fut8, provides functionally diverse N-glycosylated proteins. Aberrations of these branches are often found in cancer cells and are profoundly involved in cancer growth, invasion and metastasis. In this review, we focus on the GlcNAc and fucose branches of N-glycans and describe how their expression is dysregulated in cancer by genetic and nongenetic mechanisms including epigenetics and nucleotide sugar metabolisms. We also survey the roles that these N-glycans play in cancer progression and therapeutics. Finally, we discuss possible applications of our knowledge on basic glycobiology to the development of medicine and biomarkers for cancer therapy. PMID:27136596

  3. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer

    Directory of Open Access Journals (Sweden)

    Yasuhiko Kizuka

    2016-04-01

    Full Text Available N-glycan, a fundamental and versatile protein modification in mammals, plays critical roles in various physiological and pathological events including cancer progression. The formation of N-glycan branches catalyzed by specific N-acetylglucosaminyltransferases [GnT-III, GnT-IVs, GnT-V, GnT-IX (Vb] and a fucosyltransferase, Fut8, provides functionally diverse N-glycosylated proteins. Aberrations of these branches are often found in cancer cells and are profoundly involved in cancer growth, invasion and metastasis. In this review, we focus on the GlcNAc and fucose branches of N-glycans and describe how their expression is dysregulated in cancer by genetic and nongenetic mechanisms including epigenetics and nucleotide sugar metabolisms. We also survey the roles that these N-glycans play in cancer progression and therapeutics. Finally, we discuss possible applications of our knowledge on basic glycobiology to the development of medicine and biomarkers for cancer therapy.

  4. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer.

    Science.gov (United States)

    Kizuka, Yasuhiko; Taniguchi, Naoyuki

    2016-01-01

    N-glycan, a fundamental and versatile protein modification in mammals, plays critical roles in various physiological and pathological events including cancer progression. The formation of N-glycan branches catalyzed by specific N-acetylglucosaminyltransferases [GnT-III, GnT-IVs, GnT-V, GnT-IX (Vb)] and a fucosyltransferase, Fut8, provides functionally diverse N-glycosylated proteins. Aberrations of these branches are often found in cancer cells and are profoundly involved in cancer growth, invasion and metastasis. In this review, we focus on the GlcNAc and fucose branches of N-glycans and describe how their expression is dysregulated in cancer by genetic and nongenetic mechanisms including epigenetics and nucleotide sugar metabolisms. We also survey the roles that these N-glycans play in cancer progression and therapeutics. Finally, we discuss possible applications of our knowledge on basic glycobiology to the development of medicine and biomarkers for cancer therapy. PMID:27136596

  5. Inferring the Gene Network Underlying the Branching of Tomato Inflorescence

    NARCIS (Netherlands)

    Astola, L.; Stigter, J.D.; Dijk, van A.D.J.; Daelen, van R.; Molenaar, J.

    2014-01-01

    The architecture of tomato inflorescence strongly affects flower production and subsequent crop yield. To understand the genetic activities involved, insight into the underlying network of genes that initiate and control the sympodial growth in the tomato is essential. In this paper, we show how the

  6. Human monoamine oxidase A gene determines levels of enzyme activity.

    OpenAIRE

    Hotamisligil, G S; Breakefield, X O

    1991-01-01

    Monoamine oxidase (MAO) is a critical enzyme in the degradative deamination of biogenic amines throughout the body. Two biochemically distinct forms of the enzyme, A and B, are encoded in separate genes on the human X chromosome. In these studies we investigated the role of the structural gene for MAO-A in determining levels of activity in humans, as measured in cultured skin fibroblasts. The coding sequence of the mRNA for MAO-A was determined by first-strand cDNA synthesis, PCR amplificatio...

  7. Oscillating Gene Expression Determines Competence for Periodic Arabidopsis Root Branching

    Science.gov (United States)

    Moreno-Risueno, Miguel A.; Van Norman, Jaimie M.; Moreno, Antonio; Zhang, Jingyuan; Ahnert, Sebastian E.; Benfey, Philip N.

    2010-01-01

    Plants and animals produce modular developmental units in a periodic fashion. In plants, lateral roots form as repeating units along the root primary axis; however, the developmental mechanism regulating this process is unknown. We found that cyclic expression pulses of a reporter gene mark the position of future lateral roots by establishing prebranch sites and that prebranch site production and root bending are periodic. Microarray and promoter-luciferase studies revealed two sets of genes oscillating in opposite phases at the root tip. Genetic studies show that some oscillating transcriptional regulators are required for periodicity in one or both developmental processes. This molecular mechanism has characteristics that resemble molecular clock–driven activities in animal species. PMID:20829477

  8. Beyond branching: multiknot structured polymer for gene delivery.

    Science.gov (United States)

    Aied, Ahmed; Zheng, Yu; Newland, Ben; Wang, Wenxin

    2014-12-01

    Polymer-based transfection vectors are increasingly becoming the preferred alternative to viral vectors thanks to their safety and ease of production, but low transfection potency has limited their application. Many polycationic vectors show high efficiency in vitro, but their excessive charge density makes them toxic for in vivo applications. Herein, we demonstrate the synthesis of new and unique disulfide-reducible polymeric gene nanocarriers that exhibit significantly enhanced transfection potency and low cytotoxicity, particularly in skin cells, surpassing the efficiency of the well-known transfection reagents polyethylenimine (PEI) and Lipofectamine2000. The unique three-dimensional (3D) "multiknot" vectors were synthesized from in situ deactivation enhanced atom transfer radical (co)polymerization (DE-ATRP) of multivinyl monomers (MVMs). The high transfection levels and low toxicity of this multiknot structured polymer in vitro, combined with its ability to mediate collagen VII expression in 3D skin equivalents made from cells of recessive dystrophic epidermolysis bullosa patients, demonstrates its use as a platform nanotechnology which should be investigated further for dermatological disease therapies. Our findings suggest that the marked improvements stem from the dense multiknot architecture and degradable property, which facilitate both the binding and releasing process of the plasmid DNA. PMID:25375252

  9. Genes, enzymes and regulation of arginine biosynthesis in plants.

    Science.gov (United States)

    Slocum, Robert D

    2005-08-01

    Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate. PMID:16122935

  10. Hormonally controlled expression of the Arabidopsis MAX4 shoot branching regulatory gene.

    Science.gov (United States)

    Bainbridge, Katherine; Sorefan, Karim; Ward, Sally; Leyser, Ottoline

    2005-11-01

    The Arabidopsis MORE AXILLARY BRANCHING 4 (MAX4) gene is required for the production of a long-range, graft-transmissible signal that inhibits shoot branching. Buds of max4 mutant plants are resistant to the inhibitory effects of apically applied auxin, indicating that MAX4 is required for auxin-mediated bud inhibition. The RAMOSUS 1 (RMS1) and DECREASED APICAL DOMINANCE 1 (DAD1) genes of pea and petunia, respectively, are orthologous to MAX4 and function in a similar way. Here we show that, despite the similarities between these three genes, there are significant differences in the regulation of their expression. RMS1 is known to be upregulated by auxin in the shoot, suggesting a straightforward link between the RMS1-dependent branch-inhibiting signal and auxin, whereas we find that MAX4 is only upregulated by auxin in the root and hypocotyl, and this is not required for the inhibition of shoot branching. Furthermore, both RMS1 and DAD1 are subject to feedback regulation, for which there is no evidence for MAX4. Instead, overexpression studies and reciprocal grafting experiments demonstrate that the most functionally significant point of interaction between auxin and MAX4 is post-transcriptional and indeed post-synthesis of the MAX4-dependent graft-transmissible signal. PMID:16262707

  11. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

    Science.gov (United States)

    Shaik, Shahnoor S; Obata, Toshihiro; Hebelstrup, Kim H; Schwahn, Kevin; Fernie, Alisdair R; Mateiu, Ramona V; Blennow, Andreas

    2016-01-01

    Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD. PMID:26891365

  12. Branch-and-Bound Approach for Parsimonious Inference of a Species Tree From a Set of Gene Family Trees

    OpenAIRE

    Doyon, Jean-Philippe; Chauve, Cedric

    2010-01-01

    We describe a Branch-and-Bound algorithm for computing a parsimonious species tree given a set of gene family trees. Our algorithm can compute a parsimonious species tree for three cost measures: number of gene duplications, number of gene losses, and both combined. Moreover, to cope with intrinsic limitations of Branch-and-Bound algorithms for species trees inference regarding the number of taxa that can be considered, our algorithm can naturally take into account predefined relationships be...

  13. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    Science.gov (United States)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  14. Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

    International Nuclear Information System (INIS)

    A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both α-(1,6)- and α-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with 14C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-α-(1,6)-β-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

  15. Regulation of Expression and Evolution of Genes in Plastids of Rhodophytic Branch.

    Science.gov (United States)

    Zverkov, Oleg Anatolyevich; Seliverstov, Alexandr Vladislavovich; Lyubetsky, Vassily Alexandrovich

    2016-01-01

    A novel algorithm and original software were used to cluster all proteins encoded in plastids of 72 species of the rhodophytic branch. The results are publicly available at http://lab6.iitp.ru/ppc/redline72/ in a database that allows fast identification of clusters (protein families) both by a fragment of an amino acid sequence and by a phylogenetic profile of a protein. No such integral clustering with the corresponding functions can be found in the public domain. The putative regulons of the transcription factors Ycf28 and Ycf29 encoded in the plastids were identified using the clustering and the database. A regulation of translation initiation was proposed for the ycf24 gene in plastids of certain red algae and apicomplexans as well as a regulation of a putative gene in apicoplasts of Babesia spp. and Theileria parva. The conserved regulation of the ycf24 gene expression and specificity alternation of the transcription factor Ycf28 were shown in the plastids. A phylogenetic tree of plastids was generated for the rhodophytic branch. The hypothesis of the origin of apicoplasts from the common ancestor of all apicomplexans from plastids of red algae was confirmed. PMID:26840333

  16. Regulation of Expression and Evolution of Genes in Plastids of Rhodophytic Branch

    Science.gov (United States)

    Zverkov, Oleg Anatolyevich; Seliverstov, Alexandr Vladislavovich; Lyubetsky, Vassily Alexandrovich

    2016-01-01

    A novel algorithm and original software were used to cluster all proteins encoded in plastids of 72 species of the rhodophytic branch. The results are publicly available at http://lab6.iitp.ru/ppc/redline72/ in a database that allows fast identification of clusters (protein families) both by a fragment of an amino acid sequence and by a phylogenetic profile of a protein. No such integral clustering with the corresponding functions can be found in the public domain. The putative regulons of the transcription factors Ycf28 and Ycf29 encoded in the plastids were identified using the clustering and the database. A regulation of translation initiation was proposed for the ycf24 gene in plastids of certain red algae and apicomplexans as well as a regulation of a putative gene in apicoplasts of Babesia spp. and Theileria parva. The conserved regulation of the ycf24 gene expression and specificity alternation of the transcription factor Ycf28 were shown in the plastids. A phylogenetic tree of plastids was generated for the rhodophytic branch. The hypothesis of the origin of apicoplasts from the common ancestor of all apicomplexans from plastids of red algae was confirmed. PMID:26840333

  17. Synthesis of branched polysaccharides with tunable degree of branching

    NARCIS (Netherlands)

    Ciric, Jelena; Loos, Katja

    2013-01-01

    An in vitro enzyme-catalyzed tandem reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) to obtain branched polyglucans with tunable degree of branching (2% divided by 13%) is presented. The tunable degree of branching is obtai

  18. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  19. Mutations in Barley Row Type Genes Have Pleiotropic Effects on Shoot Branching.

    Directory of Open Access Journals (Sweden)

    Corinna Brit Liller

    Full Text Available Cereal crop yield is determined by different yield components such as seed weight, seed number per spike and the tiller number and spikes. Negative correlations between these traits are often attributed to resource limitation. However, recent evidence suggests that the same genes or regulatory modules can regulate both inflorescence branching and tillering. It is therefore important to explore the role of genetic correlations between different yield components in small grain cereals. In this work, we studied pleiotropic effects of row type genes on seed size, seed number per spike, thousand grain weight, and tillering in barley to better understand the genetic correlations between individual yield components. Allelic mutants of nine different row type loci (36 mutants, in the original spring barley varieties Barke, Bonus and Foma and introgressed in the spring barley cultivar Bowman, were phenotyped under greenhouse and outdoor conditions. We identified two main mutant groups characterized by their relationships between seed and tillering parameters. The first group comprises all mutants with an increased number of seeds and significant change in tiller number at early development (group 1a or reduced tillering only at full maturity (group 1b. Mutants in the second group are characterized by a reduction in seeds per spike and tiller number, thus exhibiting positive correlations between seed and tiller number. Reduced tillering at full maturity (group 1b is likely due to resource limitations. In contrast, altered tillering at early development (groups 1a and 2 suggests that the same genes or regulatory modules affect inflorescence and shoot branching. Understanding the genetic bases of the trade-offs between these traits is important for the genetic manipulation of individual yield components.

  20. Angiotensin converting enzyme gene polymorphism in familial hypertrophic cardiomyopathy patients

    Energy Technology Data Exchange (ETDEWEB)

    Yu, B; Peric, S.; Ross, D. [Royal Prince Alfred Hospital, Campertown (Australia)] [and others

    1994-09-01

    An insertion/deletion (I/D) polymorphism of the angiotensin I converting enzyme (ACE) gene is a useful predictor of human plasma ACE levels. ACE levels tend to be lowest in subjects with ACE genotype DD and intermediate in subjects with ACE genotype ID. Angiotensin II (Ang II) as a product of ACE is a cardiac growth factor and produces a marked hypertrophy of the chick myocyte in cell culture. Rat experiments also suggest that a small dose of ACE inhibitor that does not affect the afterload results in prevention or regression of cardiac hypertrophy. In order to study the relationship of ACE and the severity of hypertrophy, the ACE genotype has been determined in 28 patients with a clinical diagnosis of familial hypertrophic cardiomyopathy (FHC) and 51 normal subjects. The respective frequencies of I and D alleles were: 0.52 and 0.48 (in FHC patients) and 0.44 and 0.56 (in the normal controls). There was no significant difference in the allele frequencies between FHC and normal subjects ({chi}{sup 2}=0.023, p>0.05). The II, ID, and DD genotypes were present in 7, 15, and 6 FHC patients, respectively. The averages of maximal thickness of the interventricular septum measured by echocardiography or at autopsy were 18 {plus_minus}3, 19{plus_minus}4, and 19{plus_minus}3 mm in II, ID and DD genotypes, respectively. The ACE gene polymorphism did not correlate with the severity of left ventricular hypertrophy in FHC patients (r{sub s}=0.231, p>0.05). These results do not necessarily exclude the possible effect of Ang II on the hypertrophy since the latter may be produced through the action of chymase in the human ventricles. However, ACE gene polymorphism is not a useful predictor of the severity of myocardial hypertrophy in FHC patients.

  1. Molecular Genetic Analysis of Glucan Branching Enzymes from Plants and Bacteria in Arabidopsis Reveals Marked Differences in Their Functions and Capacity to Mediate Starch Granule Formation.

    Science.gov (United States)

    Lu, Kuan-Jen; Streb, Sebastian; Meier, Florence; Pfister, Barbara; Zeeman, Samuel C

    2015-11-01

    The major component of starch is the branched glucan amylopectin, the branching pattern of which is one of the key factors determining its ability to form semicrystalline starch granules. Here, we investigated the functions of different branching enzyme (BE) types by expressing proteins from maize (Zea mays BE2a), potato (Solanum tuberosum BE1), and Escherichia coli (glycogen BE [EcGLGB]) in Arabidopsis (Arabidopsis thaliana) mutant plants that are deficient in their endogenous BEs and therefore, cannot make starch. The expression of each of these three BE types restored starch biosynthesis to differing degrees. Full complementation was achieved using the class II BE ZmBE2a, which is most similar to the two endogenous Arabidopsis isoforms. Expression of the class I BE from potato, StBE1, resulted in partial complementation and high amylose starch. Expression of the glycogen BE EcGLGB restored only minimal amounts of starch production, which had unusual chain length distribution, branch point distribution, and granule morphology. Nevertheless, each type of BE together with the starch synthases and debranching enyzmes were able to create crystallization-competent amylopectin polymers. These data add to the knowledge of how the properties of the BE influence the final composition of starch and fine structure of amylopectin. PMID:26358415

  2. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  3. Mantle Branch-Specific RNA Sequences of Moon Scallop Amusium pleuronectes to Identify Shell Color-Associated Genes.

    Directory of Open Access Journals (Sweden)

    Rong-Lian Huang

    Full Text Available Amusium pleuronectes (Linnaeus that secretes red- and white-colored valves in two branches of mantle tissues is an excellent model for shell color research. High-throughput transcriptome sequencing and profiling were applied in this project to reveal the detailed molecular mechanism of this phenotype differentiation. In this study, 50,796,780 and 54,361,178 clean reads were generated from the left branch (secreting red valve, RS and right branch (secreting white valve, WS using the Illumina Hiseq 2000 platform. De novo assembly generated 149,375 and 176,652 unigenes with an average length of 764 bp and 698 bp in RS and WS, respectively. Kyoto encyclopedia of genes and genomes (KEGG metabolic pathway analysis indicated that the differentially expressed genes were involved in 228 signaling pathways, and 43 genes were significantly enriched (P<0.01. Nineteen of 20 differentially expressed vitellogenin genes showed significantly high expression in RS, which suggested that they probably played a crucial role in organic pigment assembly and transportation of the shell. Moreover, 687 crystal formation-related (or biomineralization-related genes were detected in A. pleuronectes, among which 144 genes exhibited significant difference between the two branches. Those genes could be classified into shell matrix framework participants, crystal nucleation and growth-related elements, upstream regulation factors, Ca level regulators, and other classifications. We also identified putative SNP and SSR markers from these samples which provided the markers for genetic diversity analysis, genetic linkage, QTL analysis. These results provide insight into the complexity of shell color differentiation in A. pleuronectes so as valuable resources for further research.

  4. Regulation of genes encoding enzymes involved in plant cell wall deconstruction in Trichoderma reesei

    OpenAIRE

    Ries, Laure Nicolas Annick

    2013-01-01

    This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in t...

  5. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam (NU Sinapore); (Van Andel); (IMT-India)

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  6. Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

    OpenAIRE

    Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

    2010-01-01

    In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gen...

  7. Prodrug converting enzyme gene delivery by L. monocytogenes

    International Nuclear Information System (INIS)

    Listeria monocytogenes is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that L. monocytogenes is able to colonize and replicate within solid tumors after local or even systemic injection. Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by L. monocytogenes. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria. The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although L. monocytogenes-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively PCMV-promoter with the melanoma specific P4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies. These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors

  8. Enzyme free cloning for high throughput gene cloning and expression

    OpenAIRE

    de Jong, R. N.; Daniëls, M.; Kaptein, R; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Indepe...

  9. Gene expression profiles during short-term heat stress; branching vs. massive Scleractinian corals of the Red Sea.

    Science.gov (United States)

    Maor-Landaw, Keren; Levy, Oren

    2016-01-01

    It is well-established that there is a hierarchy of susceptibilities amongst coral genera during heat-stress. However, molecular mechanisms governing these differences are still poorly understood. Here we explored if specific corals possessing different morphologies and different susceptibilities to heat stress may manifest varied gene expression patterns. We examined expression patterns of seven genes in the branching corals Stylophora pistillata and Acropora eurystoma and additionally in the massive robust coral, Porites sp. The tested genes are representatives of key cellular processes occurring during heat-stress in Cnidaria: oxidative stress, ER stress, energy metabolism, DNA repair and apoptosis. Varied response to the heat-stress, in terms of visual coral paling, algal maximum quantum yield and host gene expression was evident in the different growth forms. The two branching corals exhibited similar overall responses that differed from that of the massive coral. A. eurystoma that is considered as a susceptible species did not bleach in our experiment, but tissue sloughing was evident at 34 °C. Interestingly, in this species redox regulation genes were up-regulated at the very onset of the thermal challenge. In S. pistillata, bleaching was evident at 34 °C and most of the stress markers were already up-regulated at 32 °C, either remaining highly expressed or decreasing when temperatures reached 34 °C. The massive Porites species displayed severe bleaching at 32 °C but stress marker genes were only significantly elevated at 34 °C. We postulate that by expelling the algal symbionts from Porites tissues, oxidation damages are reduced and stress genes are activated only at a progressed stage. The differential gene expression responses exhibited here can be correlated with the literature well-documented hierarchy of susceptibilities amongst coral morphologies and genera in Eilat's coral reef. PMID:27069783

  10. Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach*

    OpenAIRE

    Wang, Qiuyan; Wu, Huili; Wang, Anming; Du, Pengfei; Pei, Xiaolin; Li, Haifeng; Yin, Xiaopu; Huang, Lifeng; Xiong, Xiaolong

    2010-01-01

    DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR...

  11. Radiation Exposure Alters Expression of Metabolic Enzyme Genes in Mice

    Science.gov (United States)

    Wotring, V. E.; Mangala, L. S.; Zhang, Y.; Wu, H.

    2011-01-01

    Most administered pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand the effects of spaceflight on the enzymes of the liver and exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. Additionally, it has been previous noted that pre-exposure to small radiation doses seems to confer protection against later and larger radiation doses. This protective power of pre-exposure has been called a priming effect or radioadaptation. This study is an effort to examine the drug metabolizing effects of radioadaptation mechanisms that may be triggered by early exposure to low radiation doses.

  12. Molecular mapping of an apical branching gene of cultivated sunflower (Helianthus annuus L.)

    Science.gov (United States)

    Commercial hybrids of cultivated sunflower (Helianthus annuus L.) are obtained by crossing a cytoplasmic male-sterile line (A-line) with a restorer pollinator (R-line). The incorporation of a recessive branching trait to extend the pollination period of R-lines during hybrid seed production is labor...

  13. Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes.

    Directory of Open Access Journals (Sweden)

    Luka Ausec

    Full Text Available Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three-domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.

  14. Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

    Science.gov (United States)

    Ausec, Luka; Zakrzewski, Martha; Goesmann, Alexander; Schlüter, Andreas; Mandic-Mulec, Ines

    2011-01-01

    Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications. PMID:22022440

  15. Change in Auxin and Cytokinin Levels Coincides with Altered Expression of Branching Genes during Axillary Bud Outgrowth in Chrysanthemum.

    Science.gov (United States)

    Dierck, Robrecht; De Keyser, Ellen; De Riek, Jan; Dhooghe, Emmy; Van Huylenbroeck, Johan; Prinsen, Els; Van Der Straeten, Dominique

    2016-01-01

    In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral

  16. Enzymes and Genes Involved in Aerobic Alkane Degradation

    Directory of Open Access Journals (Sweden)

    ZongzeShao

    2013-05-01

    Full Text Available Alkanes are major constituents of crude oil. They are also present at low concentrations in diverse non-contaminated because many living organisms produce them as chemo-attractants or as protecting agents against water loss. Alkane degradation is a widespread phenomenon in nature. The numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing alkanes as a carbon and energy source, have been isolated and characterized. This review summarizes the current knowledge of how bacteria metabolize alkanes aerobically, with a particular emphasis on the oxidation of long-chain alkanes, including factors that are responsible for chemotaxis to alkanes , transport across cell membrane of alkanes , the regulation of alkane degradation gene and initial oxidation.

  17. [Gene expression of the key enzymes controlling starch synthesis and metabolism in rice grain endosperm under effects of high temperature after anthesis].

    Science.gov (United States)

    Zhong, Lian-Jin; Dong, Hu; Cai, Xiao-Bo; Feng, Yan-Ning; Ren, Ping; Cheng, Fang-Min

    2012-03-01

    Taking an early-season indica cultivar 'Jiazao 935' whose grain quality was sensitive to temperature as test material, and by using artificial climatic chamber and real-time fluorescence quantitative PCR (FQ-PCR), this paper studied the relative expression amount and its dynamic changes of ten isoform genes of the key enzymes controlling starch synthesis and metabolism in rice grain endosperm, including sbe1, sbe3, and sbe4 of starch branching enzyme (SBE), isal, isa2, isa3, and pul of starch debranching enzyme (DBE), and Wx, sss1, and sss2a of starch synthase (SS), at the mean daily temperature 22 and 32 degrees C after anthesis. There existed obvious differences in the expression patterns of these genes under the high temperature stress, and the expression patterns were isoform-dependent. The relative expression amount of sbe1 and sbe3 under high temperature decreased significantly, and both of the genes were the sensitive isoform genes of SBE to high temperature stress. Among the DBE genes, pul was the isoform gene with high expression level, being more sensitive to high temperature stress than isa1, isa2, and isa3. Among the SS genes, sss2a had a significantly lower relative expression amount than sss1 and Wx, but sss2a and sss1 were more sensitive to high temperature than Wx, suggesting that sss2a and sss1 could be the important genes that adjusted the starch structure in rice endosperm under high temperature stress, especially at the middle and late grain filling stages. PMID:22720620

  18. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    OpenAIRE

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and derm...

  19. ANGIOTENSIN I CONVERTING ENZYME GENE POLYMORPHISM AND EXERCISE TRAINABILITY IN ELDERLY WOMEN: AN ELECTROCARDIOLOGICAL APPROACH

    OpenAIRE

    Takuro Tobina; Akira Kiyonaga; Yuko Akagi; Yukari Mori; Kojiro Ishii; Hitoshi Chiba; Munehiro Shindo; Hiroaki Tanaka

    2007-01-01

    Angiotensin I converting enzyme (ACE) gene Insertion / Deletion (I/D) polymorphism is associated with exercise trainability and exercise induced left ventricular hypertrophy. However, it is unclear whether this polymorphism influences exercise trainability in the elderly, and the electrocardiological alterations by exercise training is unknown among the genotypes. We herein investigated the association between ACE gene insertion/deletion (I/D) polymorphism, exercise trainability and the elect...

  20. The angiotensin-converting enzyme (ACE) gene family of Anopheles gambiae.

    OpenAIRE

    Isaac R Elwyn; Lee Alison J; Smith Judith A; Burnham Susan; Shirras Alan D

    2005-01-01

    Abstract Background Members of the M2 family of peptidases, related to mammalian angiotensin converting enzyme (ACE), play important roles in regulating a number of physiological processes. As more invertebrate genomes are sequenced, there is increasing evidence of a variety of M2 peptidase genes, even within a single species. The function of these ACE-like proteins is largely unknown. Sequencing of the A. gambiae genome has revealed a number of ACE-like genes but probable errors in the Ensem...

  1. Evolution of the division of labor between genes and enzymes in the RNA world

    OpenAIRE

    Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

    2014-01-01

    The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the e...

  2. Evolution of the division of labor between genes and enzymes in the RNA world.

    OpenAIRE

    Gergely Boza; András Szilágyi; Ádám Kun; Mauro Santos; Eörs Szathmáry

    2014-01-01

    The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the e...

  3. Gene expression for peroxisome-associated enzymes in hepatocellular carcinomas induced by ciprofibrate, a hypolipidemic compound

    International Nuclear Information System (INIS)

    Administration of hypolipidemic compounds leads to marked proliferation of peroxisomes and peroxisome-associated enzymes (PAE) in the livers of rodents and non-rodent species. The increase peroxisome-associated enzymes such as fatty acid β-oxidation system and catalase is shown to be due to an increase in the levels of mRNA. In this experiment they have examined hepatocellular carcinomas (HCC), induced in male F-344 rats by ciprofibrate (0.025%, w/w for 60 weeks), for gene expression of PAE. Total RNA was purified from HCC as well as from control and ciprofibrate (0.025% for 2 weeks) fed rat livers. Northern blot analysis was performed using [32/sub p/]cDNA probes for albumin, fatty acetyl-CoA oxidase, enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and catalase. mRNA levels in HCC for albumin, fatty acid β-oxidation enzymes and catalase were comparable with those levels observed in the livers of rats given ciprofibrate for 2 weeks. In control livers the mRNAs for β-oxidation enzymes were low. Albumin mRNA levels in all the 3 groups were comparable. Additional studies are necessary to determine whether the increased level of mRNAs for the β-oxidation enzymes in HCC is due to the effect of ciprofibrate or to the gene amplification

  4. Gene expression for peroxisome-associated enzymes in hepatocellular carcinomas induced by ciprofibrate, a hypolipidemic compound

    Energy Technology Data Exchange (ETDEWEB)

    Rao, M.S.; Nemali, M.R.; Reddy, J.K.

    1986-03-05

    Administration of hypolipidemic compounds leads to marked proliferation of peroxisomes and peroxisome-associated enzymes (PAE) in the livers of rodents and non-rodent species. The increase peroxisome-associated enzymes such as fatty acid ..beta..-oxidation system and catalase is shown to be due to an increase in the levels of mRNA. In this experiment they have examined hepatocellular carcinomas (HCC), induced in male F-344 rats by ciprofibrate (0.025%, w/w for 60 weeks), for gene expression of PAE. Total RNA was purified from HCC as well as from control and ciprofibrate (0.025% for 2 weeks) fed rat livers. Northern blot analysis was performed using (32/sub p/)cDNA probes for albumin, fatty acetyl-CoA oxidase, enoyl-CoA hydratase 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and catalase. mRNA levels in HCC for albumin, fatty acid ..beta..-oxidation enzymes and catalase were comparable with those levels observed in the livers of rats given ciprofibrate for 2 weeks. In control livers the mRNAs for ..beta..-oxidation enzymes were low. Albumin mRNA levels in all the 3 groups were comparable. Additional studies are necessary to determine whether the increased level of mRNAs for the ..beta..-oxidation enzymes in HCC is due to the effect of ciprofibrate or to the gene amplification.

  5. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    Science.gov (United States)

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism. PMID:23800623

  6. Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme

    Directory of Open Access Journals (Sweden)

    Kyslík Pavel

    2010-02-01

    Full Text Available Abstract Background Penicillin G acylase of Escherichia coli (PGAEc is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage, continues at crossing the cytoplasmic membrane (signal sequence removing, and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. Results Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The α-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. Conclusions Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc

  7. A branch-inducing phytoplasma in Euphorbia pulcherrima associated with changes in expression of host genes

    Science.gov (United States)

    Four Euphorbia pulcherrima genes specifically regulated during phytoplasma infection have been identified using a combination of differential display of cDNA-PCR products and microarray analysis. According to BLAST searches the possible functions of the identified genes included a histidine-containi...

  8. Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

    DEFF Research Database (Denmark)

    Østergaard, L; Lauvergeat, V; Naested, H;

    2001-01-01

    resembling the expression pattern of late embryogenic abundant ABA-responsive genes. Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli beta-glucuronidase reporter gene. This showed...... that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth. Expression of both genes is restricted to vascular tissue. Based upon their homology to proteins involved in lignin...... synthesis, we propose that AtCRL2 is involved in generating conducting tissue late in development, while AtCRL1 is involved in vascular tissue differentiation and/or synthesis in the germinating embryos....

  9. Diversity of beetle genes encoding novel plant cell wall degrading enzymes.

    Science.gov (United States)

    Pauchet, Yannick; Wilkinson, Paul; Chauhan, Ritika; Ffrench-Constant, Richard H

    2010-01-01

    Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent "disappearance" of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology. PMID:21179425

  10. Mutagenesis of Genes for Starch Debranching Enzyme Isoforms in Pea by Zinc-Finger Endonucleases

    International Nuclear Information System (INIS)

    Starch debranching enzymes in plants are divided into two groups based on their ability to hydrolyze different substrates. The first group, pullulanases, hydrolyze α-1,6-glucosidic linkages in substrates such as pullulan, amylopectin and glycogen. The second group of debranching enzymes, isoamylases, hydrolyze glycogen and amylopectin and are not active on pullulan. Three isoforms of isoamylase and a pullulanase have been isolated from a cDNA library of Pisum sativum. These isoamylases have been characterized following their heterologous expression in E. coli. Based on the DNA sequence that encodes these debranching enzymes, a specific mutagenesis targeting these enzymes will be attempted. The technique involves the homologous recombination of DNA mediated by zinc-finger endonucleases. Vectors will be constructed to include a fragment that will modify these genes. Using this technique, it is hoped that null mutants for each enzyme will be created and the exact role of these enzymes for the synthesis and degradation of starch in plants will be elucidated. (author)

  11. Polymorphisms of two histamine-metabolizing enzymes genes and childhood allergic asthma: a case control study

    Directory of Open Access Journals (Sweden)

    Sobkowiak Paulina

    2010-11-01

    Full Text Available Abstract Background Histamine-metabolizing enzymes (N-methyltransferase and amiloride binding protein 1 are responsible for histamine degradation, a biogenic amine involved in allergic inflammation. Genetic variants of HNMT and ABP1 genes were found to be associated with altered enzyme activity. We hypothesized that alleles leading to decreased enzyme activity and, therefore, decreased inactivation of histamine may be responsible for altered susceptibility to asthma. Methods The aim of this study was to analyze polymorphisms within the HNMT and ABP1 genes in the group of 149 asthmatic children and in the group of 156 healthy children. The genetic analysis involved four polymorphisms of the HNMT gene: rs2071048 (-1637T/C, rs11569723 (-411C/T, rs1801105 (Thr105Ile = 314C/T and rs1050891 (1097A/T and rs1049793 (His645Asp polymorphism for ABP1 gene. Genotyping was performed with use of PCR-RFLP. Statistical analysis was performed using Statistica software; linkage disequilibrium analysis was done with use of Haploview software. Results We found an association of TT genotype and T allele of Thr105Ile polymorphism of HNMT gene with asthma. For other polymorphisms for HNMT and ABP1 genes, we have not observed relationship with asthma although the statistical power for some SNPs might not have been sufficient to detect an association. In linkage disequilibrium analysis, moderate linkage was found between -1637C/T and -411C/T polymorphisms of HNMT gene. However, no significant differences in haplotype frequencies were found between the group of the patients and the control group. Conclusions Our results indicate modifying influence of histamine N-methyltransferase functional polymorphism on the risk of asthma. The other HNMT polymorphisms and ABP1 functional polymorphism seem unlikely to affect the risk of asthma.

  12. Phage-mediated transfer of a dextranase gene in Lactobacillus sanfranciscensis and characterization of the enzyme.

    Science.gov (United States)

    Picozzi, Claudia; Meissner, Daniel; Chierici, Margherita; Ehrmann, Matthias A; Vigentini, Ileana; Foschino, Roberto; Vogel, Rudi F

    2015-06-01

    While phages of lactobacilli are extensively studied with respect to their structure and role in the dairy environment, knowledge about phages in bacteria residing in sourdough fermentation is limited. Based on the previous finding that the Lactobacillus sanfranciscensis phage EV3 carries a putative dextranase gene (dex), we have investigated the distribution of similar dex(+) phages in L. sanfranciscensis, the chance of gene transfer and the properties of the dextranase encoded by phage EV3. L. sanfranciscensis H2A (dex(-)), originally isolated from a wheat sourdough, expressed a Dex(+) phenotype upon infection with EV3. The dextranase gene was isolated from the transductant and heterologously expressed in Escherichia coli. The gene encoded a protein of 801 amino acids with a calculated molecular weight (Mw) of 89.09 kDa and a calculated pI of 5.62. Upon purification aided by a 6-His tag, enzyme kinetic parameters were determined. The Km value was 370 mM, and the Vmax was calculated in about 16 μmol of glucose released from dextran by 1 mg of enzyme in 1 min in a buffer solution at pH 5.0. The optimum conditions were 60 °C and pH 4.5. The enzyme retained its activity for >3h at 60 °C and exhibited only 40% activity at 30 °C; the highest homology of 72% was found to a dextranase gene from Lactobacillus fermentum phage φPYB5. Within 25 L. sanfransiscensis isolates tested, the strain 4B5 carried a similar prophage encoding a dextranase gene. Our data suggest a phage-mediated transfer of dextranase genes in the sourdough environment resulting in superinfection-resistant L. sanfranciscensis Dex(+) strains with a possible ecological advantage in dextran-containing sourdoughs. PMID:25771219

  13. Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source.

    Science.gov (United States)

    Derrick, S; Large, P J

    1993-11-01

    Valine aminotransferase, a key enzyme in both biosynthesis and breakdown of branched-chain amino acids, showed consistently higher activity in Candida utilis grown in continuous culture than in Saccharomyces cerevisiae, while pyruvate decarboxylase and alcohol dehydrogenase, the other two enzymes of the Ehrlich pathway of branched-chain alcohol formation, were lower in activity. By spheroplast lysis, it was shown that valine aminotransferase followed the distribution of pyruvate decarboxylase in being located in the cytosol. Replacement of ammonium as nitrogen source by valine during conditions of carbon or nitrogen limitation caused increased specific activities of these three enzymes in S. cerevisiae, but (with one exception) decreased those of C. utilis. Of the metabolites accumulating in the culture medium, little or no ethanol or branched-chain alcohols were present during carbon-limited growth of either organism, but the change to nitrogen limitation resulted in increases in concentration of 20- to 100-fold in pyruvate, acetate and non-pyruvate keto acids as well as the accumulation of branched-chain alcohols in both organisms, and of ethanol, ethyl acetate and glycerol in S. cerevisiae. When valine was the limiting nitrogen source, there was an increase in non-pyruvate keto acids and a 10- to 16-fold increase in 2-methylpropanol. Total branched-chain alcohols formed under nitrogen limitation were 2-fold higher in S. cerevisiae than in C. utilis, irrespective of nitrogen source. Accumulation of branched-chain alcohols, ethanol, acetate and glycerol was also observed during carbon-limited growth of S. cerevisiae with valine as nitrogen source at dilution rates above the critical rate for transition to respirofermentative growth. Less than 70% of the valine carbon metabolized during growth of S. cerevisiae and only 15% of that used during growth of C. utilis was recovered in identified metabolic products. Even allowing for losses by volatilization during

  14. The angiotensin-converting enzyme (ACE gene family of Anopheles gambiae

    Directory of Open Access Journals (Sweden)

    Isaac R Elwyn

    2005-12-01

    Full Text Available Abstract Background Members of the M2 family of peptidases, related to mammalian angiotensin converting enzyme (ACE, play important roles in regulating a number of physiological processes. As more invertebrate genomes are sequenced, there is increasing evidence of a variety of M2 peptidase genes, even within a single species. The function of these ACE-like proteins is largely unknown. Sequencing of the A. gambiae genome has revealed a number of ACE-like genes but probable errors in the Ensembl annotation have left the number of ACE-like genes, and their structure, unclear. Results TBLASTN and sequence analysis of cDNAs revealed that the A. gambiae genome contains nine genes (AnoACE genes which code for proteins with similarity to mammalian ACE. Eight of these genes code for putative single domain enzymes similar to other insect ACEs described so far. AnoACE9, however, has several features in common with mammalian somatic ACE such as a two domain structure and a hydrophobic C terminus. Four of the AnoACE genes (2, 3, 7 and 9 were shown to be expressed at a variety of developmental stages. Expression of AnoACE3, AnoACE7 and AnoACE9 is induced by a blood meal, with AnoACE7 showing the largest (approximately 10-fold induction. Conclusion Genes coding for two-domain ACEs have arisen several times during the course of evolution suggesting a common selective advantage to having an ACE with two active-sites in tandem in a single protein. AnoACE7 belongs to a sub-group of insect ACEs which are likely to be membrane-bound and which have an unusual, conserved gene structure.

  15. Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny.

    Science.gov (United States)

    Mata-Sotres, José Antonio; Moyano, Francisco Javier; Martínez-Rodríguez, Gonzalo; Yúfera, Manuel

    2016-07-01

    In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes. PMID:26987267

  16. Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

    Science.gov (United States)

    Williams, Elsie M; Little, Rory F; Mowday, Alexandra M; Rich, Michelle H; Chan-Hyams, Jasmine V E; Copp, Janine N; Smaill, Jeff B; Patterson, Adam V; Ackerley, David F

    2015-10-15

    This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies. PMID:26431849

  17. Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

    Science.gov (United States)

    Toyokawa, Chihana; Shobu, Misaki; Tsukamoto, Rie; Okamura, Saki; Honda, Yoichi; Kamitsuji, Hisatoshi; Izumitsu, Kousuke; Suzuki, Kazumi; Irie, Toshikazu

    2016-09-01

    We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a β-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus. PMID:26979984

  18. Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-feng; JIANG Jian-ping; MI Zu-huang

    2005-01-01

    Background Acinetobacter baumannii is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by multi-drug resistance Acinetobacter baumannii is very difficult to treat. This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycoside-modifying enzymes including N-acetyltransferases and O-phosphotransferases in Acinetobacter baumannii. Methods Bacterial identification and antimicrobial susceptibility test were performed by PhoenixTM system in 247 strains of Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multi-drug resistant Acinetobacter baumannii were detected by agar dilution. Four aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer.Results The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50%. Imipenem and meropenem showed high antibacterial activities with resistance rates of 3.2% and 4.1%. MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multi-drug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 93.3%, respectively. But their resistance rates to tobramycin, netilmicin and neomycin were 86.7%, 93.3% and 46.7%, respectively. Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected. Their positive rates were 93.3%, 20.0% and 20.0%, respectively. These three genes existed simultaneously in No.19 strain. Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 97.9% and 99.7% identities with GenBank genes (AY307113, S68058 and AY

  19. Selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in Astragalus chrysochlorus

    OpenAIRE

    Çakir, Özgür; Turgut-Kara, Neslihan; Ari, Şule

    2016-01-01

    Astragalus sp. are used in folk medicine because of their biological activities and are known for the ability to accumulate high levels of selenium (Se). The purpose of this study was to explore gene expression of selenocysteine methyltransferase (SMT), responsible for forming MeSeCys, and activities of ascorbate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) enzymes in callus tissues of Astragalus chrysochlorus growing in different Se-containing media. Quan...

  20. Angiotensin-converting enzyme gene and retinal arteriolar narrowing: The Funagata Study

    OpenAIRE

    Tanabe, Y; Kawasaki, R.; J. J. Wang; Wong, T Y; Mitchell, P; Daimon, M; Oizumi, T; Kato, T.; Kawata, S.; Kayama, T; Yamashita, H.

    2009-01-01

    The purpose of this study is to determine whether the angiotensin-converting enzyme (ACE) gene polymorphism is associated with retinal arteriolar narrowing, a subclinical marker of chronic hypertension. The Funagata Study examined a population-based sample of Japanese aged 35+ years; 368 participants had both retinal vessel diameter measurements and ACE insertion/deletion (ACE I/D) polymorphism analyses performed. Assessment of retinal vessel diameter and retinal vessel wall signs followed th...

  1. The association between paired basic amino acid cleaving enzyme 4 gene haplotype and diastolic blood pressure

    Institute of Scientific and Technical Information of China (English)

    李建平; 王晓滨; 陈常忠; 徐新; 洪雪梅; 徐希平; 高炜; 霍勇

    2004-01-01

    Background In a previously identified locus linked to hypertension on chromosome 15q, we identified three blood pressure candidate genes: insulin-like growth factor 1 receptor gene (IGF1R), myocyte specific enhancer factor 2A gene (MEF2A), and paired basic amino acid cleaving enzyme 4 gene (PACE4). In this study, we tested their associations with hypertension using haplotype analysis.Methods A total of 288 unrelated individuals, including 163 high diastolic blood pressure (DBP) subjects and 125 normal DBP subjects were enrolled in this case-control study. Twenty single nucleotide polymorphisms (SNPs) in the three genes were genotyped using polymerase chain reaction followed by restriction enzyme digestion. Haplotype analysis was accomplished in the following stages: (1) pair-wise linkage disequilibrium test among SNPs on the same gene was performed to explore blocks in which recombination is very unlikely to happen; (2) Estimation-Maximization algorithm was applied to estimate haplotype frequencies in each block; (3) the chi-square test was used to examine the specific haplotype difference, and a permutation test was used to examine the overall haplotype profile difference between cases and controls in each block.Results An estimated haplotype "CCCCG" frequency in the haplotype block on the PACE4 gene was significantly higher in high DBP cases than in controls (P<0.01). The overall estimated haplotype profile in this block was also significantly different between the cases and the controls (P<0.001). This association indicates. Conclusions This study for the first time demonstrated that PACE4 gene may play an important role in the regulation of DBP. This association indicates that variations influencing DBP resides in or near this genomic region.

  2. Bioreducible crosslinked low molecular weight branched PEI-PBLG as an efficient gene carrier

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N’-cystaminebisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP).CPP not only inherits PEI-PBLG’s amphiphilic advantages,but also possesses reducible properties.CPP can complex with DNA to form nanoparticles.CPP/DNA complex particles were characterized by particle size and zeta potential analysis.The result showed that the complex particles have suitable size and surface charges for gene delivery.And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA.CPP has good reducible property,and also has good biocompatibility because of introducing PBLG segment.The cytotoxicity of CPP was evaluated using MTT assay,and the results showed CPP has lower cytotoxicity compared with PEI 25 K.The transfection properties were characterized in different cells by using plasmid DNA as a reporter.CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells.This was attributed to bioreducible and biocompatibility properties of the CPP.These results suggested that CPP is a promising low-toxic,highly effective non-viral gene carrier.

  3. Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

    Directory of Open Access Journals (Sweden)

    Xi Chen, Yong Liang, Jing Hua, Li Tao, Wensheng Qin, Sanfeng Chen

    2010-01-01

    Full Text Available In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1. This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei.

  4. Catabolism of Branched Chain Amino Acids Supports Respiration but Not Volatile Synthesis in Tomato Fruits

    Institute of Scientific and Technical Information of China (English)

    Andrej Kochevenko; Wagner L.Araújo; Gregory S.Maloney; Denise M.Tieman; Phuc Thi Do; Mark G.Taylor; Harry J.Klee; Alisdair R.Fernie

    2012-01-01

    The branched-chain amino acid transaminases (BCATs) have a crucial role in metabolism of the branched-chain amino acids leucine,isoleucine,and valine.These enzymes catalyze the last step of synthesis and the initial step of degradation of these amino acids.Although the biosynthetic pathways of branched chain amino acids in plants have been extensively investigated and a number of genes have been characterized,their catabolism in plants is not yet completely understood.We previously characterized the branched chain amino acid transaminase gene family in tomato,revealing both the subcellular localization and kinetic properties of the enzymes encoded by six genes.Here,we examined possible functions of the enzymes during fruit development.We further characterized transgenic plants differing in the expression of branched chain amino acid transaminases 1 and 3,evaluating the rates of respiration in fruits deficient in BCAT1 and the levels of volatiles in lines overexpressing either BCAT1 or BCAT3.We quantitatively tested,via precursor and isotope feeding experiments,the importance of the branched chain amino acids and their corresponding keto acids in the formation of fruit volatiles.Our results not only demonstrate for the first time the importance of branched chain amino acids in fruit respiration,but also reveal that keto acids,rather than amino acids,are the likely precursors for the branched chain flavor volatiles.

  5. Genes Involved in Formation and Attachment of a Two-Carbon Chain as a Component of Eurekanate, a Branched-Chain Sugar Moiety of Avilamycin A

    OpenAIRE

    Treede, Irina; Hauser, Gerd; Mühlenweg, Agnes; Hofmann, Carsten; Schmidt, Maraike; Weitnauer, Gabriele; Glaser, Steffen; Bechthold, Andreas

    2005-01-01

    Eurekanate belongs to the important class of branched-chain carbohydrates present in a wide variety of natural sources. It is a component of avilamycin A, a potent inhibitor of bacterial protein synthesis targeting the 50S ribosomal subunit. The present work provides experimental proof for the function of two genes of the avilamycin biosynthetic gene cluster, aviB1 and aviO2, that are both involved in avilamycin structure modification. The functions of both genes were identified by gene inact...

  6. The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect molting hormone

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.; Gilbert, Lawrence I.

    2006-01-01

    The developmental events occurring during molting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the molting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20-hydroxyecdysone (20E......), are mediated by four cytochrome P450 enzymes, encoded by genes in the Halloween family. Orthologs of the Drosophila Halloween genes phantom (phm: CYP306A1), disembodied (dib: CYP302A1), shadow (sad: CYP315A1) and shade (shd: CYP314A1) were obtained from the endocrinological model insect, the tobacco...... hornworm Manduca sexta. Expression of these genes was studied and compared with changes in ecdysteroid titer that controls transition from the larval to pupal stage. Phm, dib and sad (mediating the final hydroxylations in the biosynthesis of ecdysone; E) were selectively expressed in the prothoracic gland...

  7. Identification and characterization of a ubiquitinconjugating enzyme UBE2A gene from lamprey.

    Science.gov (United States)

    Chen, Liyong; Wu, Fenfang; Feng, Bo

    2016-02-01

    Ubiquitin-conjugating enzymes (E2s) play an important role in the mechanism of ubiquitin transfer. Although in most species many of these enzymes share high sequence and structural conservation, their existence and functions in the lamprey remain unknown. In this study, we identified and characterized a ubiquitin-conjugating enzyme (UBE2A)-like gene in lamprey. The gene, designated as LaUBE2A, contained a 456-bp open reading frame encoding a 152-amino acid protein with a typical UBC domain. Real-time PCR assay showed that LaUBE2A was expressed in various tissues of the adult lamprey, with higher levels in the leukocytes and muscle and lower levels in the skin and liver. The high conservation in amino acid sequence between LaUBE2A and UBE2As from Homo sapiens, Mus musculus, Cavia porcellus, and Alligator sinensi implied that the function of LaUBE2A may be similar to that of UBE2A. PMID:26463350

  8. Duplication of the dystroglycan gene in most branches of teleost fish

    Directory of Open Access Journals (Sweden)

    Giardina Bruno

    2007-05-01

    Full Text Available Abstract Background The dystroglycan (DG complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. A single copy of the DG gene (DAG1 has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. Similarly, D. rerio (zebrafish seems to have a single copy of DAG1, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD event, many teleost fish acquired multiple copies of several genes (paralogues. Results Data mining of pufferfish (T. nigroviridis and T. rubripes and other teleost fish (O. latipes and G. aculeatus available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in T. nigroviridis. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by DAG1 in mammals and D. rerio. A similar scenario emerged also in D. labrax (sea bass, from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. Western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost Antarctic species (T. bernacchii and C. hamatus. Conclusion Our evolutionary analysis has shown that the whole-genome duplication event in the Class Actinopterygii (ray-finned fish involved also DAG1. We unravelled new important molecular genetic details

  9. Comparative transcriptomics as a tool for the identification of root branching genes in maize.

    Science.gov (United States)

    Jansen, Leentje; Hollunder, Jens; Roberts, Ianto; Forestan, Cristian; Fonteyne, Philippe; Van Quickenborne, Charlotte; Zhen, Rui-Guang; McKersie, Bryan; Parizot, Boris; Beeckman, Tom

    2013-12-01

    The root system is fundamental for plant development, is crucial for overall plant growth and is recently being recognized as the key for future crop productivity improvement. A major determinant of root system architecture is the initiation of lateral roots. While knowledge of the genetic and molecular mechanisms regulating lateral root initiation has mainly been achieved in the dicotyledonous plant Arabidopsis thaliana, only scarce data are available for major crop species, generally monocotyledonous plants. The existence of both similarities and differences at the morphological and anatomical level between plant species from both clades raises the question whether regulation of lateral root initiation may or may not be conserved through evolution. Here, we performed a targeted genome-wide transcriptome analysis during lateral root initiation both in primary and in adventitious roots of Zea mays and found evidence for the existence of common transcriptional regulation. Further, based on a comparative analysis with Arabidopsis transcriptome data, a core of genes putatively conserved across angiosperms could be identified. Therefore, it is plausible that common regulatory mechanisms for lateral root initiation are at play in maize and Arabidopsis, a finding that might encourage the extrapolation of knowledge obtained in Arabidopsis to crop species at the level of root system architecture. PMID:23941360

  10. Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

    OpenAIRE

    Dolferus, R; Osterman, J. C.; Peacock, W. J.; Dennis, E.S.

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved fr...

  11. Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis.

    OpenAIRE

    Pang, A S; Nathoo, S; Wong, S L

    1991-01-01

    Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were clones and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease ca...

  12. Recent progress in gene-directed enzyme prodrug therapy: an emerging cancer treatment.

    Science.gov (United States)

    Both, Gerald W

    2009-08-01

    The principle of gene-directed enzyme prodrug therapy (GDEPT) has existed for many years but, while simple in concept, the effective practical application of this therapy has proven to be challenging. Improvements in the efficacy of GDEPT have been achieved principally through the choice and development of more effective vectors, by optimizing and controlling gene expression and by increasing the activity of the delivered enzyme through mutation. While innovation continues in this field, the pioneering GDEPT systems designed to treat glioma and prostate cancer have completed or are now entering late-stage clinical trials, respectively. As the pace of innovation in GDEPT technology far exceeds its clinical application, these initial products are anticipated to be replaced by next-generation biologicals. This review highlights recent progress in the strategies and development of GDEPT and summarizes the status of current clinical trials. With the first GDEPT product for treatment of resected gliomas poised to gain marketing approval, a new era in cancer gene medicine is emerging. PMID:19649987

  13. Identification of Genes Coding Aminoglycoside Modifying Enzymes in E. coli of UTI Patients in India

    Directory of Open Access Journals (Sweden)

    Abdul Rouf Mir

    2016-01-01

    Full Text Available This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR. Out of 98 isolates, 71 (72.45% isolates were identified as E. coli and the remaining 27 (27.55% as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.

  14. Betacyanin biosynthetic genes and enzymes are differentially induced by (abiotic stress in Amaranthus hypochondriacus.

    Directory of Open Access Journals (Sweden)

    Gabriela Casique-Arroyo

    Full Text Available An analysis of key genes and enzymes of the betacyanin biosynthetic pathway in Amaranthus hypochondriacus (Ah was performed. Complete cDNA sequence of Ah genes coding for cyclo-DOPA 5-O glucosyltransferase (AhcDOPA5-GT, two 4, 5-DOPA-extradiol-dioxygenase isoforms (AhDODA-1 and AhDODA-2, respectively, and a betanidin 5-O-glucosyltransferase (AhB5-GT, plus the partial sequence of an orthologue of the cytochrome P-450 R gene (CYP76AD1 were obtained. With the exception AhDODA-2, which had a closer phylogenetic relationship to DODA-like genes in anthocyanin-synthesizing plants, all genes analyzed closely resembled those reported in related Caryophyllales species. The measurement of basal gene expression levels, in addition to the DOPA oxidase tyrosinase (DOT activity, in different tissues of three Ah genotypes having contrasting pigmentation levels (green to red-purple was determined. Additional analyses were performed in Ah plants subjected to salt and drought stress and to two different insect herbivory regimes. Basal pigmentation accumulation in leaves, stems and roots of betacyanic plants correlated with higher expression levels of AhDODA-1 and AhB5-GT, whereas DOT activity levels coincided with pigment accumulation in stems and roots and with the acyanic nature of green plants, respectively, but not with pigmentation in leaves. Although the abiotic stress treatments tested produced changes in pigment levels in different tissues, pigment accumulation was the highest in leaves and stems of drought stressed betacyanic plants, respectively. However, tissue pigment accumulation in stressed Ah plants did not always correlate with betacyanin biosynthetic gene expression levels and/or DOT activity. This effect was tissue- and genotype-dependent, and further suggested that other unexamined factors were influencing pigment content in stressed Ah. The results obtained from the insect herbivory assays, particularly in acyanic plants, also support the

  15. Expression of the Isoamylase Gene of Flavobacterium odoratum KU in Escherichia coli and Identification of Essential Residues of the Enzyme by Site-Directed Mutagenesis

    OpenAIRE

    Abe, Jun-ichi; Ushijima, Chiaki; HIZUKURI, Susumu

    1999-01-01

    The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109. The promoter of the gene was successful in E. coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same Mr, heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumula...

  16. Evolution of the division of labor between genes and enzymes in the RNA world.

    Directory of Open Access Journals (Sweden)

    Gergely Boza

    2014-12-01

    Full Text Available The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA.

  17. Evolution of the division of labor between genes and enzymes in the RNA world.

    Science.gov (United States)

    Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

    2014-12-01

    The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA. PMID:25474573

  18. IMPACT OF ANGIOTENSIN-CONVERTING ENZYME GENE POLYMORPHISM ON THE DEVELOPMENT OF INSULIN RESISTANCE SYNDROME

    Directory of Open Access Journals (Sweden)

    G. E. Roitberg

    2013-01-01

    Full Text Available Objective: to analyze the distribution of components of insulin resistance (IR syndrome and to study the frequency of their combinations in relation to the genotypes and allelic variants of the angiotensin-converting enzyme (ACE gene.Subjects and methods. A group of clinically healthy patients (50 women and 42 men with different genotypes of the ACE gene was examined.The distribution of IR syndrome components and the frequency of their combinations were analyzed in relation to the genotypes and allelicvariants of the ACE gene.Results. A group of D allele carriers compared to A allele ones showed a pronounced tendency for the frequency of IR to reduce due to thehigher proportion of patients with complete IR syndrome. This observation becomes statistically significant in the assessment of homozygous variants of the ACE gene. At the same time dyslipidemia and hypertension in the presence of IR significantly more frequently occurred in patients with the DD genotype than in those with genotype II.Conclusion. There was a marked predominance of the manifestations of IR syndrome with a complete set of components in the DD genotypicgroup, which confirms the significant strong association between ACE gene polymorphism and IR syndrome.

  19. Comparison of the Gene Coding Contents and Other Unusual Features of the GC-Rich and AT-Rich Branch Probosciviruses.

    Science.gov (United States)

    Ling, Paul D; Long, Simon Y; Zong, Jian-Chao; Heaggans, Sarah Y; Qin, Xiang; Hayward, Gary S

    2016-01-01

    Nearly 100 cases of lethal acute hemorrhagic disease in young Asian elephants have been reported worldwide. All tested cases contained high levels of elephant endotheliotropic herpesvirus (EEHV) DNA in pathological blood or tissue samples. Seven known major types of EEHVs have been partially characterized and shown to all belong to the novel Proboscivirus genus. However, the recently determined 206-kb EEHV4 genome proved to represent the prototype of a GC-rich branch virus that is very distinct from the previously published 180-kb EEHV1A, EEHV1B, and EEHV5A genomes, which all fall within an alternative AT-rich branch. Although EEHV4 retains the large family of 7xTM and vGPCR-like genes, six are unique to either just one or the other branch. While both branches display a highly enriched distribution of A and T tracts in intergenic domains, they are generally much larger within the GC-rich branch. Both branches retain the vGCNT1 acetylglucosamine transferase and at least one vOX-2 gene, but the two branches differ by 25 genes overall, with the AT-rich branch encoding a fucosyl transferase (vFUT9) plus two or three more vOX2 proteins and an immunoglobulin-like gene family that are all absent from the GC-rich branch. Several envelope glycoproteins retain only 15 to 20% protein identity or less across the two branches. Finally, the two plausible predicted transcriptional regulatory proteins display no homology at all to those in the alpha-, beta-, or gammaherpesvirus subfamilies. These results reinforce our previous proposal that the probosciviruses should be designated a new subfamily of mammalian herpesviruses. IMPORTANCE Multiple species of herpesviruses from three different lineages of the Proboscivirus genus (EEHV1/6, EEHV2/5, and EEHV3/4/7) infect either Asian or African elephants, but the highly lethal hemorrhagic disease is largely confined to Asian elephant calves and is predominantly associated with EEHV1. In the accompanying paper [P. D. Ling et al., m

  20. Characterization of the Ubiquitin E2 Enzyme Variant Gene Family in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Pei Wen; On-Sun Lau; Xing-Wang Deng

    2007-01-01

    Ubiquitin E2 enzyme variant (UEV) proteins are similar to ubiquitin-conjugating enzyme (E2) in both sequence and structure, but the lack of a catalytic cysteine residue renders them incapable of forming a thiolester linkage with ubiquitin. While the functional roles of several UEVs have been defined in yeast and animal systems, Arabidopsis COP10, a photomorphogenesis repressor, is the only UEV characterized in plants. Phylogenetic analysis revealed that the eight Arabidopsis UEV genes belong to three subfamilies.The expression of those genes is supported by either the presence of ESTs or RT-PCR analysis. We also characterized the other members of the COP10 subfamily, UEV2. Semi-quantitative RT-PCR analysis indicated that the UEV2 transcripts can be detected in most organs of Arabidopsis. Analysis of UEV2::GUS transgenic lines also showed its ubiquitous expression in nearly all the developmental stages of Arabidopsis.Transient expression analysis indicated that the sGFP-UEV2 fusion protein can localize to both the cytoplasm and nucleus. A T-DNA insertion mutant, uev2-1, which abolished the transcription of UEV2, displays no visible phenotype. Further, the cop10-4 uev2-1 double mutant exhibits the same phenotype as the cop10-4mutant in darkness. UEV2 is therefore not functionally redundant with COP10.

  1. Linoleic acid-induced expression of defense genes and enzymes in tobacco.

    Science.gov (United States)

    Sumayo, Marilyn S; Kwon, Duck-Kee; Ghim, Sa-Youl

    2014-11-15

    Linoleic acid (LA) is a naturally occurring fatty acid (FA) found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (PCC). In this study, we examined effects of six doses of exogenous LA on the induction of defense genes and enzymes. The optimum ISR activity was observed in plants treated with 0.1mM LA where the effect of LA on membrane permeability was minimal. The application of LA as a root drench enhanced the activity of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) and induced the expression of β-glucuronidase (GUS). PAL and POD activities were increased in a concentration dependent manner while the maximum PPO activity was observed after treatment with 0.01mM LA. An RT-PCR analysis of the defense-related genes, Coi1, NPR1, PR-1a and PR-1b, of tobacco plants treated with 0.1mM LA revealed an association of LA with elicitation of ISR in tobacco. PMID:25238656

  2. Analysis of the Relationship between Antioxidant Enzyme Gene Polymorphisms and Their Activity in Post-Traumatic Gonarthrosis.

    Science.gov (United States)

    Vnukov, V V; Panina, S B; Milyutina, N P; Krolevets, I V; Zabrodin, M A

    2016-05-01

    Analysis of polymorphisms of genes encoding antioxidant enzymes SOD1 (G7958A), SOD2 (T58C), CAT (C-262T), and GSTP1 (Ile105Val) in 93 patients with post-traumatic gonarthrosis showed that GSTP1 Ile105Val polymorphism is often associated with heterozygous mutation in catalase gene CAT C-262T. In gonarthrosis, catalase activity in peripheral blood mononuclear cells in patients with CT genotype of the C-262T locus of CAT gene more than 2-fold surpassed that in CC genotype and more than 50% surpassed the normal. Changes in the balance of activity of antioxidant enzymes can affect viability of mononuclear cells. PMID:27270931

  3. Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

    Directory of Open Access Journals (Sweden)

    Abderrazak Kitsy

    Full Text Available The essential branched-chain amino acids (BCAA, leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2 and branched-chain alpha keto acid dehydrogenase (Bckdha was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4 compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our

  4. Dynamic Linkages between Denitrification Functional Genes/Enzymes and Biogeochemical Reaction Rates of Nitrate and Its Reduction Products

    Science.gov (United States)

    Li, M.; Shi, L.; Qian, W.; Gao, Y.; Liu, Y.; Liu, C.

    2015-12-01

    Denitrification is a respiratory process in which oxidized nitrogen compounds are used as alternative electron acceptors for energy production when oxygen is limited. Denitrification is an important process that not only accounts for the significant loss of nitrogen fertilizers from soils but also leads to NO, N2O and CO2 emissions, which are important greenhouse gas species. In this study, denitrification was investigated in Columbia River sediments, focusing on the dynamic linkages between functional genes/enzymes and biogeochemical reaction rates of nitrate and its reduction products. NO3-, NO2- and N2O were assayed in different incubation time. DNA was extracted from the sediments and functional genes were quantified as a function of time during the denitrification. Functional enzymes were extracted from the sediments and measured using a newly developed, targeted protein method. The biogeochemical, functional gene, and enzyme data were collectively used to establish the dynamic correlation of functional genes/enzymes and biogeochemical reaction rates. The results provide fundamental insights regarding the dynamic regulation of functional genes and enzymes in the processes of denitrification and greenhouse gas production, and also provide experimental data critical for the development of biogeochemical reaction models that incorporate genome-scale insights and describe macroscopic biogeochemical reaction rates in ecosystems.

  5. Carotid remodeling of hypertensive subjects and polymorphism of the angiotensin-converting enzyme gene

    Institute of Scientific and Technical Information of China (English)

    李世军; 孙宁玲; 周素敏

    2004-01-01

    Background This study was designed to investigate the relationships between changes in the structure and function of carotid arteries and angiotensin converting enzyme (ACE) gene polymorphism in Chinese hypertensive subjects. Methods Multiplex polymerase chain reaction amplification was used to evaluate the ACE gene insertion/deletion (I/D) polymorphism. High-resolution B-mode ultrasound examinations were performed to detect parameters of carotid artery remodeling. Results Intima-media thickness (IMT) was significantly different among the DD, ID and II genotypes of ACE (DD>ID>II, P0.05) in hypertensive subjects. The frequency of the DD gene and D allele of ACE were higher in patients with thickening carotid than in patients with normal carotid (70.4% vs 24.1%, and 79.5% vs 40.5%, respectively, P<0.001). In multiple stepwise regression analysis, independent risk factors for increased carotid IMT in hypertensive subjects were ACE genotypes (P<0.001), age (P<0.001) and carotid internal diameter (P=0.032). Moreover, triglycerides and total cholesterol were higher in patients with the DD genotype than in those with the II genotype (P<0.05). Conclusions The I/D polymorphism of the ACE gene was related to IMT, but not to internal diameter, distensibility and stiffness of the carotid in Chinese hypertensive subjects. ACE gene polymorphism was a main risk factor for increased carotid IMT. These results may imply that there is a link between lipid metabolism and ACE genotype polymorphism in Chinese hypertensive subjects.

  6. Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway.

    Science.gov (United States)

    Lillo, Cathrine; Lea, Unni S; Ruoff, Peter

    2008-05-01

    The content of flavonoids increases in response to nitrogen and phosphorus depletion in plants. Manipulation of these macronutrients may therefore be used to control the levels of desirable compounds and improve plant quality. Key enzymes in the shikimate pathway, which feeds precursors into the flavonoid pathway, are regulated post-translationally by feedback from aromatic amino acids, and possibly by redox control through photosynthesis. Use of microarrays for global transcript analysis in Arabidopsis has revealed that transcript levels are less influenced by mineral nutrients in the shikimate pathway compared with the flavonoid pathway. The responses in the shikimate pathway appear complex, whereas in the flavonoid pathway, a single gene often responds similarly to mineral depletion, high light intensity and sucrose. MYB [production of anthocyanin pigment 1 (PAP1)/production of anthocyanin pigment 2 (PAP2)] and bHLH [GLABRA3 (GL3)] transcription factors are important for the nutrient depletion response. PAP1/2 stimulate gross activation of the flavonoid pathway, and different investigations support merging signal transduction chains for various abiotic treatments on PAP1/2. Flavonol synthase is not part of the PAP1/2 regulon, and expression is mainly enhanced by high light intensity and sucrose, not mineral depletion. Nevertheless, both cyanidin and flavonol derivatives increase in response to nitrogen depletion. Kaempferols are the dominating flavonols in Arabidopsis leaves under normal cultivation conditions, but quercetin accumulation can be triggered by nitrogen depletion in combination with other abiotic factors. PMID:18031469

  7. Targeted Expression of Stromelysin-1 in Mammary Gland Provides Evidence for a Role of Proteinases in Branching Morphogenesis and the Requirement for an Intact Basement Membrane for Tissue-specific Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sympson, Carolyn J; Talhouk, Rabih S; Alexander, Caroline M; Chin, Jennie R; Cliff, Shirley M; Bissell, Mina J; Werb, Zena

    1994-05-01

    The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene

  8. Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains

    OpenAIRE

    Kralj, S.; van Geel-Schutten, G.H.; Dondorff, M.M.G.; Kirsanovs, S.; van der Maarel, M.J.E.C.; Dijkhuizen, L.

    2004-01-01

    Members of the genera Streptococcus and Leuconostoc synthesize various α-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly α-(1→4) linkages together with α-(1→6) and α-(1→4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. ...

  9. Coordination of gene expression of arachidonic and docosahexaenoic acid cascade enzymes during human brain development and aging.

    Directory of Open Access Journals (Sweden)

    Veronica H Ryan

    Full Text Available The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades.AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging.The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism.Expression patterns were split into Development (0 to 20 years and Aging (21 to 78 years intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2, cyclooxygenases (COX-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA and PTGS2 (COX-2 genes at 1q25, highly inter-correlated genes were at distant chromosomal loci.Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease.

  10. Enzyme-responsive destabilization of stabilized plasmid-lipid nanoparticles as an efficient gene delivery.

    Science.gov (United States)

    Song, Su Jeong; Lee, Seulgi; Lee, Yan; Choi, Joon Sig

    2016-08-25

    Stabilized plasmid-lipid particles (SPLPs) have been developed to overcome the low stability issue of cationic liposomes, however, SPLPs that are too stable result in unsatisfactory transfection efficiency. In this article, we prepared enzyme-responsive SPLPs (eSPLPs) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG-GLFG-K-(C16)2, a PEG lipid with an enzymatically-cleavable linker (glycine-phenylalanine-leucine-glycine, GFLG). eSPLPs were successfully prepared with plasmid DNA (pDNA) encapsulation efficiency of over 80%, using the detergent dialysis method. The PEG shell stabilized eSPLPs and maintained a hydrodynamic diameter of around 200nm. Although typical SPLPs were relatively intact in endosomal condition, the PEG shell of eSPLPs was cleaved following the degradation of the GFLG linker by cathepsin B in the endosome. Then, eSPLPs collapsed and induced endosomal disruption triggering the controlled release of the encapsulated pDNA into cytoplasm. Owing to the enzyme-responsive destabilization, eSPLPs showed a 10 to 100-fold higher transfection efficiency than control SPLPs, which was confirmed using luciferase assay. These results suggest that eSPLPs might be promising candidates for practical use as gene delivery systems, with both stability and high transfection efficiency for future in vivo applications. PMID:27240779

  11. Multiple horizontally acquired genes from fungal and prokaryotic donors encode cellulolytic enzymes in the bdelloid rotifer Adineta ricciae.

    Science.gov (United States)

    Szydlowski, L; Boschetti, C; Crisp, A; Barbosa, E G G; Tunnacliffe, A

    2015-07-25

    The bdelloid rotifer, Adineta ricciae, an anhydrobiotic microinvertebrate, exhibits a high rate of horizontal gene transfer (HGT), with as much as 10% of its transcriptome being of foreign origin. Approximately 80% of these foreign transcripts are involved in metabolic processes, and therefore bdelloids represent a useful model for assessing the contribution of HGT to biochemical diversity. To validate this concept, we focused on cellulose digestion, an unusual activity in animals, which is represented by at least 16 genes encoding cellulolytic enzymes in A. ricciae. These genes have been acquired from a variety of different donor organisms among the bacteria and fungi, demonstrating that bdelloids use diverse genetic resources to construct a novel biochemical pathway. A variable complement of the cellulolytic gene set was found in five other bdelloid species, indicating a dynamic process of gene acquisition, duplication and loss during bdelloid evolution. For example, in A. ricciae, gene duplications have led to the formation of three copies of a gene encoding a GH45 family glycoside hydrolase, at least one of which encodes a functional enzyme; all three of these gene copies are present in a close relative, Adineta vaga, but only one copy was found in each of four Rotaria species. Furthermore, analysis of expression levels of the cellulolytic genes suggests that a bacterial-origin cellobiase is upregulated upon desiccation. In summary, bdelloid rotifers have apparently developed cellulolytic functions by the acquisition and domestication of multiple foreign genes. PMID:25863176

  12. Regulation of Glucose Metabolism in Pseudomonas: THE PHOSPHORYLATIVE BRANCH AND ENTNER-DOUDOROFF ENZYMES ARE REGULATED BY A REPRESSOR CONTAINING A SUGAR ISOMERASE DOMAIN*

    OpenAIRE

    Daddaoua, Abdelali; Krell, Tino; Ramos, Juan-Luis

    2009-01-01

    In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes. Divergently with respect to the edd gene is the gap-1 gene. Expression from Pzwf, Pedd, and Pgap is modulated by HexR in response to the availability of glucose in the medium. To study the regulatory process in greater detail we purified HexR and showed that ...

  13. Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    de Villena Fernando

    2010-05-01

    Full Text Available Abstract Background The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (Pgk2, Aldoart1, and Aldoart2. Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes. Results We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except Pfk. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and Gpi1 transcript in mouse spermatogenic cells. Conclusions Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.

  14. Angiotensin Converting Enzyme Gene Polymorphism in Egyptian Patients with Systemic Lupus Erythematosus

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    El-Shafeey M.M., El-Shayeb M., Othman E. and Elfawy N

    2005-12-01

    Full Text Available Systemic lupus erythematosus (SLE shows various clinical manifestations with various immunological abnormalities. The development of lupus nephritis and vasculitis is common in patients with SLE. As angiotensin I-converting enzyme (ACE has been reported to be associated with various immunological phenomena, we investigated the correlation between insertion(I / deletion(D polymorphism of the ACE gene and SLE. Fifty Egyptian patients with SLE and thirty healthy control persons were involved in this study. ACE gene was detected by the polymerase chain reaction (PCR. In SLE patients, there is a significant difference when comparing DD and II genotypes (P<0.05,being higher in the DD genotype. And a highly significant difference when comparing ID and II genotypes (P=0.001, being much higher in ID genotype than II genotype. According to vasculitis, there is a significant relationship between vasculitis and patients genotypes when comparing ID genotype with both II and DD genotypes (P<0.05, being highest in ID genotype. There is a significant relationship found when comparing ID genotype with both II and DD genotypes, being highest in ID genotype in patients with score 21. These results suggest that the ACE genotype could be associated with SLE.

  15. Xenobiotic metabolizing enzyme gene polymorphisms predict response to lung volume reduction surgery

    Directory of Open Access Journals (Sweden)

    DeMeo Dawn L

    2007-08-01

    Full Text Available Abstract Background In the National Emphysema Treatment Trial (NETT, marked variability in response to lung volume reduction surgery (LVRS was observed. We sought to identify genetic differences which may explain some of this variability. Methods In 203 subjects from the NETT Genetics Ancillary Study, four outcome measures were used to define response to LVRS at six months: modified BODE index, post-bronchodilator FEV1, maximum work achieved on a cardiopulmonary exercise test, and University of California, San Diego shortness of breath questionnaire. Sixty-four single nucleotide polymorphisms (SNPs were genotyped in five genes previously shown to be associated with chronic obstructive pulmonary disease susceptibility, exercise capacity, or emphysema distribution. Results A SNP upstream from glutathione S-transferase pi (GSTP1; p = 0.003 and a coding SNP in microsomal epoxide hydrolase (EPHX1; p = 0.02 were each associated with change in BODE score. These effects appeared to be strongest in patients in the non-upper lobe predominant, low exercise subgroup. A promoter SNP in EPHX1 was associated with change in BODE score (p = 0.008, with the strongest effects in patients with upper lobe predominant emphysema and low exercise capacity. One additional SNP in GSTP1 and three additional SNPs in EPHX1 were associated (p Conclusion Genetic variants in GSTP1 and EPHX1, two genes encoding xenobiotic metabolizing enzymes, were predictive of response to LVRS. These polymorphisms may identify patients most likely to benefit from LVRS.

  16. ANGIOTENSIN I CONVERTING ENZYME GENE POLYMORPHISM AND EXERCISE TRAINABILITY IN ELDERLY WOMEN: AN ELECTROCARDIOLOGICAL APPROACH

    Directory of Open Access Journals (Sweden)

    Takuro Tobina

    2007-06-01

    Full Text Available Angiotensin I converting enzyme (ACE gene Insertion / Deletion (I/D polymorphism is associated with exercise trainability and exercise induced left ventricular hypertrophy. However, it is unclear whether this polymorphism influences exercise trainability in the elderly, and the electrocardiological alterations by exercise training is unknown among the genotypes. We herein investigated the association between ACE gene insertion/deletion (I/D polymorphism, exercise trainability and the electrocardiological alternations by exercise in elderly women. Eighty four elderly women participated in this study. In all subjects the leg extension power (LEP and lactate threshold (LT were determined in order to evaluate the muscle strength, aerobic capacity and to also select the appropriate training intensity for each individual. They performed bench step exercise training for 12 weeks. A resting electrocardiogram was recorded for the obtained QTc interval in before and after the program. The baseline of aerobic capacity was higher in I/I than that in I/D, and the QTc interval was shorter in I/I than that in I/D. All other characteristics were similar among the genotypes. The QTc interval tended to be shorten only in the D/D. Furthermore, the value of the QTc interval change showed a significant difference between the I/I and D/D genotype after the program. The LT and LEP demonstrated a similar response among the genotypes. The D allele of ACE gene I/D polymorphism may therefore play a role in the electrocardiological aspect during exercise training, however, it was not found to influence the aerobic capacity

  17. Association of polymorphisms in angiotensin-converting enzyme gene with gestational diabetes mellitus in Indian women

    Science.gov (United States)

    Aggarwal, Parul; Agarwal, Nutan; Das, Nibhriti; Dalal, Krishna

    2016-01-01

    Background: Numerous genes have been reported in relation with gestational diabetes mellitus (GDM), but the findings were not consistently replicated across populations, or there have been no detailed studies on them. Previous literatures suggested that, out of all angiotensin converting enzyme (ACE) gene polymorphisms, only ACE insertion/deletion (I/D) gene polymorphism has a strong association with GDM in Asian Indian women. Aim: This study was devoted to evaluate the association of four single nucleotide polymorphisms (SNPs) ACE A240T, C1237T, G2350A and I/D with GDM and Type 2 diabetes mellitus. Materials and Methods: This study recruited 105 GDM cases, 119 Type 2 diabetes mellitus subjects and 120 controls. PCR-RFLP was used for identifying genotypes of ACE A240T, C1237T and G2350A and PCR was performed in the case of ACE I/D. Results: Significant associations of ACE SNP's, C1237T, and G2350A with GDM were observed. Haplotype analysis revealed the remarkably significant evidence of association with SNP combination ACE A240T, C1237T, G2350A, and I/D with GDM patients (P = 0.024). Individuals possessing haplotype “TTAI” (frequency 30% in GDM and 0 in controls) derived from these SNPs had 185 fold increased risk of developing GDM (95% of confidence interval: 11.13–3102.15), which was highest when compared with other 15 haplotypes. Conclusion: Shorter-range haplotypes were also significant, but the only consistently associated alleles were found to be in ACE C1237T, G2350A, and I/D. These results suggested that the variant in close proximity to ACE C1237T, G2350A and/or I/D modulates susceptibility to GDM and noninsulin dependent diabetes mellitus in Indian women. PMID:26958520

  18. DNA methylation analysis of the angiotensin converting enzyme (ACE gene in major depression.

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    Peter Zill

    Full Text Available BACKGROUND: The angiotensin converting enzyme (ACE has been repeatedly discussed as susceptibility factor for major depression (MD and the bi-directional relation between MD and cardiovascular disorders (CVD. In this context, functional polymorphisms of the ACE gene have been linked to depression, to antidepressant treatment response, to ACE serum concentrations, as well as to hypertension, myocardial infarction and CVD risk markers. The mostly investigated ACE Ins/Del polymorphism accounts for ~40%-50% of the ACE serum concentration variance, the remaining half is probably determined by other genetic, environmental or epigenetic factors, but these are poorly understood. MATERIALS AND METHODS: The main aim of the present study was the analysis of the DNA methylation pattern in the regulatory region of the ACE gene in peripheral leukocytes of 81 MD patients and 81 healthy controls. RESULTS: We detected intensive DNA methylation within a recently described, functional important region of the ACE gene promoter including hypermethylation in depressed patients (p = 0.008 and a significant inverse correlation between the ACE serum concentration and ACE promoter methylation frequency in the total sample (p = 0.02. Furthermore, a significant inverse correlation between the concentrations of the inflammatory CVD risk markers ICAM-1, E-selectin and P-selectin and the degree of ACE promoter methylation in MD patients could be demonstrated (p = 0.01 - 0.04. CONCLUSION: The results of the present study suggest that aberrations in ACE promoter DNA methylation may be an underlying cause of MD and probably a common pathogenic factor for the bi-directional relationship between MD and cardiovascular disorders.

  19. Frequency of Polymorphism in Aromatase Enzyme Coding Gene with Prostate Cancer Risk in North Indian Population

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    KH Onsory

    2015-10-01

    Full Text Available Background: A series of biochemical reactions are involved in the endogenous production of estrogens. Their final and rate-limiting step is catalyzed by aromatase belonging to the class XIX of cytochrome P450. CYP19 is a key enzyme for estrogen synthesis in males. It catalyzes the irreversible conversion of androstenedione and testosterone to estrone and estradiol-17β, respectively. Aromatase P450 is present in the endoplasmic reticulum of estrogen-reproducing cells in which it is expressed. The effects of the resulting estrogens are mediated through the estrogen receptor. One of the most important polymorphism, is a C to T variation in exon 7 resulting in an Arg264Cys amino acid exchange, has been shown to be very common in Asia. The purpose of this study was to determine the association of CYP19 gene polymorphism with the prostate cancer risk among the studied population. Methods: PCR-RFLP analysis of CYP19 gene was on 100 prostate cancer patients and an equal number of matching controls. The data was analyzed using the computer software SPSS for windows (version 19. Results: The frequency of CT genotype was higher in patients (37% as compared to controls (21.2% and this incidence was statistically significant (OR, 2.10; 95 % CI, 1.02-4.34; P=0.044. Stratification of patients according to the risk factors, resulted in a slightly improved OR in individuals carrying CT compared to CC genotype (OR, 2.35 95% CI, 1.11-4.96; P=0.024. The TT genotype was not significantly associated with prostate cancer risk (OR, 0.63; 95% CI, 0.16-2.50; P=0.519. Conclusion: It seems that CT genotype is more associated with cancer prostate compare with other genotypes. It appears to be an increased risk of prostate cancer associated with the Arg264Cys substitution in the CYP19 gene.

  20. Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

    OpenAIRE

    Dong, G.; Vieille, C; Zeikus, J G

    1997-01-01

    The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three protei...

  1. Gene-gene-environment interactions between drugs, transporters, receptors, and metabolizing enzymes: Statins, SLCO1B1, and CYP3A4 as an example.

    Science.gov (United States)

    Sadee, Wolfgang

    2013-09-01

    Pharmacogenetic biomarker tests include mostly specific single gene-drug pairs, capable of accounting for a portion of interindividual variability in drug response and toxicity. However, multiple genes are likely to contribute, either acting independently or epistatically, with the CYP2C9-VKORC1-warfarin test panel, an example of a clinically used gene-gene-dug interaction. I discuss here further instances of gene-gene-drug interactions, including a proposed dynamic effect on statin therapy by genetic variants in both a transporter (SLCO1B1) and a metabolizing enzyme (CYP3A4) in liver cells, the main target site where statins block cholesterol synthesis. These examples set a conceptual framework for developing diagnostic panels involving multiple gene-drug combinations. PMID:23436703

  2. Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains.

    Science.gov (United States)

    Kralj, S; van Geel-Schutten, G H; Dondorff, M M G; Kirsanovs, S; van der Maarel, M J E C; Dijkhuizen, L

    2004-11-01

    Members of the genera Streptococcus and Leuconostoc synthesize various alpha-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly alpha-(1-->4) linkages together with alpha-(1-->6) and alpha-(1-->4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. This paper reports, for the first time, isolation and characterization of dextransucrase and mutansucrase genes and enzymes from various Lactobacillus species and the characterization of the glucan products synthesized, which mainly have alpha-(1-->6)- and alpha-(1-->3)-glucosidic linkages. The four GTF enzymes characterized from three different Lb. reuteri strains are highly similar at the amino acid level, and consequently their protein structures are very alike. Interestingly, these four Lb. reuteri GTFs have relatively large N-terminal variable regions, containing RDV repeats, and relatively short putative glucan-binding domains with conserved and less-conserved YG-repeating units. The three other GTF enzymes, isolated from Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri, contain smaller variable regions and larger putative glucan-binding domains compared to the Lb. reuteri GTF enzymes. PMID:15528655

  3. Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

    Science.gov (United States)

    Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

    2014-03-15

    To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis. PMID:24365593

  4. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Science.gov (United States)

    Busk, Peter K; Lange, Mette; Pilgaard, Bo; Lange, Lene

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls. PMID:25461894

  5. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  6. Prognostic impact of carboxylesterase 1 gene variants in patients with congestive heart failure treated with angiotensin-converting enzyme inhibitors

    DEFF Research Database (Denmark)

    Nelveg-Kristensen, Karl E.; Madsen, Majbritt B.; Torp-Pedersen, Christian;

    2016-01-01

    OBJECTIVE: Most angiotensin-converting enzyme inhibitors (ACEIs) are prodrugs activated by carboxylesterase 1 (CES1). We investigated the prognostic importance of CES1 gene (CES1) copy number variation and the rs3815583 single-nucleotide polymorphism in CES1 among ACEI-treated patients with conge...

  7. Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

    Science.gov (United States)

    Houck, C M; Pear, J R; Elliott, R; Perchorowicz, J T

    1987-01-01

    Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases. Images PMID:3112128

  8. Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

    DEFF Research Database (Denmark)

    Jensen, A T; Gaafar, A; Ismail, A;

    1996-01-01

    An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...

  9. End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Rizk, Mazen, E-mail: mazen.rizk@tuhh.de [Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg (Germany); Antranikian, Garabed, E-mail: antranikian@tuhh.de [Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg (Germany); Elleuche, Skander, E-mail: skander.elleuche@tuhh.de [Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg (Germany)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Multifunctional enzymes offer an interesting approach for biomass degradation. Black-Right-Pointing-Pointer Size and conformation of separate constructs play a role in the effectiveness of chimeras. Black-Right-Pointing-Pointer A connecting linker allows for maximal flexibility and increased thermostability. Black-Right-Pointing-Pointer Genes with functional similarities are the best choice for fusion candidates. -- Abstract: The reduction of fossil fuels, coupled with its increase in price, has made the search for alternative energy resources more plausible. One of the topics gaining fast interest is the utilization of lignocellulose, the main component of plants. Its primary constituents, cellulose and hemicellulose, can be degraded by a series of enzymes present in microorganisms, into simple sugars, later used for bioethanol production. Thermophilic bacteria have proven to be an interesting source of enzymes required for hydrolysis since they can withstand high and denaturing temperatures, which are usually required for processes involving biomass degradation. However, the cost associated with the whole enzymatic process is staggering. A solution for cost effective and highly active production is through the construction of multifunctional enzyme complexes harboring the function of more than one enzyme needed for the hydrolysis process. There are various strategies for the degradation of complex biomass ranging from the regulation of the enzymes involved, to cellulosomes, and proteins harboring more than one enzymatic activity. In this review, the construction of multifunctional biomass degrading enzymes through end-to-end gene fusions, and its impact on production and activity by choosing the enzymes and linkers is assessed.

  10. Hepatic xenobiotic metabolizing enzyme and transporter gene expression through the life stages of the mouse.

    Directory of Open Access Journals (Sweden)

    Janice S Lee

    Full Text Available BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs. No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD 19, neonatal (postnatal day (PND 7, prepubescent (PND32, middle age (12 months, and old age (18 and 24 months in the C57BL/6J (C57 mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I, conjugation (Phase II and excretion (Phase III. In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs was observed at early (GD19, PND7 and to a lesser extent, later life stages (18 and 24 months. A number of female-specific XMETs exhibited a spike in expression centered at PND7. CONCLUSIONS: The analysis revealed dramatic differences in the expression of the XMETs, especially in the fetus and neonate that are partially dependent on gender-dependent factors. XMET expression can be used to predict life stage-specific responses to environmental chemicals and drugs.

  11. Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

    International Nuclear Information System (INIS)

    This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring

  12. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  13. Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.

    Directory of Open Access Journals (Sweden)

    Joshua T Schiffer

    Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

  14. Angiotensin-converting enzyme gene (ACE) insertion/deletion polymorphism in Mexican populations.

    Science.gov (United States)

    Vargas-Alarcón, Gilberto; Hernández-Pacheco, Guadalupe; Rodríguez-Pérez, José Manuel; Pérez-Hernández, Nonanzit; Pavón, Zinnia; Fragoso, José Manuel; Juarez-Cedillo, Teresa; Villarreal-Garza, Cynthia; Granados, Julio

    2003-12-01

    The angiotensin-converting enzyme gene (ACE) insertion/deletion polymorphism was determined in 211 Mexican healthy individuals belonging to different Mexican ethnic groups (98 Mestizos, 64 Teenek, and 49 Nahuas). ACE polymorphism differed among Mexicans with a high frequency of the D allele and the D/D genotype in Mexican Mestizos. The D/D genotype was absent in Teenek and present in only one Nahua individual (2.0%). When comparisons were made, we observed that Caucasian, African, and Asian populations presented the highest frequencies of the D allele, whereas Amerindian (Teenek and Pima) and Australian Aboriginals showed the highest frequencies of the I allele. The distribution of I/D genotype was heterogeneous in all populations: Australian Aboriginals presented the lowest frequency (4.9%), whereas Nahuas presented the highest (73.4%). The present study shows the frequencies of a polymorphism not analyzed previously in Mexican populations and establishes that this polymorphism distinguishes the Amerindian populations of other groups. On the other hand, since ACE alleles have been associated with genetic susceptibility to developing cardiovascular diseases and hypertension, knowledge of the distribution of these alleles could help to define the true significance of ACE polymorphism as a genetic susceptibility marker in the Amerindian populations. PMID:15018037

  15. Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

    Science.gov (United States)

    Cachón-González, M Begoña; Wang, Susan Z; McNair, Rosamund; Bradley, Josephine; Lunn, David; Ziegler, Robin; Cheng, Seng H; Cox, Timothy M

    2012-08-01

    The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of β-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human β-hexosaminidase α (HEXA) and β (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of β-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system. PMID:22453766

  16. Investigation of the genes involved in antigenic switching at the vlsE locus in Borrelia burgdorferi: an essential role for the RuvAB branch migrase.

    Directory of Open Access Journals (Sweden)

    Ashley R Dresser

    2009-12-01

    Full Text Available Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et

  17. Use of a Microarray to Detect Expression of Genes for Lignin-Degrading Enzymes in Soil Fungi

    Science.gov (United States)

    Bailey, V. L.; Smith, J. L.; Bolton, H.

    2003-12-01

    Lignin is a complex biopolymer that is degraded by fungi. Several extracellular enzymes have been implicated in degradation and include lignin peroxidases, laccases, manganese peroxidases, and glyoxal oxidases. Versions of these enzymes are produced by multiple species of fungi, and in some cases, multiple versions of a single enzyme may be produced by the same species of fungus. Previous research has indicated changes in fungal activity and diversity along a tallgrass prairie restoration chronosequence (Fermi National Lab, IL). A cDNA microarray was designed to interrogate the expression and microbial source of these lignin degrading enzymes in the chronosequence soils. We hypothesized that less diversity in gene expression would be detected in a farmed soil than in a restored prairie soil. The array had 46 oligonucleotides (15-25mer) that represent each of the enzymes listed above. Messenger RNA was extracted from 32 one-gram subsamples of the target soils then all of the extracts were pooled prior to RNA precipitation and mRNA purification. Aminoallyl modified dUTPs were incorporated during reverse transcription, after which the cDNA was labeled with Alexa-555 dye. The labeled cDNA was hybridized with the microarray for 24 hours and then imaged. Preliminary results support the hypothesis that fewer genes were expressed in the farmed soil than in the restored soil.

  18. Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family.

    Science.gov (United States)

    Vuillemin, Marlène; Claverie, Marion; Brison, Yoann; Séverac, Etienne; Bondy, Pauline; Morel, Sandrine; Monsan, Pierre; Moulis, Claire; Remaud-Siméon, Magali

    2016-04-01

    Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1→6) dextrans through the specific formation of α-(1→3) linkages. To date, BRS-B is the first reported α-(1→3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1→3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family. PMID:26763236

  19. The Functional Angiotensin Converting Enzyme Gene I/D Polymorphism Does not Alter Susceptibility to Chronic Pancreatitis

    Directory of Open Access Journals (Sweden)

    Whitcomb DC

    2004-11-01

    Full Text Available CONTEXT: Alterations of the renin-angiotensin system have been implicated in the pathogenesis of various diseases. The angiotensin converting enzyme is a key enzyme in the renin-angiotensin system. A deletion polymorphism of a 287-bp fragment of intron 16 of the angiotensin converting enzyme gene allele results in higher levels of circulating enzyme. ACE deletion genotype has been linked to heart diseases, sarcoidosis and liver fibrosis. The pancreatic renin-angiotensin system plays a role in the development of pancreatic fibrosis and ACE inhibitors decrease pancreatic fibrosis in experimental models. OBJECTIVES: We investigated the frequency of the ACE gene insertion/deletion polymorphism in chronic pancreatitis patients and controls. PATIENTS: Subjects with familial pancreatitis (n=51, sporadic chronic pancreatitis (n=104, and healthy controls (n=163 were evaluated. MAIN OUTCOME MEASURE: The presence of ACE insertion/deletion polymorphism. RESULTS: The frequency of the ACE gene deletion allele was similar in familial pancreatitis (49.0% sporadic pancreatitis (51.0% and controls (55.8%. Furthermore, there was no significant difference in clinical features between patients with ACE-insertion or insertion/deletion genotypes vs. patients with ACE-deletion genotype. CONCLUSION: We conclude that the ACE deletion genotype does not make a significant contribution to the pathogenesis and the progression of chronic pancreatitis.

  20. Effect of Maturity Stage on the Gene Expression of Antioxidative Enzymes in Cucumber (Cucumis sativus L.) Fruits Under Chilling Stress

    Institute of Scientific and Technical Information of China (English)

    QIAN Chun-lu; MI Hong-bo; ZHAO Yu-ying; HE Zhi-ping; MAO Lin-chun

    2013-01-01

    The gene expression patterns of antioxidative enzymes in cucumber (Cucumis sativus L.) fruits at four different maturity stages, immature (3-8 d after anthesis (DAA), mature (9-16 DAA), breaker (17-22 DAA), and yellow (35-40 DAA), were determined before and after cold storage at 2°C for 9 d and after subsequent rewarming at 20°C for 2 d. The electrolyte leakage and malondialdehyde content in cucumber fruits were increased after cold storage and subsequent rewarming. Increased expressions of peroxidase, ascorbate peroxidase (APX), and monodehydroascorbate reductase after cold storage played an important role in cucumber fruits to cope with chilling injury. The elevated cyt-superoxide dismutase, catalase, APX and dehydroascorbate reductase after subsequent rewarming in cucumber fruits facilitated the recovery from chilling stress. The highest expression levels of all the seven antioxidative enzyme genes in yellow fruits might be responsible for the enhanced chilling tolerance. Cucumber fruits at earlier developmental stages was more susceptible to chilling stress than those at later stages. The relative higher gene expressions of antioxidative enzymes genes at earlier developmental stages may be the responses to the sever oxidative stress caused by chilling injury.

  1. Localization of genes for lateral branch and female sex expression and construction of a molecular linkage map in cucumber (Cucumis sativus L. ) with RAPD markers

    Institute of Scientific and Technical Information of China (English)

    LI Xiaozun; PAN Junsong; WANG Gang; TIAN Libo; SI Longting; WU Aizhong; CAI Run

    2005-01-01

    A cucumber ( Cucumis sativus L. ) molecular linkage map, including 79 random-amplified polymorphic DNAs (RAPD)and two genes , lb for lateral branch and f for female sex expression, is constructed from a cross between a line, S52, with weak lateral growing ability and staminate from Dabieshan Mountains area in China and another line, S06, with strong lateral growing ability and gynoecious from Europe. The map contains nine linkage groups and spans 1110.0 cM with an average distance of 13.7 cM between loci. The lb locus is located in a longer linkage group LG-2 and flanked by two markers, OP-Q5-1 and OP-M-2-2, at 9.3 cM and 15.9 cM, respectively. In the meantime, the RAPD loci, OP-Q5-2 and BC151, in a short linkage group were found to flank f at 13.7 cM and 13.4 cM,respectively. The construction of RAPD map has paved a way for further study of the genes for lateral branch, female sex expression and other agronomic traits in cucumber.

  2. Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

    Science.gov (United States)

    The analysis of a wheat lysine ketoglutarate reductase – saccharopine dehydrogenase (LKR/SDH) gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes...

  3. Regulation of genes coding for enzyme constituents of the bacterial phosphotransferase system.

    OpenAIRE

    Rephaeli, A W; Saier, M H

    1980-01-01

    Regulation of the synthesis of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system was systematically studied in wild-type and mutant strains of Salmonella typhimurium and Escherichia coli. The results suggest that enzyme I and HPr as well as the glucose-specific and the mannose-specific enzymes II are synthesized by a mechanism which depends on (i) cyclic adenosine monophosphate and its receptor protein; (ii) extracellular inducer; (iii) the sugar-specific enzyme II compl...

  4. Branching out

    CERN Document Server

    Biggins, J D

    2010-01-01

    Results on the behaviour of the rightmost particle in the $n$th generation in the branching random walk are reviewed and the phenomenon of anomalous spreading speeds, noticed recently in related deterministic models, is considered. The relationship between such results and certain coupled reaction-diffusion equations is indicated.

  5. Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

    Energy Technology Data Exchange (ETDEWEB)

    Pinheiro, D.F.; Pacheco, P.D.G.; Alvarenga, P.V.; Buratini, J. Jr; Castilho, A.C.S.; Lima, P.F.; Sartori, D.R.S.; Vicentini-Paulino, M.L.M. [Departamento de Fisiologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP (Brazil)

    2013-03-15

    This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g{sup -1}·min{sup -1}) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g{sup -1}·min{sup -1}) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

  6. YANA – a software tool for analyzing flux modes, gene-expression and enzyme activities

    Directory of Open Access Journals (Sweden)

    Engels Bernd

    2005-06-01

    Full Text Available Abstract Background A number of algorithms for steady state analysis of metabolic networks have been developed over the years. Of these, Elementary Mode Analysis (EMA has proven especially useful. Despite its low user-friendliness, METATOOL as a reliable high-performance implementation of the algorithm has been the instrument of choice up to now. As reported here, the analysis of metabolic networks has been improved by an editor and analyzer of metabolic flux modes. Analysis routines for expression levels and the most central, well connected metabolites and their metabolic connections are of particular interest. Results YANA features a platform-independent, dedicated toolbox for metabolic networks with a graphical user interface to calculate (integrating METATOOL, edit (including support for the SBML format, visualize, centralize, and compare elementary flux modes. Further, YANA calculates expected flux distributions for a given Elementary Mode (EM activity pattern and vice versa. Moreover, a dissection algorithm, a centralization algorithm, and an average diameter routine can be used to simplify and analyze complex networks. Proteomics or gene expression data give a rough indication of some individual enzyme activities, whereas the complete flux distribution in the network is often not known. As such data are noisy, YANA features a fast evolutionary algorithm (EA for the prediction of EM activities with minimum error, including alerts for inconsistent experimental data. We offer the possibility to include further known constraints (e.g. growth constraints in the EA calculation process. The redox metabolism around glutathione reductase serves as an illustration example. All software and documentation are available for download at http://yana.bioapps.biozentrum.uni-wuerzburg.de. Conclusion A graphical toolbox and an editor for METATOOL as well as a series of additional routines for metabolic network analyses constitute a new user

  7. Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus

    OpenAIRE

    Tahlan, Kapil; Park, Hyeon Ung; Wong, Annie; Beatty, Perrin H.; Jensen, Susan E.

    2004-01-01

    Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and β-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upst...

  8. Construction of a cucumber genetic linkage map with SRAP markers and location of the genes for lateral branch traits

    Institute of Scientific and Technical Information of China (English)

    WANG; Gang; PAN; Junsong; LI; Xiaozun; HE; Huanle; WU; Aizh

    2005-01-01

    Using SRAP (sequence-related amplified polymorphism) markers a genetic linkage map of cucumber was constructed with a population consisting of 138 F2 individuals derived from a cross of the two cucumber lines, SO6 and S52. In the survey of parental polymorphisms with 182 primer combinations, 64 polymorphism-revealing primer pairs were screened out, which generated totally 108 polymorphic bands with an average of 1.7 bands per primer pair and at most 6 bands from one primer pair. The constructed molecular linkage map included 92 loci,distributed in seven linkage groups and spanning 1164.2 cM in length with an average genetic distance of 12.6 cM between two neighboring loci. Based on this linkage map, the quantitative trait loci (QTL) for the lateral branch number (Ibn) and the lateral branch average length (Ibl) in cucumber were identified by QTLMapper1.6. A major QTL Ibnl located between ME11SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from SO6. A major QTL Ibll located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S06.

  9. PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing.

    Science.gov (United States)

    Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

    2014-01-01

    Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the

  10. PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Florian Barbi

    Full Text Available Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5 and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2, active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may

  11. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens.

    OpenAIRE

    Navarro, F.; Courvalin, P

    1994-01-01

    Using degenerate oligonucleotides complementary to sequences encoding conserved amino acid motifs in D-alanine-D-alanine (Ddl) ligases, we have amplified ca. 600-bp fragments from Enterococcus casseliflavus ATCC 25788 and Enterococcus flavescens CCM439. Sequence analysis of the amplification products indicated that each strain possessed two genes, ddlE. cass. and vanC-2, and ddlE. flav. and vanC-3, respectively, encoding Ddl-related enzymes. The fragments internal to the vanC genes were 98.3%...

  12. Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo.

    OpenAIRE

    Lim, B; Williams, D A; Orkin, S H

    1987-01-01

    Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free r...

  13. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    OpenAIRE

    Elza Ibrahim Auerkari; Mamoru Ouchida; Mehmet Gunduz

    2015-01-01

    DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR), DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose g...

  14. A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

    OpenAIRE

    Cazzonelli, Christopher I.; Nisar, Nazia; Roberts, Andrea C.; Murray, Kevin D.; Borevitz, Justin O; Pogson, Barry J.

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzym...

  15. The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

    Science.gov (United States)

    Jaramillo-Gutierrez, Giovanna; Molina-Cruz, Alvaro; Kumar, Sanjeev; Barillas-Mury, Carolina

    2010-01-01

    Background OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. Methodology/Principal Findings OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response. Conclusion The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection. PMID:20567517

  16. The Anopheles gambiae oxidation resistance 1 (OXR1 gene regulates expression of enzymes that detoxify reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Giovanna Jaramillo-Gutierrez

    Full Text Available BACKGROUND: OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. METHODOLOGY/PRINCIPAL FINDINGS: OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT and glutathione peroxidase (Gpx expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response. CONCLUSION: The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.

  17. Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

    Institute of Scientific and Technical Information of China (English)

    刘梅; 孙宗修; 朱洁; 徐同; HARMANGaryE; LORITOMatteo

    2004-01-01

    Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

  18. Mutagenesis of genes for starch debranching enzyme isoforms in pea by means of zinc-finger endonucleases

    International Nuclear Information System (INIS)

    Starch debranching enzymes in plants are divided into two groups based on their ability to hydrolyse different substrates. The first group, pullulanases, hydrolyses α-1,6-glucosidic linkages in substrates such as pullulan, amylopectin and glycogen. The second group of debranching enzymes, isoamylases, hydrolyse glycogen and amylopectin and are not active on pullulan. Three isoforms of isoamylase and a pullulanase have been isolated from cDNA library of Pisum sativum. These isoamylases have been characterised based on the their heterologous expression in E coli. Based on the DNA sequence that encodes these debranching enzyme, a specific mutagenesis targeting at these DNA will be attempted. The method that will be employed are based on the techniques developed by Wright et al. (2005). This technique involves the homologous recombination of DNA that is mediated by zinc-finger endonucleases. Vectors will be constructed to include a fragment that will modify these genes. Microinjection technique will be used to insert these vectors into pollen which then will be fertilized. Using this technique, it is hoped that null mutant for each enzyme will be created and the exact role of these enzymes for the synthesis and degradation of starch in plants will be elucidate. (author)

  19. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    Science.gov (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  20. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    Science.gov (United States)

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

  1. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Directory of Open Access Journals (Sweden)

    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

  2. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  3. Rv2131c gene product: An unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

    International Nuclear Information System (INIS)

    Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K m of 0.22 ± 0.03 mM for inositol-1-phosphate and K m of 0.45 ± 0.05 mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC5 ∼ 60 mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV)

  4. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

    International Nuclear Information System (INIS)

    Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

  5. Diversity of beetle genes encoding novel plant cell wall degrading enzymes

    OpenAIRE

    Pauchet, Y.; Wilkinson, P.; Chauhan, R.; Ffrench-Constant, R.

    2010-01-01

    Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterost...

  6. Insulin-like growth factor-I gene therapy increases hippocampal neurogenesis, astrocyte branching and improves spatial memory in female aging rats.

    Science.gov (United States)

    Pardo, Joaquín; Uriarte, Maia; Cónsole, Gloria M; Reggiani, Paula C; Outeiro, Tiago F; Morel, Gustavo R; Goya, Rodolfo G

    2016-08-01

    In rats, learning and memory performance decline during aging, which makes this rodent species a suitable model to evaluate therapeutic strategies of potential value for correcting age-related cognitive deficits. Some of these strategies involve neurotrophic factors like insulin-like growth factor-I (IGF-I), a powerful neuroprotective molecule in the brain. Here, we implemented 18-day long intracerebroventricular (ICV) IGF-I gene therapy in 28 months old Sprague-Dawley female rats, and assessed spatial memory performance in the Barnes maze. We also studied hippocampal morphology using an unbiased stereological approach. Adenovectors expressing the gene for rat IGF-I or the reporter DsRed were used. Cerebrospinal fluid (CSF) samples were taken and IGF-I levels determined by radioimmunoassay. At the end of the study, IGF-I levels in the CSF were significantly higher in the experimental group than in the DsRed controls. After treatment, the IGF-I group showed a significant improvement in spatial memory accuracy as compared with DsRed counterparts. In the dentate gyrus (DG) of the hippocampus, the IGF-I group showed a higher number of immature neurons than the DsRed controls. The treatment increased hippocampal astrocyte branching and reduced their number in the hippocampal stratum radiatum. We conclude that the ependymal route is an effective approach to increase CSF levels of IGF-I and that this strategy improves the accuracy of spatial memory in aging rats. The favorable effect of the treatment on DG neurogenesis and astrocyte branching in the stratum radiatum may contribute to improving memory performance in aging rats. PMID:27188415

  7. Gene expression in the DpnI and DpnII restriction enzyme systems of Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.; Sabelnikov, A.G.; Chen, Jau-Der; Greenberg, B.

    1992-12-31

    Although a number of bacterial species are naturally transformable, that is, their cells are able to take up external DNA in substantial amounts and integrate it into the chromosome without artificial manipulation of the cell surface, Streptococcus pneumoniae, the first species in which this phenomenon was detected, remains a prototype of such transformation. Cells of S. pneumonias also contain potent restriction endonucleases able to severely restrict DNA introduced during viral infection. Our current understanding of the genetic basis of the complementary DpnI and DpnII restriction systems and of the biochemistry of their component enzymes are briefly reviewed. The manner in which these enzymes impinge on the transfer of chromosomal genes and of plasmeds will be examined in detail. It will be seen that far from acting against foreign DNA in general, the restriction systems seem to be designed to exclude only infecting viral DNA The presence of complementary restriction systems in different cells of S. pneumonias enhances their effectiveness in blocking viral infection and promoting species survival. This enhanced effectiveness requires the expression of alternative restriction systems. Therefore, the ability of the cells to transfer the restriction enzyme genes and to regulate their expression are important for survival of the species.

  8. Coal-burning endemic fluorosis is associated with reduced activity in antioxidative enzymes and Cu/Zn-SOD gene expression.

    Science.gov (United States)

    Wang, Qi; Cui, Kang-ping; Xu, Yuan-yuan; Gao, Yan-ling; Zhao, Jing; Li, Da-sheng; Li, Xiao-lei; Huang, Hou-jin

    2014-02-01

    To study the effect of fluorine on the oxidative stress in coal-burning fluorosis, we investigated the environmental characteristics of coal-burning endemic fluorosis combined with fluorine content surveillance in air, water, food, briquette, and clay binder samples from Bijie region, Guizhou Province, southwest of China. The activities of antioxidant enzymes including copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and level of lipid peroxidation such as malondialdehyde (MDA) were measured in serum samples obtained from subjects residing in the Bijie region. Expression of the Cu/Zn-SOD gene was assessed by quantitative reverse transcriptase PCR (qRT-PCR). Our results showed that people suffering from endemic fluorosis (the high and low exposure groups) had much higher MDA level. Their antioxidant enzyme activities and Cu/Zn-SOD gene expression levels were lower when compared to healthy people (the control group). Fluorosis can decrease the activities of antioxidant enzymes, which was associated with exposure level of fluorine. Down-regulation of Cu/Zn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis. PMID:23567976

  9. Identification of Synchronized Role of Transcription Factors, Genes, and Enzymes in Arabidopsis thaliana under Four Abiotic Stress Responsive Pathways

    Directory of Open Access Journals (Sweden)

    Samsad Razzaque

    2014-01-01

    Full Text Available Microarray datasets are widely used resources to predict and characterize functional entities of the whole genomics. The study initiated here aims to identify overexpressed stress responsive genes using microarray datasets applying in silico approaches. The target also extended to build a protein-protein interaction model of regulatory genes with their upstream and downstream connection in Arabidopsis thaliana. Four microarray datasets generated treating abiotic stresses like salinity, cold, drought, and abscisic acid (ABA were chosen. Retrieved datasets were firstly filtered based on their expression comparing to control. Filtered datasets were then used to create an expression hub. Extensive literature mining helped to identify the regulatory molecules from the expression hub. The study brought out 42 genes/TF/enzymes as the role player during abiotic stress response. Further bioinformatics study and also literature mining revealed that thirty genes from those forty-two were highly correlated in all four datasets and only eight from those thirty genes were determined as highly responsive to the above abiotic stresses. Later their protein-protein interaction (PPI, conserved sequences, protein domains, and GO biasness were studied. Some web based tools and software like String database, Gene Ontology, InterProScan, NCBI BLASTn suite, etc. helped to extend the study arena.

  10. Composition and expression of genes encoding carbohydrate-active enzymes in the straw-degrading mushroom Volvariella volvacea.

    Directory of Open Access Journals (Sweden)

    Bingzhi Chen

    Full Text Available Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation and GH43 (hemicellulose and pectin degradation, and the lyase families PL1, PL3 and PL4 (pectin degradation but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3'-tag digital gene expression (DGE reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.

  11. PTM-Switchboard--a database of posttranslational modifications of transcription factors, the mediating enzymes and target genes.

    Science.gov (United States)

    Everett, Logan; Vo, Antony; Hannenhalli, Sridhar

    2009-01-01

    Gene transcription is largely regulated by sequence-specific transcription factors (TFs). The TF activity is significantly regulated by its posttranslational modifications (PTMs). TF-PTMs serve as 'molecular switchboards' that map multiple upstream signaling events, in response to various environmental perturbations, to the downstream transcriptional events. While many instances of TF-PTMs and their effect on gene regulation have been experimentally determined, a systematic meta-analysis or a quantitative model-based investigation of this process has not been undertaken. A prerequisite to such analyses is a database of known instances of TF-PTMs affecting transcriptional regulation. The PTM-Switchboard database meets this need by cataloging such instances in the model system Saccharomyces cerevisiae. The database stores triplets of genes such that the ability of one gene (TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene (modifying enzyme). The database currently includes a large sample of experimentally characterized instances curated from the literature. In addition to providing a framework for searching and analyzing the data, the database will serve to benchmark computational methods. In the future, the database will be expanded to mammalian organisms, and will also include triplets predicted from computational approaches. The database can be accessed at http://cagr.pcbi.upenn.edu/PTMswitchboard. PMID:18927104

  12. Insertion/deletion polymorphism of the angiotensin-converting enzyme gene and the risk of hypertension among residents of two cities, South-South Nigeria

    Directory of Open Access Journals (Sweden)

    Mary Esien Kooffreh

    2014-01-01

    Conclusion: The I/D polymorphism of the angiotensin-converting enzyme gene was a risk factor for hypertension in the sample population of Calabar and Uyo. This research will form baseline information for subsequent molecular studies in this population.

  13. Phylogenomic relationships between amylolytic enzymes from 85 strains of fungi.

    Science.gov (United States)

    Chen, Wanping; Xie, Ting; Shao, Yanchun; Chen, Fusheng

    2012-01-01

    Fungal amylolytic enzymes, including α-amylase, gluocoamylase and α-glucosidase, have been extensively exploited in diverse industrial applications such as high fructose syrup production, paper making, food processing and ethanol production. In this paper, amylolytic genes of 85 strains of fungi from the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota were annotated on the genomic scale according to the classification of glycoside hydrolase (GH) from the Carbohydrate-Active enZymes (CAZy) Database. Comparisons of gene abundance in the fungi suggested that the repertoire of amylolytic genes adapted to their respective lifestyles. Amylolytic enzymes in family GH13 were divided into four distinct clades identified as heterologous α-amylases, eukaryotic α-amylases, bacterial and fungal α-amylases and GH13 α-glucosidases. Family GH15 had two branches, one for gluocoamylases, and the other with currently unknown function. GH31 α-glucosidases showed diverse branches consisting of neutral α-glucosidases, lysosomal acid α-glucosidases and a new clade phylogenetically related to the bacterial counterparts. Distribution of starch-binding domains in above fungal amylolytic enzymes was related to the enzyme source and phylogeny. Finally, likely scenarios for the evolution of amylolytic enzymes in fungi based on phylogenetic analyses were proposed. Our results provide new insights into evolutionary relationships among subgroups of fungal amylolytic enzymes and fungal evolutionary adaptation to ecological conditions. PMID:23166747

  14. Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome.

    Science.gov (United States)

    Stoetzel, Corinne; Muller, Jean; Laurier, Virginie; Davis, Erica E; Zaghloul, Norann A; Vicaire, Serge; Jacquelin, Cecile; Plewniak, Frederic; Leitch, Carmen C; Sarda, Pierre; Hamel, Christian; de Ravel, Thomy J L; Lewis, Richard Alan; Friederich, Evelyne; Thibault, Christelle; Danse, Jean-Marc; Verloes, Alain; Bonneau, Dominique; Katsanis, Nicholas; Poch, Olivier; Mandel, Jean-Louis; Dollfus, Helene

    2007-01-01

    Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ~70% of affected families. We have combined single-nucleotide-polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for approximately 5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family. PMID:17160889

  15. Differential display identifies overexpression of the USP36 gene, encoding a deubiquitinating enzyme, in ovarian cancer

    OpenAIRE

    Li, Jianduan; Olson, Lisa M.; Zhang, Zhengyan; LI, LINA; Bidder, Miri; Nguyen, Loan; Pfeifer, John; Rader, Janet S.

    2008-01-01

    Objectives. To find potential diagnostic markers or therapeutic targets, we used differential display technique to identify genes that are over or under expressed in human ovarian cancer. Methods. Genes were initially identified by differential display between two human ovarian surface epithelium cultures and two ovarian cancer cell lines, A2780 and Caov-3. Genes were validated by relative quantitative RT-PCR and RNA in situ hybridization. Results. Twenty-eight non-redundant sequences were ex...

  16. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    Science.gov (United States)

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa. PMID:25393679

  17. Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

    Directory of Open Access Journals (Sweden)

    L. Gavrilovic

    2009-12-01

    Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

  18. Psychiatric and cognitive symptoms in Huntington's disease are modified by polymorphisms in catecholamine regulating enzyme genes

    DEFF Research Database (Denmark)

    Vinther-Jensen, T; Nielsen, T T; Budtz-Jørgensen, E;

    2016-01-01

    -described cohort of Danish HD gene-expansion carriers. We show that cognitive impairment and psychiatric symptoms in HD are modified by polymorphisms in the monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT) genes and by the 4p16.3 B haplotype. These results support the theory of dopamine imbalance...

  19. Gene Regulation by Metabolic Enzyme GMP Synthetase and Chromatin Remodeler NuRD

    NARCIS (Netherlands)

    B.A. Reddy (Ashok)

    2012-01-01

    textabstractSpatial and temporal control of the gene expression is crucial for normal growth and development of an organism. Environmental stress factors pose a constant threat to normal development of an organism by causing altered gene expression. Cells have evolved counteractive mechanisms to ove

  20. Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved From Different Invertases. Expression of Fructan Genes Throughout Chicory Development

    Directory of Open Access Journals (Sweden)

    Wim Van den Ende

    2002-01-01

    Full Text Available Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The biochemistry of fructan biosynthesis in dicots has been resolved, and the respective cDNAs have been cloned. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar-type invertase, fructan exohydrolases (FEHs seem to have evolved from a cell-wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Expression analysis reveals that fructan enzymes are controlled mainly at the transcriptional level. Using chicory as a model system, northern analysis was consistent with enzymatic activity measurements and observed carbohydrate changes throughout its development.

  1. Effect of n-3 polyunsaturated fatty acid on gene expression of the critical enzymes involved in homocysteine metabolism

    Directory of Open Access Journals (Sweden)

    Huang Tao

    2012-01-01

    Full Text Available Abstract Background Previous studies showed that plasma n-3 polyunsaturated fatty acid (PUFA was negatively associated with plasma homocysteine (Hcy. Objective We investigated the regulatory effect of n-3 PUFA on mRNA expression of the critical genes encoding the enzymes involved in Hcy metabolism. Methods HepG2 cells were treated with docosahexaenoic acid (DHA, eicosapentaenoic acid (EPA, alpha-linolenic acid (ALA respectively for 48 h. The cells were collected and total RNA was isolated. The mRNA expression levels of the genes were determined by using Real Time-PCR. Results Compared with controls, the mRNA expression levels of 5-methyltetrahydrofolate reductase (MTHFR were significantly increased in the DHA group (p Conclusions Our results suggest that DHA up-regulates CSE and MTHFR mRNA expression and down-regulates MAT mRNA expression involved in Hcy metabolism.

  2. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris

    Energy Technology Data Exchange (ETDEWEB)

    Qian Haifeng; Chen Wei; Sheng, G. Daniel; Xu Xiaoyan; Liu Weiping [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Fu Zhengwei [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China)], E-mail: azwfu2003@yahoo.com.cn

    2008-07-30

    Greater exposure to herbicide increases the likelihood of harmful effects in humans and the environment. Glufosinate, a non-selective herbicide, inhibits glutamine synthetase (GS) and thus blocks ammonium assimilation in plants. In the present study, the aquatic unicellular alga Chlorella vulgaris was chosen to assess the effects of acute glufosinate toxicity. We observed physiological changes during 12-96 h of exposure, and gene transcription during 6-48 h of exposure. Exposure to glufosinate increased malondialdehyde content by up to 2.73 times compared with the control, suggesting that there was some oxidative damage. Electron microscopy also showed that there were some chloroplast abnormalities in response to glufosinate. The activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) also increased markedly in the presence of glufosinate. Maximum activities of SOD, POD, and CAT were 2.90, 2.91, and 2.48 times that of the control, respectively. These elevated activities may help alleviate oxidative damage. A real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL. The results showed that glufosinate reduced the transcript abundances of the three genes after 12 h exposure. The lowest abundances of psaB, psbC and rbcL transcripts in response to glufosinate exposure were 38%, 16% and 43% of those of the control, respectively. Our results demonstrate that glufosinate affects the activities of antioxidant enzymes, disrupts chloroplast ultrastructure, and reduces transcription of photosynthesis-related genes in C. vulgaris.

  3. Novel variants of major drug-metabolising enzyme genes in diverse African populations and their predicted functional effects

    Directory of Open Access Journals (Sweden)

    Matimba Alice

    2009-01-01

    Full Text Available Abstract Pharmacogenetics enables personalised therapy based on genetic profiling and is increasingly applied in drug discovery. Medicines are developed and used together with pharmacodiagnostic tools to achieve desired drug efficacy and safety margins. Genetic polymorphism of drug-metabolising enzymes such as cytochrome P450s (CYPs and N-acetyltransferases (NATs has been widely studied in Caucasian and Asian populations, yet studies on African variants have been less extensive. The aim of the present study was to search for novel variants of CYP2C9, CYP2C19, CYP2D6 and NAT2 genes in Africans, with a particular focus on their prevalence in different populations, their relevance to enzyme functionality and their potential for personalised therapy. Blood samples from various ethnic groups were obtained from the AiBST Biobank of African Populations. The nine exons and exon-intron junctions of the CYP genes and exon 2 of NAT2 were analysed by direct DNA sequencing. Computational tools were used for the identification, haplotype analysis and prediction of functional effects of novel single nucleotide polymorphisms (SNPs. Novel SNPs were discovered in all four genes, grouped to existing haplotypes or assigned new allele names, if possible. The functional effects of non-synonymous SNPs were predicted and known African-specific variants were confirmed, but no significant differences were found in the frequencies of SNPs between African ethnicities. The low prevalence of our novel variants and most known functional alleles is consistent with the generally high level of diversity in gene loci of African populations. This indicates that profiles of rare variants reflecting interindividual variability might become the most relevant pharmacodiagnostic tools explaining Africans' diversity in drug response.

  4. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris

    International Nuclear Information System (INIS)

    Greater exposure to herbicide increases the likelihood of harmful effects in humans and the environment. Glufosinate, a non-selective herbicide, inhibits glutamine synthetase (GS) and thus blocks ammonium assimilation in plants. In the present study, the aquatic unicellular alga Chlorella vulgaris was chosen to assess the effects of acute glufosinate toxicity. We observed physiological changes during 12-96 h of exposure, and gene transcription during 6-48 h of exposure. Exposure to glufosinate increased malondialdehyde content by up to 2.73 times compared with the control, suggesting that there was some oxidative damage. Electron microscopy also showed that there were some chloroplast abnormalities in response to glufosinate. The activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) also increased markedly in the presence of glufosinate. Maximum activities of SOD, POD, and CAT were 2.90, 2.91, and 2.48 times that of the control, respectively. These elevated activities may help alleviate oxidative damage. A real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL. The results showed that glufosinate reduced the transcript abundances of the three genes after 12 h exposure. The lowest abundances of psaB, psbC and rbcL transcripts in response to glufosinate exposure were 38%, 16% and 43% of those of the control, respectively. Our results demonstrate that glufosinate affects the activities of antioxidant enzymes, disrupts chloroplast ultrastructure, and reduces transcription of photosynthesis-related genes in C. vulgaris

  5. Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

    OpenAIRE

    Manna, Sam; Harman, Ashley; Accari, Jessica; Barth, Christian

    2013-01-01

    Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice,...

  6. Variations in angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian populations of different ethnic origins

    Indian Academy of Sciences (India)

    M A Qadar Pasha; Amjad P Khan; Ratan Kumar; Rekh B Ram; Surinder K Grover; Kaushal K Srivastava; William Selvamurthy; Samir K Brahmachari

    2002-02-01

    The pattern of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the Indian population is poorly known. In order to determine the status of the polymorphism, young unrelated male army recruits were screened. The population had cultural and linguistic differences and lived in an environment that varied significantly from one region to another. Analysis of the genotype, showed higher frequency of the insertion allele in four of the five groups i.e. I allele frequency was significantly higher ( < 0.05) in Dogras, Assamese and Kumaonese. The deletion allele frequency was comparatively higher in the fifth group that belonged to Punjab. A correlation was observed between the genotype and enzyme activity. Involvement of a single D allele in the genotype enhanced the activity up to 37.56 ± 3.13%. The results suggested ethnic heterogeneity with a significant gene cline with higher insertion allele frequency. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.

  7. Gene polymorphisms of fibrinolytic enzymes in coal workers' pneumoconiosis

    Energy Technology Data Exchange (ETDEWEB)

    Chang, L.C.; Tseng, J.C.; Hua, C.C.; Liu, Y.C.; Shieh, W.B.; Wu, H.P. [Chang Gung Memorial Hospital, Chilung (Taiwan)

    2006-03-15

    The authors assessed the gene polymorphisms of missense C/T polymorphism in exon 6 of the urokinase-plasminogen activator (PLAU) gene (PLAU P141L), A/u-repeat in intron 8 of the tissue-type plasminogen activator (PLAT) gene (PLAT TPA25 Alu insertion), and 4G/5G in the promoter region of the serine proteinase inhibitor, clade E (SERPINE) or plasminogen activator inhibitor type 1 gene (SERPINE1 -675 4G/5G) in 153 healthy volunteers and 154 retired coal miners with coal miners' pneumoconiosis (CWP). The CWP subjects included 94 individuals with simple pneumoconiosis and 60 individuals with progressive massive fibrosis presenting with worse pulmonary function. The distributions of genotypes of these three genes did not differ between the control and CWP subjects or between subjects with simple pneumoconiosis and those with progressive massive fibrosis. However, by assessing duration of work and its interaction with genotypes by means of logistic regression, the authors found the missense C/T polymorphism in exon 6 of the PLAU gene to be an effect modifier of the association between work duration and the development of progressive massive fibrosis.

  8. Functional Gene Markers for Fumarate-Adding and Dearomatizing Key Enzymes in Anaerobic Aromatic Hydrocarbon Degradation in Terrestrial Environments.

    Science.gov (United States)

    von Netzer, Frederick; Kuntze, Kevin; Vogt, Carsten; Richnow, Hans H; Boll, Matthias; Lueders, Tillmann

    2016-01-01

    Anaerobic degradation is a key process in many environments either naturally or anthropogenically exposed to petroleum hydrocarbons. Considerable advances into the biochemistry and physiology of selected anaerobic degraders have been achieved over the last decades, especially for the degradation of aromatic hydrocarbons. However, researchers have only recently begun to explore the ecology of complex anaerobic hydrocarbon degrader communities directly in their natural habitats, as well as in complex laboratory systems using tools of molecular biology. These approaches have mainly been facilitated by the establishment of a suite of targeted marker gene assays, allowing for rapid and directed insights into the diversity as well as the identity of intrinsic degrader populations and degradation potentials established at hydrocarbon-impacted sites. These are based on genes encoding either peripheral or central key enzymes in aromatic compound breakdown, such as fumarate-adding benzylsuccinate synthases or dearomatizing aryl-coenzyme A reductases, or on aromatic ring-cleaving hydrolases. Here, we review recent advances in this field, explain the different detection methodologies applied, and discuss how the detection of site-specific catabolic gene markers has improved the understanding of processes at contaminated sites. Functional marker gene-based strategies may be vital for the development of a more elaborate population-based assessment and prediction of aromatic degradation potentials in hydrocarbon-impacted environments. PMID:26959523

  9. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue.

    Science.gov (United States)

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males' subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  10. P25-S Gene Expression Profiling from Formalin-Fixed, Paraffin-Embedded Tissues Using the QuantiGene Branched DNA Assay

    OpenAIRE

    Davies, J.; Maqsodi, B.; Yang, W.; Ma, Y.; Luo, Y.; McMaster, G.

    2007-01-01

    Large numbers of formalin-fixed, paraffin-embedded (FFPE) human tissue specimens with known clinical outcome are archived worldwide, representing a vast resource for biomarker and gene-disease association studies. However, RNA quality in FFPE tissues is compromised by chemical modifications and extensive fragmentation caused by formalin fixation. As a result, quantifying RNA in FFPE samples can be problematic.

  11. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl

    OpenAIRE

    Yadav, Narendra; McDevitt, Raymond E.; Benard, Susan; Falco, S. Carl

    1986-01-01

    Sulfometuron methyl, a sulfonylurea herbicide, blocks growth of bacteria, yeast, and higher plants by inhibition of acetolactate synthase (EC 4.1.3.18), the first common enzyme in the biosynthesis of branched-chain amino acids. Spontaneous mutations that confer increased resistance to the herbicide were obtained in cloned genes for acetolactate synthase from Escherichia coli and Saccharomyces cerevisiae. The DNA sequence of a bacterial mutant gene and a yeast mutant gene revealed single nucle...

  12. Glycolytic enzyme activities and gene expression in Cicer arietinum exposed to water-deficit stress.

    Science.gov (United States)

    Khanna, Suruchi M; Taxak, Pooja Choudhary; Jain, Pradeep K; Saini, Raman; Srinivasan, R

    2014-08-01

    The specific activities and transcript levels of glycolytic enzymes were examined in shoots of chickpea (Cicer arietinum L.) cultivars, Pusa362 (drought tolerant) and SBD377 (drought sensitive), subjected to water-deficit stress 30 days after sowing. Water-deficit stress resulted in decrease in relative water content, chlorophyll content, plant dry weight, and NADP/NADPH ratio and increase in NAD/NADH ratio in both the cultivars. A successive decline in the specific activities of fructose-1,6-bisphosphate aldolase (aldolase), 3-phosphoglycerate kinase (PGK), and NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and elevation in the specific activities of phosphoglycerate mutase (PGM) and triosephosphate isomerase (TPI) was observed in both the cultivars under stress as compared to their respective control plants. The specific activities of hexokinase, fructose-6-phosphate kinase (PFK), and NAD-GAPDH were least affected. The transcript levels of PGK and NADP-GAPDH decreased and that of glucose-6-phosphate isomerase (GPI), PGM, and PFK increased in response to water-deficit stress while water-deficit stress had no effect on the steady-state transcript levels of hexokinase, aldolase, TPI, and NAD-GAPDH. The results suggest that under water-deficit stress, the activities and transcript levels of most of the glycolytic enzymes are not significantly affected, except the increased activity and transcript level of PGM and decreased activities and transcript levels of PGK and NADP-GAPDH. Further, the glycolytic enzymes do not show much variation between the tolerant and sensitive cultivars under water deficit. PMID:25008554

  13. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    Science.gov (United States)

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  14. 187-gene phylogeny of protozoan phylum Amoebozoa reveals a new class (Cutosea) of deep-branching, ultrastructurally unique, enveloped marine Lobosa and clarifies amoeba evolution.

    Science.gov (United States)

    Cavalier-Smith, Thomas; Chao, Ema E; Lewis, Rhodri

    2016-06-01

    Monophyly of protozoan phylum Amoebozoa, and subdivision into subphyla Conosa and Lobosa each with different cytoskeletons, are well established. However early diversification of non-ciliate lobose amoebae (Lobosa) is poorly understood. To clarify it we used recently available transcriptomes to construct a 187-gene amoebozoan tree for 30 species, the most comprehensive yet. This robustly places new genus Atrichosa (formerly lumped with Trichosphaerium) within lobosan class Tubulinea, not Discosea as previously supposed. We identified an earliest diverging lobosan clade comprising marine amoebae armoured by porose scaliform cell-envelopes, here made a novel class Cutosea with two pseudopodially distinct new families. Cutosea comprise Sapocribrum, ATCC PRA-29 misidentified as 'Pessonella', plus from other evidence Squamamoeba. We confirm that Acanthamoeba and ATCC 50982 misidentified as Stereomyxa ramosa are closely related. Discosea have a strongly supported major subclade comprising Thecamoebida plus Glycostylida (suborders Dactylopodina, Stygamoebina; Vannellina) phylogenetically distinct from Centramoebida. Stygamoeba is sister to Dactylopodina. Himatismenida are either sister to Centramoebida or deeper branching. Discosea usually appear holophyletic (rarely paraphyletic). Paramoeba transcriptomes include prokinetoplastid Perkinsela-like endosymbiont sequences. Cunea, misidentified as Mayorella, is closer to Paramoeba than Vexillifera within holophyletic Dactylopodina. Taxon-rich site-heterogeneous rDNA trees confirm cutosan distinctiveness, allow improved conosan taxonomy, and reveal previous dictyostelid tree misrooting. PMID:27001604

  15. Hypoxia-Inducible Regulation of a Prodrug-Activating Enzyme for Tumor-Specific Gene Therapy

    Directory of Open Access Journals (Sweden)

    Toru Shibata

    2002-01-01

    Full Text Available Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

  16. Angiotensin-converting enzyme gene I/D genotype affected metoprolol-induced reduction in 24-hour average heart rate

    Institute of Scientific and Technical Information of China (English)

    LIU Li-wei; LIU Hong; CHEN Guo-liang; HUANG Yi-ling; HAN Lu-lu; XU Zhi-min; JIANG Xiong-jing; LI Yi-shi

    2010-01-01

    Background Genetic factors can influence antihypertensive response to metoprolol, and many studies focused on the relationship between the genotype in β1-adrenergic receptor and blood pressure (BP), little was known about the association of angiotensin-converting enzyme (ACE) genotype with the therapeutic result of metoprolol. The present study aimed to investigate whether the ACE gene insertion (I) / deletion (D) polymorphism Is related to the response to metoprolol in Chinese Han hypertensive patients.Methods Ninety-six patients with essential hypertension received metoprolol (100 mg once daily) as monotherapy for 8 weeks. Twenty-four hours ambulatory blood pressure monitoring and dynamic electrocardiogram were performed before and after treatment. Genotyping analysis was performed using PCR. The association of the ACE gene I/D polymorphism with variations in BP and heart rate (HR) was observed after the 8-week treatment.Results The patients with ACE gene II polymorphism showed greater reduction in 24-hour average HR than those with ID or DD polymorphisms (P=0.045), no effect of this genotype on the reduction in seating HR or in BP was observed. After adjusting for age, gender, body mass index, BP and HR at baseline, the ACE gene I/D polymorphism was still an independent predictor for variations in 24-hour average HR.Conclusions The II polymorphism in ACE gene could be a candidate predictor for greater reduction in 24-hour average HR in Chinese Han hypertensive patients treated by metoprolol. Greater benefits would be obtained by patients with II polymorphism from the treatment with metoprolol. Larger studies are warranted to validate this finding.

  17. Effect of angiotensin converting enzyme gene I/D polymorphism in patients with metabolic syndrome in North Indian population

    Institute of Scientific and Technical Information of China (English)

    Gaurav Mittal; Vibhanshu Gupta; Shahzad F Haque; Anwer S Khan

    2011-01-01

    Background Numerous studies have investigated the effect of angiotensin converting enzyme (ACE) gene I/D polymorphism and various cardiovascular risk factors in different populations with varied results. Currently, the association of ACE gene polymorphism with metabolic syndrome has not been studied in North Indians. While studies assessing the effect with polymorphism on each of the components of metabolic syndrome separately are present, data regarding the metabolic syndrome per se are sparse. The present study evaluated the effect of ACE gene I/D polymorphism in patients with metabolic syndrome in North Indian population at a tertiary care centre.Methods Fifty subjects, with thirty cases of metabolic syndrome (NCEP/ATP Ⅲ guidelines, 2004) and twenty age and gender matched healthy controls were chosen. Detailed history was reviewed and clinical examination of the subjects was carried out. Relevant investigations including blood glucose (fasting and post prandial), blood urea, serum creatinine and serum lipids were done. DNA of cases and controls was analysed for I/D polymorphism using polymerase chain reaction.Results D/D genotype was more frequent in patients with metabolic syndrome as compared with healthy controls (P<0.05). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) was significantly higher in the D/D genotype than I/D and I/I genotypes (P <0.05). Our study also showed positive association between obesity, fasting blood glucose and ACE gene polymorphism while no association was found with triglycerides and high density lipoprotein cholesterol.The I/I group was significantly associated with waist circumference and fasting blood glucose (P <0.05).Conclusion Our study clearly showed that metabolic syndrome was associated with ACE gene polymorphism.However due to less number of subjects in the study further studies are needed to corroborate our results.

  18. Transcriptome analysis of the Tan sheep testes: Differential expression of antioxidant enzyme-related genes and proteins in response to dietary vitamin E supplementation.

    Science.gov (United States)

    Xu, Chenchen; Zuo, Zhaoyun; Liu, Kun; Jia, Huina; Zhang, Yuwei; Luo, Hailing

    2016-03-15

    Gene-chip technology was employed to study the effect of dietary vitamin E on gene expression in sheep testes based on our previous research. Thirty-five male Tan sheep (20-30 days after weaning) with similar body weight were randomly allocated into five groups and supplemented 0, 20, 100, 200 and 2,000 IU sheep(-1)day(-1) vitamin E (treatments denoted as E0, E20, E100, E200, and E2000, respectively) for 120 days. At the end of the study the sheep were slaughtered and the testis samples were immediately collected and stored in liquid nitrogen. Differences in gene expression between different treated groups were identified. Based on GO enrichment analysis and the KEGG database to evaluate the gene expression data we found that vitamin E might affect genes in the testes by modulating the oxidation level, by affecting the expression of various receptors and transcription factors in biological pathways, and by regulating the expression of metabolism-associated genes. The effect of vitamin E supplementation on the expression of oxidative enzyme-related genes was detected by quantitative real-time PCR (qRT-PCR) and Western blot. The results show that dietary vitamin E, at various doses, can significantly increase (P<0.05) the mRNA and protein expression of Glutathione peroxidase 3 and Glutathione S-transferase alpha 1. In addition, the results of qRT-PCR of the antioxidant enzyme genes were consistent with those obtained using the gene chip microarray analysis. In summary, the dietary vitamin E treatment altered the expression of a number of genes in sheep testes. The increase in the mRNA and protein levels of antioxidant enzyme genes, coupled with the elevation in the activity of the antioxidant enzymes were primarily responsible for the improved reproductive performance promoted by dietary vitamin E. PMID:26723511

  19. Gene expression analysis and enzyme assay reveal a potential role of the carboxylesterase gene CpCE-1 from Cydia pomonella in detoxification of insecticides.

    Science.gov (United States)

    Yang, Xue-Qing

    2016-05-01

    Carboxylesterases (CarEs) are responsible for metabolism of xenobiotics including insecticides in insects. Understanding the expression patterns of a such detoxifying gene and effect of insecticides on its enzyme activity are important to clarify the function of this gene relevant to insecticides-detoxifying process, but little information is available in the codling moth Cydia pomonella (L.). In this study, we investigated the expression profiles of CarE gene CpCE-1 at different developmental stages and in different tissues of C. pomonella, as well as the larvae exposed to chlorpyrifos-ethyl and lambda-cyhalothrin by using absolute real-time quantitative PCR (absolute RT-qPCR). Results indicated that CpCE-1 expression was significantly altered during C. pomonella development stages, and this expression differed between sexes, with a higher transcript in females than males. Meanwhile, CpCE-1 is overexpressed in cuticle, midgut and head than silk gland, fat body and Malpighian tubules. Exposure of third instar larvae to a non-lethal dosage of chlorpyrifos-ethyl and lambda-cyhalothrin resulted in induction of CpCE-1 transcript. The total carboxylesterase enzyme activity was inhibited by chlorpyrifos-ethyl in vivo; in contrast, the activity of Escherichia coli produced recombinant CpCE-1 was significantly inhibited by both lambda-cyhalothrin and chlorpyrifos-ethyl in vitro. These results suggested that CpCE-1 in C. pomonella is potentially involved in the development and in detoxification of chlorpyrifos-ethyl and lambda-cyhalothrin. PMID:27017882

  20. Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme.

    Science.gov (United States)

    Zakhartsev, Maxim; Lucassen, Magnus; Kulishova, Liliya; Deigweiher, Katrin; Smirnova, Yuliya A; Zinov'eva, Rina D; Mugue, Nikolay; Baklushinskaya, Irina; Pörtner, Hans O; Ozernyuk, Nikolay D

    2007-03-01

    Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 degrees C) and high (18 degrees C) temperatures leads to differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5'-UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3'-UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH-A(alpha) and LDH-A(beta) subunits. It is expected that the beta-subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH-A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm-acclimated fish the relative expression between alpha and beta isoforms of the LDH-A mRNA is around 5 : 1, whereas in cold-acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH-A is more homogeneous in terms of alpha/beta-subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine-tuning of the properties of skeletal

  1. Insertion/Insertion Genotype of Angiotensin I-Converting-Enzyme Gene Predicts Risk of Myocardial Infarction in North East India.

    Science.gov (United States)

    Baruah, Sukanya; Chaliha, Mriganka S; Borah, Prasanta K; Rajkakati, Rashmi; Borua, Prodeep K; Mahanta, Jagadish

    2016-04-01

    Myocardial infarction (MI) is common in India and the disease occurs at a relatively younger age. We wanted to look for association of Angiotensin I-converting enzyme (ACE) gene with MI in North East India. We also wanted to examine possible environmental interaction of ACE gene with established cardiovascular risk factors in causation of MI. In the study carried out in Assam Medical College, 200 consecutive confirmed cases of MI were recruited. Equal numbers of age- and sex-matched control subjects from hospital workers and patients attending the hospital for diseases unrelated to cardiovascular disease were enrolled. Structured questionnaires were used to note demographic and clinical factors. Cardiovascular risk factors were determined from history, physical examination and biochemical investigations. ACE insertion/deletion (I/D) polymorphism was determined by PCR method. Interaction of ACE gene with other risk factors was noted. The study identified ACE II genotype (odds ratio = 3.02; 95% CI 1.40-6.51), smoking, hypertension, diabetes and serum triglyceride > 150 mg/dl as independent risk factors for MI. ACE II genotype showed greater risk in non-smokers, non-hypertensives, non-diabetics and in subjects with LDL-C < 130 mg/dl. Low HDL cholesterol enhanced the genetic risk. Subjects with ACE II genotype have an independent risk of developing MI, specially in low cardiovascular risk subjects. PMID:26687160

  2. Distribution of genetic polymorphisms of genes encoding drug metabolizing enzymes & drug transporters - a review with Indian perspective

    Directory of Open Access Journals (Sweden)

    Gurusamy Umamaheswaran

    2014-01-01

    Full Text Available Phase I and II drug metabolizing enzymes (DME and drug transporters are involved in the absorption, distribution, metabolism as well as elimination of many therapeutic agents, toxins and various pollutants. Presence of genetic polymorphisms in genes encoding these proteins has been associated with marked inter-individual variability in their activity that could result in variation in drug response, toxicity as well as in disease predisposition. The emergent field pharmacogenetics and pharmacogenomics (PGx is a promising discipline, as it predicts disease risk, selection of proper medication with regard to response and toxicity, and appropriate drug dosage guidance based on an individual′s genetic make-up. Consequently, genetic variations are essential to understand the ethnic differences in disease occurrence, development, prognosis, therapeutic response and toxicity. For that reason, it is necessary to establish the normative frequency of these genes in a particular population before unraveling the genotype-phenotype associations. Although a fair amount of allele frequency data are available in Indian populations, the existing pharmacogenetic data have not been compiled into a database. This review was intended to compile the normative frequency distribution of the variants of genes encoding DMEs (CYP450s, TPMT, GSTs, COMT, SULT1A1, NAT2 and UGTs and transporter proteins (MDR1, OCT1 and SLCO1B1 with Indian perspective.

  3. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

    Science.gov (United States)

    Kan, O; Griffiths, L; Baban, D; Iqball, S; Uden, M; Spearman, H; Slingsby, J; Price, T; Esapa, M; Kingsman, S; Kingsman, A; Slade, A; Naylor, S

    2001-07-01

    Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. PMID:11498768

  4. Relationship between angiotensin-converting enzyme gene polymorphism and cardio-brain complications in patients with NIDDM (type 2 diabetes mellitus)

    International Nuclear Information System (INIS)

    Objective: To investigate the relationship between angiotensin-converting enzyme gene polymorphism and cardio-brain complications in patients with NIDDM. Methods: The angiotensin-converting enzyme (ACE) gene insertion/deletion polymorphism in 174 patients with NIDDM and 62 controls were examined with PCR. Results: ACE gene I/D polymorphism was closely related to coronary heart disease (angina, cardiac infarction) and cerebral infarction in diabetic patients but not with hypertension. Plasma renin activity and plasma angiotensin II levels in complicated diabetic patients with ACE D/D gene were significantly higher than those in the controls (p < 0.01). Their aldosterone and endothelin contents were not significantly different. Conclusion: Examination of ACE gene I/D polymorphism was useful for the primary prevention of cardio-brain complications in diabetic patients and helpful in the early diagnosis and therapy of coronary heart disease and cerebral infarction

  5. The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (mshA)

    OpenAIRE

    Newton, Gerald L.; Koledin, Teresa; Gorovitz, Batia; Rawat, Mamta; Fahey, Robert C.; Av-Gay, Yossef

    2003-01-01

    Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1d-myo-inosityl 2-acetamido-2-deoxy-α-d-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.

  6. Restriction enzyme-free construction of random gene mutagenesis libraries in E. coli

    OpenAIRE

    Pai, Jen C.; Entzminger, Kevin C.; Maynard, Jennifer A.

    2011-01-01

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in E. coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restri...

  7. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform

    OpenAIRE

    Kazuki Nakamura; Ryo Iizuka; Shinro Nishi; Takao Yoshida; Yuji Hatada; Yoshihiro Takaki; Ayaka Iguchi; Dong Hyun Yoon; Tetsushi Sekiguchi; Shuichi Shoji; Takashi Funatsu

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental ...

  8. Differential display identifies overexpression of the USP36 gene, encoding a deubiquitinating enzyme, in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Jianduan Li, Lisa M. Olson, Zhengyan Zhang, Lina Li, Miri Bidder, Loan Nguyen, John Pfeifer, Janet S. Rader

    2008-01-01

    Full Text Available Objectives. To find potential diagnostic markers or therapeutic targets, we used differential display technique to identify genes that are over or under expressed in human ovarian cancer. Methods. Genes were initially identified by differential display between two human ovarian surface epithelium cultures and two ovarian cancer cell lines, A2780 and Caov-3. Genes were validated by relative quantitative RT-PCR and RNA in situ hybridization. Results. Twenty-eight non-redundant sequences were expressed differentially in the normal ovarian epithelium and ovarian cancer cell lines. Seven of the 28 sequences showed differential expression between normal ovary and ovarian cancer tissue by RT-PCR. USP36 was over-expressed in ovarian cancer cell lines and tissues by RT-PCR and RNA in situ hybridization. Northern blot analysis and RT-PCR revealed two transcripts for USP36 in ovarian tissue. The major transcript was more specific for ovarian cancer and was detected by RT-PCR in 9/9 ovarian cancer tissues, 3/3 cancerous ascites, 5/14 (36% sera from patients with ovarian cancer, and 0/7 sera from women without ovarian cancer. Conclusion. USP36 is overexpressed in ovarian cancer compared to normal ovary and its transcripts were identified in ascites and serum of ovarian cancer patients.

  9. Psychiatric and cognitive symptoms in Huntington's disease are modified by polymorphisms in catecholamine regulating enzyme genes.

    Science.gov (United States)

    Vinther-Jensen, T; Nielsen, T T; Budtz-Jørgensen, E; Larsen, I U; Hansen, M M; Hasholt, L; Hjermind, L E; Nielsen, J E; Nørremølle, A

    2016-03-01

    Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder characterized by motor, psychiatric, and cognitive manifestations. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene but the exact pathogenesis remains unknown. Dopamine imbalance has previously been shown in HD, and furthermore dopamine is thought to be implicated in cognition, behavioral and motor disturbances. A substantiated inverse correlation between motor onset and the elongated CAG repeat in the HTT has been established. This relation does not account for the full variability of the motor onset, and efforts have been put into finding genetic modifiers of motor onset, however, mostly with unsuccessful outcome. In this study, we took an alternative approach focusing on symptom complexes and searched for modifiers of cognitive impairment and psychiatric symptoms in a well-described cohort of Danish HD gene-expansion carriers. We show that cognitive impairment and psychiatric symptoms in HD are modified by polymorphisms in the monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT) genes and by the 4p16.3 B haplotype. These results support the theory of dopamine imbalance in HD, and point toward more personalized treatment modalities of HD in the future. PMID:26081309

  10. The angiotensin-converting enzyme gene insertion–deletion polymorphism in a white British patient cohort with obstetric cholestasis

    Science.gov (United States)

    Müllenbach, Roman; Tetlow, Natasha; Bennett, Amanda; Pipkin, Fiona Broughton; Morgan, Linda; Williamson, Catherine

    2009-01-01

    The DD genotype of the angiotensin-converting enzyme (ACE) gene is over-represented in Finnish patients with obstetric cholestasis (OC). The purpose of this study was to establish whether this genotype is associated with cholestasis in UK cases. In a retrospective case-control study, we determined the ACE insertion/deletion frequencies in 166 British cases and 100 control women by polymerase chain reaction analysis. No significant difference in allele frequencies was found between these groups, but allele frequencies differed significantly between Finnish and UK OC cases (P = 0.0005). The prevalence of the DD genotype is lower in UK cases than in controls (χ2 [1 d.f.] = 4.32, P = 0.05) and the odds ratio for OC associated with the DD genotypeis 0.54, 95% confidence interval 0.30–0.97. In contrast to Finnish OC cases, the DD genotype of the ACE is not increased in UK cases.

  11. A plant gene for photolyase: an enzyme catalyzing the repair of UV-light-induced DNA damage

    International Nuclear Information System (INIS)

    Photolyases are thought to be critical components of the defense of plants against damage to DNA by solar ultraviolet light, but nothing is known about their molecular or enzymatic nature. The molecular cloning of a photolyase from mustard (Sinapis alba) described here is intended to increase the knowledge about this important repair mechanism in plant species at a molecular level. The gene encodes a polypeptide of 501 amino acids with a predicted molecular mass of 57 kDa. There is a strong sequence similarity to bacterial and yeast photolyases, with a close relationship to enzymes with a deazaflavin chromophor. The plant photolyase is shown to be functional in Escherichia coli which also indicates conservation of photolyases during evolution. It is demonstrated that photolyase expression in plants is light induced, thus providing good evidence for the adaptation of plants to their environment in order to diminish the harmful effects of sunlight. (author)

  12. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments.

    Science.gov (United States)

    Lanfranconi, Mariana P; Alvarez, Adrián F; Alvarez, Héctor M

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest. PMID:26228353

  13. I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.

    Science.gov (United States)

    Ouedraogo, Jean Paul; Arentshorst, Mark; Nikolaev, Igor; Barends, Sharief; Ram, Arthur F J

    2016-03-20

    We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site. PMID:26860210

  14. Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme

    OpenAIRE

    Atanasiu, C.; Su, T J; Sturrock, S. S.; Dryden, D T F

    2002-01-01

    The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over similar to24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional r...

  15. Interactions between urinary 4-tert-octylphenol levels and metabolism enzyme gene variants on idiopathic male infertility.

    Directory of Open Access Journals (Sweden)

    Yufeng Qin

    Full Text Available Octylphenol (OP and Trichlorophenol (TCP act as endocrine disruptors and have effects on male reproductive function. We studied the interactions between 4-tert-Octylphenol (4-t-OP, 4-n- Octylphenol (4-n-OP, 2,3,4-Trichlorophenol (2,3,4-TCP, 2,4,5-Trichlorophenol (2,4,5-TCP urinary exposure levels and polymorphisms in selected xenobiotic metabolism enzyme genes among 589 idiopathic male infertile patients and 396 controls in a Han-Chinese population. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS was used to measure alkylphenols and chlorophenols in urine. Polymorphisms were genotyped using the SNPstream platform and the Taqman method. Among four phenols that were detected, we found that only exposure to 4-t-OP increased the risk of male infertility (P(trend = 1.70×10(-7. The strongest interaction was between 4-t-OP and rs4918758 in CYP2C9 (P(inter = 6.05×10(-7. It presented a significant monotonic increase in risk estimates for male infertility with increasing 4-t-OP exposure levels among men with TC/CC genotype (low level compared with non-exposed, odds ratio (OR = 2.26, 95% confidence intervals (CI = 1.06, 4.83; high level compared with non-exposed, OR = 9.22, 95% CI = 2.78, 30.59, but no associations observed among men with TT genotype. We also found interactions between 4-t-OP and rs4986894 in CYP2C19, and between rs1048943 in CYP1A1, on male infertile risk (P(inter = 8.09×10(-7, P(inter = 3.73×10(-4, respectively.We observed notable interactions between 4-t-OP exposure and metabolism enzyme gene polymorphisms on idiopathic infertility in Han-Chinese men.

  16. Association of Angiotensin-Converting Enzyme (ACE Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients.

    Directory of Open Access Journals (Sweden)

    Laila Rashed

    Full Text Available Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D polymorphism of the ACE gene was reported be associated with the development of vitiligo.Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL-6 and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide in vitiligo patients.This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively.The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026. However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115. There were statistically significant higher VIDA score (P = 0.007, and serum IL-6 (P < 0.001 in patients with the DD genotype when compared to other genotypes. Serum nitrite in patients with the DD genotype was significantly higher (P = 0.007 when compared to patients with II genotype. Serum levels of ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls.As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases.

  17. Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton (Gossypium hirsuturm L.)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene (GhDWF1) involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton (Gossypium hirsutum L.) cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5'-untranslated region, an ORF of 1692 bp, and a 120 bp 3'-untranslated region.The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea,tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD-dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA (day post anthesis) ovule (with fibers) while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules (with fibers) of wildtype (Xuzhou 142) than in ovules of fuzzless-lintless mutant at the same developmental stages (0 and 4 DPA). The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

  18. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

    Science.gov (United States)

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes. PMID:26915788

  19. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform

    Science.gov (United States)

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes. PMID:26915788

  20. Sensitivity of human cells defective of DNA repair enzyme genes to radiation and medical agents

    International Nuclear Information System (INIS)

    Homologous recombination and non-homologous end-joining (NHEJ) are the known mechanisms of repairing DNA with double strand break (DSB) yielded by radiation and cell-cycle independent NHEJ is thought to be major in higher eukaryotes. Recognized now are 7 proteins like Artemis and XRCC4 concerned in NHEJ, but little is known for functions of those proteins in human cells. Authors have developed a method to destroy the specific gene by targeting for the study of the responses to DNA damage in human Artemis-/- and XRCC4-/- cells, which is described in this paper. Parent cell strain is a human colorectal cancer-derived epithelial HCT116, and those defective cells are obtained by targeting with puromycin and neomycin resistant vectors. Their sensitivities to X-ray (0.6 Gy/min), to etoposide and to other anti-cancers are examined by survival vs dose; and the relationship between the sensitivity to damaged DNA stress and DSB production is tested by chromosome aberration frequency and by γH22AX focus formation (a measure of DSB yield) after X-exposure. Results obtained show the important role of Artemis and XRCC4 also in human cell DSB response. With reactive oxygen species (H2O2), those cells are further used in similar experiments to above, which suggesting a different mechanism of DSB induction by H2O2 from that by radiation. Other genes than the two here in NHEJ will be investigated in future with gene targeting techniques for systematic, molecular elucidation of radiation effects in humans. (K.T.)

  1. The amylose-free potato mutant as a model plant to study gene expression and gene silencing.

    NARCIS (Netherlands)

    Flipse, E.

    1995-01-01

    In this thesis, gene-expression and gene silencing were examined for Granule Bound Starch Synthase (GBSS) which catalyses the formation of amylose and Branching Enzyme (BE) which catalyses the formation of amylopectin. The (GBSS) deficient, with iodine, red staining amylose-free (amf) potato mutant

  2. Future cereal starch bioengineering: Cereal ancestors encounter gene technology and designer enzymes

    DEFF Research Database (Denmark)

    Blennow, Andreas; Jensen, Susanne Langgård; Shaik, Shahnoor Sultana;

    2013-01-01

    genomes and the exploding advancement in whole genome sequencing now paves the road for identifying new genes to be exploited to generate a multitude of completely new starch functionalities directly in the cereal grain converting cereal crops to production plants. Newly released genome data from cereal...... functionalize or stabilize the starch polymers. Importantly, such products can be multifunctional in the sense of combined food/material or food/pharma purposes e.g. edible plastics, shape memory materials and cycloamylose carriers and stabilizers for diverse bioactives....

  3. Auxin levels and MAX1–4 and TAC1 gene expression in different growth habits of peach

    Science.gov (United States)

    Branch orientation and distribution establish a fruit tree’s canopy architecture; tree architecture is a core factor for orchard management including novel mechanized technologies. Endogenous hormone concentrations and gene expression of a key branching enzyme in herbaceous species, MAX4, were dete...

  4. Melatonin biosynthesizing enzyme genes and clock genes in ovary and whole brain of zebrafish (Danio rerio): Differential expression and a possible interplay.

    Science.gov (United States)

    Khan, Zeeshan Ahmad; Yumnamcha, Thangal; Rajiv, Chongtham; Devi, Haobijam Sanjita; Mondal, Gopinath; Devi, Sh Dharmajyoti; Bharali, Rupjyoti; Chattoraj, Asamanja

    2016-07-01

    The present study on zebrafish (Danio rerio) is the first attempt to demonstrate the circadian mRNA expression of melatonin biosynthesizing enzyme genes (Tph1a, Aanat1, Aanat2 and Hiomt) and clock associated genes (Bmal1a, Clock1a, Per1b, Per2 and Cry2a) in the ovary with a comparison to whole brain in normal (LD=12h L:12h D) and altered photic conditions (continuous dark, DD; continuous light, LL). Moreover, the present study also confirmed the ability of zebrafish ovary to biosynthesize melatonin both in vivo and in vitro with a significant difference at day and night. qRT-PCR analysis of genes revealed a dark acrophase of Aanat2 in both organs while Tph1 is in whole brain in LD condition. On the contrary, Bmal1a and Clock1a giving their peak in light, thereby showing a negative correlation with Tph1a and Aanat2. In LD-ovary, the acrophase of Tph1a, Bmal1a and Clock1a is in light and thus display a positive correlation. This trend of relationship in respect to Tph1a is not changing in altered photic conditions in both organs (except in DD-ovary). On the other hand this association for Aanat2 is varying in ovary under altered photic conditions but only in DD-whole brain. Both in LD and LL the expression of Aanat2 in brain presenting an opposite acrophase with both Bmal1a and Clock1a of ovary and consequently displaying a strong negative correlation among them. Interestingly, all ovarian clock associated genes become totally arrhythmic in DD, representing a loss of correlation between the melatonin synthesizing genes in brain and clock associated genes in ovary. The result is also indicating the formation of two heterodimers namely Clock1a:Bmal1a and Per2:Cry2a in the functioning of clock genes in both organs, irrespective of photic conditions, as they are exhibiting a strong significant positive correlation. Collectively, our data suggest that ovary of zebrafish is working as peripheral oscillator having its own melatonin biosynthesizing machinery and signifying a

  5. Jasmonic acid Modulates the Physio-Biochemical Attributes, Antioxidant Enzyme Activity and Gene Expression in Glycine max under Nickel Toxicity

    Directory of Open Access Journals (Sweden)

    Geetika eSirhindi

    2016-05-01

    Full Text Available In present study, we evaluated the effects of Jasmonic acid (JA on physio-biochemical attributes, antioxidant enzyme activity and gene expression in soybean (Glycine max L. plants subjected to nickel (Ni stress. Ni stress decreases the shoot and root length and chlorophyll content by 37.23%, 38.31% and 39.21% respectively over the control. However, application of JA was found to improve the chlorophyll content and growth of Ni-stressed seedlings in terms of root and shoot length. Plants supplemented with Jasmonate restores the chlorophyll fluorescence, which was disturbed by Ni stress. The present study demonstrated increase in proline, glycinebetaine, total protein and total soluble sugar (TSS by 33.09%, 51.26%, 22.58% and 49.15% respectively under Ni toxicity as compared to control. Supplementation of JA to Ni stressed plants further enhanced the above parameters. Ni stress increases hydrogen peroxide (H2O2 by 68.49%, lipid peroxidation (MDA by 50.57% and NADPH oxidase by 50.92% over the control. Supplementation of JA minimizes the accumulation of H2O2, MDA and NADPH oxidase, which helps in stabilization of biomolecules. The activities of superoxide dismutase (SOD, peroxidase (POD, catalase (CAT and ascorbate peroxidase (APX increases by 40.04%, 28.22%, 48.53% and 56.79% respectively over the control in Ni treated seedlings and further enhancement in the antioxidant activity was observed by the application of JA. Ni treated soybean seedlings showed increase in expression of Fe-SOD by 77.62%, CAT by 15.25%, POD by 58.33% and APX by 80.58% over the control. Nevertheless, application of JA further enhanced the expression of the above genes in the present study. Our results signified that Ni stress caused negative impacts on soybean seedlings, but, co-application of JA facilitate the seedlings to combat the detrimental effects of Ni through enhanced osmolytes and osmoprotectants, antioxidant enzyme activity and gene expression.

  6. Jasmonic Acid Modulates the Physio-Biochemical Attributes, Antioxidant Enzyme Activity, and Gene Expression in Glycine max under Nickel Toxicity

    Science.gov (United States)

    Sirhindi, Geetika; Mir, Mudaser Ahmad; Abd-Allah, Elsayed Fathi; Ahmad, Parvaiz; Gucel, Salih

    2016-01-01

    In present study, we evaluated the effects of Jasmonic acid (JA) on physio-biochemical attributes, antioxidant enzyme activity, and gene expression in soybean (Glycine max L.) plants subjected to nickel (Ni) stress. Ni stress decreases the shoot and root length and chlorophyll content by 37.23, 38.31, and 39.21%, respectively, over the control. However, application of JA was found to improve the chlorophyll content and length of shoot and root of Ni-fed seedlings. Plants supplemented with JA restores the chlorophyll fluorescence, which was disturbed by Ni stress. The present study demonstrated increase in proline, glycinebetaine, total protein, and total soluble sugar (TSS) by 33.09, 51.26, 22.58, and 49.15%, respectively, under Ni toxicity over the control. Addition of JA to Ni stressed plants further enhanced the above parameters. Ni stress increases hydrogen peroxide (H2O2) by 68.49%, lipid peroxidation (MDA) by 50.57% and NADPH oxidase by 50.92% over the control. Supplementation of JA minimizes the accumulation of H2O2, MDA, and NADPH oxidase, which helps in stabilization of biomolecules. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) increases by 40.04, 28.22, 48.53, and 56.79%, respectively, over the control in Ni treated seedlings and further enhancement in the antioxidant activity was observed by the application of JA. Ni treated soybean seedlings showed increase in expression of Fe-SOD by 77.62, CAT by 15.25, POD by 58.33, and APX by 80.58% over the control. Nevertheless, application of JA further enhanced the expression of the above genes in the present study. Our results signified that Ni stress caused negative impacts on soybean seedlings, but, co-application of JA facilitate the seedlings to combat the detrimental effects of Ni through enhanced osmolytes, activity of antioxidant enzymes and gene expression. PMID:27242811

  7. Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2.

    Science.gov (United States)

    Chen, Huizhong; Hopper, Sherryll L; Li, Xin-Liang; Ljungdahl, Lars G; Cerniglia, Carl E

    2006-11-01

    An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 beta-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA). PMID:17019643

  8. Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement

    International Nuclear Information System (INIS)

    The functional human adenine phosphoribosyltransferase (APRT) gene is <2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other housekeeping genes, lacks TATA and CCAAT boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function

  9. Effect of angiotensin I-converting enzyme and α-actinin-3 gene polymorphisms on sport performance.

    Science.gov (United States)

    Gunel, Tuba; Gumusoglu, Ece; Hosseini, Mohammad Kazem; Yilmazyildirim, Eda; Dolekcap, Ismail; Aydinli, Kilic

    2014-04-01

    Genetic polymorphism is considered to be associated with human physical performance. The angiotensin I-converting enzyme insertion/deletion (ACE I/D) and the α-actinin-3 gene (ACTN3) R577X polymorphisms have been widely investigated for such associations, and functional ACE I/D and ACTN3 R577X polymorphisms have been associated with sprinter performance. The aim of this study was to determine the effect of these polymorphisms on sport performance among 37 elite athletes and 37 healthy controls. The ACE II genotype was identified in 32.43% of the control group and 8.11% of elite athletes, the DD genotype in 37.84% of the control group and 51.35% of the elite athletes, and the ID genotype in 29.73% of the control group and 40.54% of the elite athletes. With regard to the ACTN3 gene, the XX genotype, which confers an advantage for endurance activities, was identified in 10.81% of the control group and 35.14% of the elite athletes. The XX genotype was observed more frequently than the RR genotype (advantageous for sprinting), which was identified in 2.70% of the control group and 10.81% of elite athletes. The RX genotype (observed in 86.48% of the control group and in 54.05% of the elite athletes) was the most common genotype of the individuals in the present study. The study showed that ACTN3 and ACE gene polymorphisms have an effect on muscle power; however, larger studies are required. PMID:24566537

  10. Polymorphisms of angiotensin-converting enzyme 2 gene confer a risk to lone atrial fibrillation in Chinese male patients

    Institute of Scientific and Technical Information of China (English)

    WANG Shu-xia; TAO Tao; FU Zhi-qing; XIE Xiang-zhu; WANG Hao; WANG Yu-tang

    2013-01-01

    Background Growing epidemiologic evidence has indicated that genetics can predispose individuals to the occurrence of lone atrial fibrillation (AF).The angiotensin-converting enzyme 2 (ACE2) gene has been established to be associated with hypertension and left ventricular hypertrophy.The objective of our study was to investigate the association of ACE2 gene polymorphisms with lone AF.Methods A total of 265 consecutive lone AF patients and 289 healthy controls were successfully investigated.The polymorphisms rs2106809 and rs2285666 were genotyped by polymerase chain reaction (PCR) and direct sequencing.A Logistic regression model was used to determine the odds ratio (OR) and 95% confidence intervals (CI) of variations of ACE2 for lone AF.Results The T allele of rs2106809 conferred an increased risk for lone AF (OR 1.24,95% CI 1.01-1.52,P=0.03) in males after adjustment for conventional risk factors.SNP at rs2285666 in males was not significantly different between AF patients and controls.No association was found between the two polymorphisms in the female population with lone AF.After (36.3±4.5) months of follow-up,the end point data were obtained:death (cardiac and noncardiac),ischemic stroke,and heart failure.In the male subgroup,the associations between rs2106809 T male carriers and combined end points including ischemic stroke,heart failure,and death in our study were of significance (OR 3.6,95% CI 1.0-13.1,P=0.04).Conclusions The results indicate that polymorphism at ACE2 gene is associated with male lone AF in a Chinese Han population.Lone AF males who carry the rs2106809 T allele are associated with adverse cardiac events.

  11. Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti

    OpenAIRE

    Han, Q; Calvo, E.; Marinotti, O.; Fang, J; Rizzi, M; James, A A; Li, J.

    2003-01-01

    Kynurenine 3-monooxygenase (KMO) catalyses the hydroxylation of kynurenine to 3-hydroxykynurenine. KMO has a key role in tryptophan catabolism and synthesis of ommochrome pigments in mosquitoes. The gene encoding this enzyme in the yellow fever mosquito, Aedes aegypti, is called kynurenine hydroxylase (kh) and a mutant allele that produces white eyes has been designated khw. A number of cDNA clones representative of wild-type and mutant genes were isolated. Sequence analyses of the wild-type ...

  12. The Gene CBO0515 from Clostridium botulinum Strain Hall A Encodes the Rare Enzyme N5-(Carboxyethyl) Ornithine Synthase, EC 1.5.1.24▿

    Science.gov (United States)

    Thompson, John; Hill, Karen K.; Smith, Theresa J.; Pikis, Andreas

    2010-01-01

    Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N5-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum. PMID:19933367

  13. Two novel soluble trehalase genes cloned from Harmonia axyridis and regulation of the enzyme in a rapid changing temperature.

    Science.gov (United States)

    Shi, Zuokun; Liu, Xiaojun; Xu, Qingye; Qin, Zi; Wang, Su; Zhang, Fan; Wang, Shigui; Tang, Bin

    2016-08-01

    In previous studies, we have cloned two soluble trehalase genes (HaTreh1-1 and HaTreh1-2) from the harlequin ladybird Harmonia axyridis. Here, we obtained the other two novel genes (HaTreh1-3 and HaTreh1-4) by transcriptome sequencing and rapid amplification of cDNA ends. Generally, anabolism enhancement and catabolism inhibition together contribute to accumulation of trehalose, and trehalase is the key enzyme to start the catabolism of trehalose. To characterize the metabolism of trehalose in H. axyridis and how these trehalase genes are regulated under cold stress conditions, a comparison of trehalose content and trehalase levels in two different rapidly changing temperature environments was carried out to explore the regulation of these genes. We found that an accumulation of trehalose could be observed at 5°C, 0°C and -5°C and trehalase was suppressed in these temperature points during a gradually cooling environment. Then, in a gradually warming environment, trehalose levels increased slightly from -5°C to 15°C and then decreased at 25°C; however, no significant negative association was observed between trehalase and trehalose. Additionally, we found that glycogen could be converted into trehalose to help the individual resist the low temperature. Analysis of the expression of soluble trehalase showed that HaTreh1-1, HaTreh1-2, HaTreh1-3 and HaTreh1-4 were involved in trehalose metabolism; but the gene HaTreh1-4 plays the most important role in the cooling process, and HaTreh1-2 and HaTreh1-4 play the most important role in the warming process. Finally, we found that 5°C might be a temperature signal for H. axyridis; prior to this temperature, individuals must make enough physical preparations to resist cold stress during the winter. PMID:26969108

  14. The prevalence of aminoglycoside-modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Farzam Vaziri

    2011-01-01

    Full Text Available INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6'-II (36% was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2"-I, aph (3'-VI, and aac (6'-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

  15. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.;

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

  16. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

    Science.gov (United States)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

  17. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T;

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...

  18. Pharmacokinetics and pharmacodynamics of phase II drug metabolizing/antioxidant enzymes gene response by anticancer agent sulforaphane in rat lymphocytes.

    Science.gov (United States)

    Wang, Hu; Khor, Tin Oo; Yang, Qian; Huang, Ying; Wu, Tien-Yuan; Saw, Constance Lay-Lay; Lin, Wen; Androulakis, Ioannis P; Kong, Ah-Ng Tony

    2012-10-01

    This study assesses the pharmacokinetics (PK) and pharmacodynamics (PD) of Nrf2-mediated increased expression of phase II drug metabolizing enzymes (DME) and antioxidant enzymes which represents an important component of cancer chemoprevention in rat lymphocytes following intravenous (iv) administration of an anticancer phytochemical sulforaphane (SFN). SFN was administered intravenously to four groups of male Sprague-Dawley JVC rats each group comprising four animals. Blood samples were drawn at selected time points. Plasma were obtained from half of each of the blood samples and analyzed using a validated LC-MS/MS method. Lymphocytes were collected from the remaining blood samples using Ficoll-Paque Plus centrifuge medium. Lymphocyte RNAs were extracted and converted to cDNA, quantitative real-time PCR analyses were performed, and fold changes were calculated against those at time zero for the relative expression of Nrf2-target genes of phase II DME/antioxidant enzymes. PK-PD modeling was conducted based on Jusko's indirect response model (IDR) using GastroPlus and bootstrap method. SFN plasma concentration declined biexponentially and the pharmacokinetic parameters were generated. Rat lymphocyte mRNA expression levels showed no change for GSTM1, SOD, NF-κB, UGT1A1, or UGT1A6. Moderate increases (2-5-fold) over the time zero were seen for HO-1, Nrf2, and NQO1, and significant increases (>5-fold) for GSTT1, GPx1, and Maf. PK-PD analyses using GastroPlus and the bootstrap method provided reasonable fitting for the PK and PD profiles and parameter estimates. Our present study shows that SFN could induce Nrf2-mediated phase II DME/antioxidant mRNA expression for NQO1, GSTT1, Nrf2, GPx, Maf, and HO-1 in rat lymphocytes after iv administration, suggesting that Nrf2-mediated mRNA expression in lymphocytes may serve as surrogate biomarkers. The PK-PD IDR model simultaneously linking the plasma concentrations of SFN and the PD response of lymphocyte mRNA expression is

  19. Lysine 92 amino acid residue of USP46, a gene associated with 'behavioral despair' in mice, influences the deubiquitinating enzyme activity.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available Deubiquitinating enzymes (DUBs regulate diverse cellular functions by their activity of cleaving ubiquitin from specific protein substrates. Ubiquitin-Specific Protease 46 (USP46 has recently been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with a lysine codon (Lys 92 deletion in USP46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. However, whether this deletion affects enzyme activity is unknown. Here we show that USP46 has deubiquitinating enzyme activity detected by USP cleavage assay using GST-Ub52 as a model substrate. Interestingly, compared to wild type, the Lys 92 deletion mutant resulted in a decreased deubiquitinating enzyme activity of 27.04%. We also determined the relative expression levels of Usp46 in rat tissues using real-time RT-PCR. Usp46 mRNA was expressed in various tissues examined including brain, with the highest expression in spleen. In addition, like rat USP46, both human and mouse USP46 are active toward to the model substrate, indicating the USP cleavage assay is a simple method for testing the deubiquitinating enzyme activity of USP46. These results suggest that the Lys 92 deletion of USP46 could influence enzyme activity and thereby provide a molecular clue how the enzyme regulating the pathogenesis of mental illnesses.

  20. Clock genes, melanopsins, melatonin, and dopamine key enzymes and their modulation by light and glutamate in chicken embryonic retinal cells.

    Science.gov (United States)

    Lima, Leonardo Henrique Ribeiro Graciani de; Santos, Kátia Pereira dos; Lauro Castrucci, Ana Maria de

    2011-03-01

    The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 µM glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 µM glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. PMID:21231870

  1. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    OpenAIRE

    Margaret Ann Carpenter; Nigel eJoyce; Russell eGenet; Rebecca eCooper; Sarah eMurray; Alasdair eNoble; Ruth eButler; Gail eTimmerman-Vaughan

    2015-01-01

    Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch ...

  2. Concordant association of insulin degrading enzyme gene (IDE variants with IDE mRNA, Abeta, and Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Minerva M Carrasquillo

    Full Text Available BACKGROUND: The insulin-degrading enzyme gene (IDE is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD. METHODOLOGY/PRINCIPAL FINDINGS: We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5x10(-8, fold-increase = 2.12,; the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003. Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2-C (p = 0.006 and HepG2 (p = 0.02 cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879, we identified a proxy for rs6583817 that associated significantly with decreased plasma Abeta40 levels (ss = -0.124, p = 0.011 and total measured plasma Abeta levels (b = -0.130, p = 0.009. Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03, and the eleven IDE haplotypes (global p = 0.02 also showed significant association. CONCLUSIONS: Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Abeta40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect.

  3. Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli.

    Science.gov (United States)

    Kim, Jae-Eung; Huang, Rui; Chen, Hui; You, Chun; Zhang, Y-H Percival

    2016-09-01

    A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids. PMID:27367290

  4. Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses

    Institute of Scientific and Technical Information of China (English)

    LU Shao-wei; LI Tian-lai; JIANG Jing

    2009-01-01

    Changes in sucrose metabolism in response to salt (NaCl) and water (polyethylene glycol,PEG6000) iso-osmotic stresses were measured in tomato cultivar Liaoyuan Duoli (Solanum lycopersicum L.)and the objective was to provide a new evidence for the relationship between salt and osmotic stresses.The carbohydrate contents,as well as sucrose metabolizing enzymes activities and transcript levels were determined.The results indicated that soluble sugar and hexoses accumulated to higher levels and the contents of sucrose and starch were lower in mature fruit under the two stress treatments.Salt and water stresses can enhance the invertase and sucrose synthase activities of tomato fruit in a long period of time (45-60 days after anthesis),and elevate the expression of soluble acid invertase mRNA.It showed that two different stresses could also regulate the soluble acid invertase activity by controlling its gene expression.The activity of sucrose synthase was linked to the changes in soluble sugar levels but not with transcript levels.The effects of salt and water stress treatments on sucrose phosphate synthase activities were weak.

  5. Use of PCR-RFLP (Polymerase Chain Reaction - Restricted Fragment Length Polymorphism in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    H. Tonhati

    2010-02-01

    Full Text Available The milk is an important food because it contents Conjugated Linoleic Acids (CLA. These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD´s gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z43D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren’t genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

  6. Gene expression and enzyme activity of lipoprotein lipase correlate with intramuscular fat content in Guangxi san-huang and Arbor Acres chickens.

    Science.gov (United States)

    Huang, Y N; Wang, J; Chen, B J; Jiang, Q Y; Guo, Y F; Lan, G Q; Jiang, H S

    2016-01-01

    Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. This study investigated LPL gene expression, LPL enzyme activity, and the correlation of each with intramuscular fat (IMF) in Chinese Guangxi san-huang (GXSH) and Arbor Acres (AA) chickens. The results showed that age and breed had significant effects on LPL expression and enzyme activity. Correlation analyses showed significant positive correlations between LPL expression levels and IMF contents in the breast and thigh tissues of both GXSH (r = 0.712, P = 0.001; r = 0.792, P < 0.001, respectively) and AA (r = 0.644, P < 0.001; r = 0.545, P < 0.001, respectively) chickens. The results also indicated a significant positive correlation between LPL enzyme activity and IMF contents in the breast and thigh tissues of both GXSH (r = 0.615, P = 0.001; r = 0.685, P < 0.001, respectively) and AA (r = 0.600, P = 0.001; r = 0.528, P = 0.003, respectively) chickens. The results indicated that the LPL gene was significantly correlated with IMF in these two breeds. The results presented here could contribute to knowledge of LPL mRNA developmental expression patterns and enzyme activity, and it could facilitate further research on the molecular mechanisms underlying IMF deposition in chickens. PMID:27323106

  7. Effects of supplementation with branched-chain amino acids to low-protein diets on expression of genes related to lipid metabolism in skeletal muscle of growing pigs.

    Science.gov (United States)

    Duan, Yehui; Duan, Yangmiao; Li, Fengna; Li, Yinghui; Guo, Qiuping; Ji, Yujiao; Tan, Bie; Li, Tiejun; Yin, Yulong

    2016-09-01

    Branched-chain amino acids (BCAA), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in energy homeostasis and lipid metabolism in addition to their other functions, such as in protein metabolism. This study investigated the effects of different dietary BCAA ratios on the intramuscular fat (IMF) content and fatty acid composition in different location of skeletal muscles, including the longissimus dorsi (LD), biceps femoris (BF), and psoas major (PM) muscles of growing pigs, and also examined the mRNA expression levels of genes involved in lipid metabolism in these muscle tissues. The experiment was performed on 40 growing pigs (Large White × Landrace) with a similar initial weight (9.85 ± 0.35 kg). The pigs were randomly assigned to one of five diets: diet A was a positive control and contained 20 % crude protein (CP) with a Leu:Ile:Val ratio of 1:0.51:0.63 according to the recommendation of the National Research Council (NRC); for diets B to E, the CP level was reduced to 17 %, and the Leu:Ile:Val ratios were 1:1:1, 1:0.75:0.75, 1:0.51:0.63, and 1:0.25:0.25, respectively. No significant difference was observed in the average feed intake and feed efficiency of the pigs fed the low protein diet (17 % CP) with BCAA treatments relative to the positive control. However, there was a tendency for increased feed efficiency of the 1:0.75:0.75 group compared with the 1:1:1 group (P = 0.09). The BCAA ratio of 1:0.75:0.75 (17 % CP) increased the IMF content of BF muscle (P protein level had different effects on the fatty acid composition of the LD, BF, and PM muscles. The BCAA ratio of 1:0.51:0.63-1:0.75:0.75 (17 % CP) significantly lowered the ratio of n-6 to n-3 polyunsaturated fatty acid in these muscles compared with the positive control group (20 % CP). This effect was associated with an increase in mRNA expression levels of acetyl-CoA carboxylase, lipoprotein lipase, fatty acid transport protein, and fatty acid binding

  8. Antibody directed enzyme prodrug therapy: Discovery of novel genes, isolation of novel gene variants and production of long acting drugs for efficient cancer treatment

    NARCIS (Netherlands)

    Goda, S.K.; AlQahtani, A.; Rashidi, F.A.; Dömling, A.

    2015-01-01

    Background: Cancer accounts for 13% of the mortality rate worldwide. Antibody-Directed Enzyme Prodrug Therapy (ADEPT) is a novel strategy to improve the selectivity of cancer treatment. The ADEPT uses the bacterial enzyme, glucarpidase to produce the antibody-enzyme complex. Also the glucarpidase is

  9. Seasonal alteration in amounts of lignans and their glucosides and gene expression of the relevant biosynthetic enzymes in the Forsythia suspense leaf.

    Science.gov (United States)

    Morimoto, Kinuyo; Satake, Honoo

    2013-01-01

    Lignans of Forsythia spp. are essential components of various Chinese medicines and health diets. However, the seasonal alteration in lignan amounts and the gene expression profile of lignan-biosynthetic enzymes has yet to be investigated. In this study, we have assessed seasonal alteration in amounts of major lignans, such as pinoresinol, matairesinol, and arctigenin, and examined the gene expression profile of pinoresinol/lariciresinol reductase (PLR), pinoresinol-glucosylating enzyme (UGT71A18), and secoisolariciresinol dehydrogenase (SIRD) in the leaf of Forsythia suspense from April to November. All of the lignans in the leaf continuously increased from April to June, reached the maximal level in June, and then decreased. Ninety percent of pinoresinol and matairesinol was converted into glucosides, while approximately 50% of arctigenin was aglycone. PLR was stably expressed from April to August, whereas the PLR expression was not detected from September to November. In contrast, the UGT71A18 expression was found from August to November, but not from April to July. The SIRD expression was prominent from April to May, not detected in June to July, and then increased again from September to November. These expression profiles of the lignan-synthetic enzymes are largely compatible with the alteration in lignan contents. Furthermore, such seasonal lignan profiles are in good agreement with the fact that the Forsythia leaves for Chinese medicinal tea are harvested in June. This is the first report on seasonal alteration in lignans and the relevant biosynthetic enzyme genes in the leaf of Forsythia species. PMID:23832493

  10. Ethanol and supercritical fluid extracts of hemp seed (Cannabis sativa L.) increase gene expression of antioxidant enzymes in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Sunghyun Hong; Kandhasamy Sowndhararajan; Taewoo Joo; Chanmook Lim; Haeme Cho; Songmun Kim; Gur-Yoo Kim; Jin-Woo Jhoo

    2015-01-01

    Objective: To determine the gene expression of antioxidant enzymes by hemp seed extracts in human hepatoma (HepG2) cells. Methods: Ethanol and supercritical fluid (SF) extracts obtained from de-hulled hemp seed were used for the evaluation of in vitro antioxidant activity and gene expression of antioxidant enzymes. In vitro antioxidant activities of the samples evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging assays. The expression of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in HepG2cells was evaluated by real-time PCR. Results:In the antioxidant assay, SF extract of hemp seed exhibited higher ABTS and DPPH radical scavenging activities (IC50 of 66.6 µg/mL and 9.2 mg/mL, respectively) than ethanol extract. The results of antioxidant enzyme expression in real-time PCR study revealed the H2O2 (200 µM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells treated with ethanol and SF extracts were up-regulated the expression of antioxidant enzymes in concentration dependent manner. When compared to ethanol extract, the SF extract exhibited higher activity in the expression of all the antioxidant enzymes at the concentration of 500 µg/mL. Conclusion: In conclusion, the findings of our study demonstrated that the hemp seed effectively inhibited H2O2 mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases.

  11. The CanOE strategy: integrating genomic and metabolic contexts across multiple prokaryote genomes to find candidate genes for orphan enzymes.

    Directory of Open Access Journals (Sweden)

    Adam Alexander Thil Smith

    2012-05-01

    Full Text Available Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes, a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short. The first step locates "genomic metabolons", i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12.

  12. Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefit from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carcinoma

    International Nuclear Information System (INIS)

    Little is known of the function and clinical significance of intratumoral dysregulation of xenobiotic-metabolizing enzyme expression in breast cancer. One molecular mechanism proposed to explain tamoxifen resistance is altered tamoxifen metabolism and bioavailability. To test this hypothesis, we used real-time quantitative RT-PCR to quantify the mRNA expression of a large panel of genes coding for the major xenobiotic-metabolizing enzymes (12 phase I enzymes, 12 phase II enzymes and three members of the ABC transporter family) in a small series of normal breast (and liver) tissues, and in estrogen receptor alpha (ERα)-negative and ERα-positive breast tumors. Relevant genes were further investigated in a well-defined cohort of 97 ERα-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Seven of the 27 genes showed very weak or undetectable expression in both normal and tumoral breast tissues. Among the 20 remaining genes, seven genes (CYP2A6, CYP2B6, FMO5, NAT1, SULT2B1, GSTM3 and ABCC11) showed significantly higher mRNA levels in ERα-positive breast tumors than in normal breast tissue, or showed higher mRNA levels in ERα-positive breast tumors than in ERα-negative breast tumors. In the 97 ERα-positive breast tumor series, most alterations of these seven genes corresponded to upregulations as compared with normal breast tissue, with an incidence ranging from 25% (CYP2A6) to 79% (NAT1). Downregulation was rare. CYP2A6, CYP2B6, FMO5 and NAT1 emerged as new putative ERα-responsive genes in human breast cancer. Relapse-free survival was longer among patients with FMO5-overexpressing tumors or NAT1-overexpressing tumors (P = 0.0066 and P = 0.000052, respectively), but only NAT1 status retained prognostic significance in Cox multivariate regression analysis (P = 0.0013). Taken together, these data point to a role of genes coding for xenobiotic-metabolizing enzymes in breast tumorigenesis, NAT1 being an

  13. Colored light-quality selective plastic films affect anthocyanin content, enzyme activities, and the expression of flavonoid genes in strawberry (Fragaria × ananassa) fruit.

    Science.gov (United States)

    Miao, Lixiang; Zhang, Yuchao; Yang, Xiaofang; Xiao, Jinping; Zhang, Huiqin; Zhang, Zuofa; Wang, Yuezhi; Jiang, Guihua

    2016-09-15

    The influence of colored light-quality selective plastic films (red, yellow, green, blue, and white) on the content of anthocyanin, the activities of the related enzymes and the transcripts of the flavonoid gene was studied in developing strawberry fruit. The results indicated that colored films had highly significant effects on the total anthocyanin content (TAC) and proportions of individual anthocyanins. Compared with the white control film, the red and yellow films led to the significant increase of TAC, while the green and blue films caused a decrease of TAC. Colored film treatments also significantly affected the related enzyme activity and the expression of structural genes and transcription factor genes, which suggested that the enhancement of TAC by the red and yellow films might have resulted from the activation of related enzymes and transcription factor genes in the flavonoid pathway. Treatment with red and yellow light-quality selective plastic films might be useful as a supplemental cultivation practice for enhancing the anthocyanin content in developing strawberry fruit. PMID:27080884

  14. Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-alpha-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli.

    Science.gov (United States)

    Goda, S K; Eissa, O; Akhtar, M; Minton, N P

    1997-10-01

    An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1.8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57,184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequences identity was apparent with the 4-alpha-glucano-transferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated maIQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MaIP). The translation stop codon of MaIQ overlapped the translation start codon of the putative maIP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-alpha-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MaIQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves. PMID:9353929

  15. Branched polynomial covering maps

    DEFF Research Database (Denmark)

    Hansen, Vagn Lundsgaard

    1999-01-01

    A Weierstrass polynomial with multiple roots in certain points leads to a branched covering map. With this as the guiding example, we formally define and study the notion of a branched polynomial covering map. We shall prove that many finite covering maps are polynomial outside a discrete branch...... set. Particular studies are made of branched polynomial covering maps arising from Riemann surfaces and from knots in the 3-sphere....

  16. Branched polynomial covering maps

    DEFF Research Database (Denmark)

    Hansen, Vagn Lundsgaard

    2002-01-01

    A Weierstrass polynomial with multiple roots in certain points leads to a branched covering map. With this as the guiding example, we formally define and study the notion of a branched polynomial covering map. We shall prove that many finite covering maps are polynomial outside a discrete branch...... set. Particular studies are made of branched polynomial covering maps arising from Riemann surfaces and from knots in the 3-sphere. (C) 2001 Elsevier Science B.V. All rights reserved....

  17. ROLE OF ANGIOTENSIN-CONVERTING ENZYME AND VITAMIN D RECEPTOR GENE POLYMORPHISMS IN CANCER ANOREXIA-CACHEXIA SYNDROME

    Directory of Open Access Journals (Sweden)

    Ariele Fabris

    2012-01-01

    Full Text Available The ubiquitin-proteasome pathway is a crucial connection between aberrant immune system activation, systemic inflammation and Cancer Anorexia-Cachexia Syndrome (CACS, a syndrome that culminates in hyper-activation of the ubiquitin-proteasome pathway. Angiotensin directly up-regulates this pathway, while vitamin D down-regulates it indirectly through the insulin-like growth factor-1 pathway. We investigated the genetic predisposition towards CACS in a cancer population, examining Insertion/Deletion (I/D polymorphism of angiotensin-converting enzyme gene and FokI and BsmI polymorphisms of vitamin D receptor gene. Sixty-two cancer patients were recruited and divided into three groups: primary cachectic (C1, n = 14; dysmetabolic body weight loss ≥5% in 6 months; secondary cachectic (C2, n = 34; similar weight loss, mechanic or iatrogenic origin; and non-cachectic (NC, n = 16. C2+NC were merged in the control group. The three groups showed significant differences in average prognostic inflammatory nutritional index (C1: 26.4±23.4; C2: 5.4±5.6; NC: 0.37±0.5, C-reactive protein serum levels (C1: 6.6±2.1; C2: 2.4±2.2; NC: 1.0±2.0 mg/dL, albumin serum levels (C1: 3.1±0.6; C2: 3.5±0.4; NC 3.7±0.6 g/dL, weight loss (C1: 22±8; C2: 15±6.7; NC 5±6% and life expectancy (C1: 6.4±3.3; C2: 25±28; NC: 45±25 months. However, none of the chosen polymorphisms showed any statistically significant correlation with CACS. The complexity of the changes of the immune system in the chronic inflammation state associated with CACS is far greater than expected and further studies are required to identify genetic independent markers of progression toward CACS."

  18. A Meta-analysis on the correlation between the polymorphism of angiotensin converting enzyme gene and hypertrophic cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ling CHEN

    2014-01-01

    Full Text Available Objective To systematically investigate the correlation between the polymorphism of angiotensin converting enzyme (ACE gene I/D and hypertrophic cardiomyopathy. Methods The databases, such as PubMed, Embase, OVID, Web of Science, Cochrane library, CNKI, WanFang Data and VIP, were searched to collect the studies on the correlation between ACE I/D polymorphism and hypertrophic cardiomyopathy susceptibility. Studies that met the inclusion criteria were Meta-analyzed using Stata 11.0 software. Results Fifteen articles were collected including 1114 cases and 1648 controls. The Meta-analysis indicated that there was significant correlation between the 4 models of ACE I/D polymorphism and hypertrophic cardiomyopathy susceptibility [D vs I: OR=1.49, 95%CI (1.20, 1.84; DD vs (ID+II: OR=1.56, 95%CI (1.17, 2.08; (DD+ID vs II: OR=1.76, 95%CI (1.30, 2.38; DD vs II: OR=2.20, 95%CI (1.44, 3.37]. In subgroup analysis, the significant difference existed in Asian population, but no significance was found in European population (P<0.05. Conclusions There is a positive correlation between hypertrophic cardiomyopathy and ACE I/D polymorphism in population, and D allele and DD genotype are likely to be the risk factors of hypertrophic cardiomyopathy. But such correlation does not exist in European population. DOI: 10.11855/j.issn.0577-7402.2013.12.07

  19. Regulation of the malic enzyme gene malE by the transcriptional regulator MalR in Corynebacterium glutamicum.

    Science.gov (United States)

    Krause, Jens P; Polen, Tino; Youn, Jung-Won; Emer, Denise; Eikmanns, Bernhard J; Wendisch, Volker F

    2012-06-15

    Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR. PMID:22261175

  20. A Methanocaldococcus jannaschii archaeal signature gene encodes for a 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate synthetase. A new enzyme in purine biosynthesis.

    Science.gov (United States)

    Ownby, Katie; Xu, Huimin; White, Robert H

    2005-03-25

    We have identified and characterized a new member of the ATP-grasp enzyme family that catalyzes the ATP- and formate-dependent formylation of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (AICAR) to 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (FAICAR) in the absence of folates. The enzyme, which we designate as PurP, is the product of the Methanocaldococcus jannaschii purP gene (MJ0136), which is a signature gene for Archaea. As is characteristic of reactions catalyzed by this family of enzymes, the other products of the reaction, ADP and P(i), were produced stoichiometrically with the amount of ATP, formate, and AICAR used. Formyl phosphate was found to substitute for ATP and formate in the reaction, yet the methylene analog, phosphonoacetaldehyde, was not an inhibitor or substrate for the reaction. The enzyme, along with PurO, which catalyzes the cyclization of FAICAR to inosine 5'-monophosphate, catalyzes the same overall transformation in purine biosynthesis as is accomplished by PurH in bacteria and eukaryotes. No homology exists between PurH and either PurO or PurP. 1H NMR and gas chromatography-mass spectrometry analysis of an M. jannaschii cell extract showed the presence of free formate that can be used by the enzyme for purine biosynthesis. This formate arises by the reduction of CO2 with hydrogen; this was demonstrated by incorporating 13C into the formate when M. jannaschii cell extracts were incubated with H13CO3- and hydrogen gas. The presence of this signature gene in all of the Archaea indicates the presence of a purine biosynthetic pathway proceeding in the absence of folate coenzymes. PMID:15623504

  1. Glucan synthesis in the genus Lactobacillus: Isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains

    NARCIS (Netherlands)

    Kralj, S.; Geel-Schutten, G.H. van; Dondorff, M.M.G.; Kirsanovs, S.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2004-01-01

    Members of the genera Streptococcus and Leuconostoc synthesize various α-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reutera

  2. Glucan synthesis in the genus Lactobacillus : isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains

    NARCIS (Netherlands)

    Kralj, S.; Geel-Schutten, G.H. van; Dondorff, M.M.G.; Kirsanovs, S.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2004-01-01

    Members of the genera Streptococcus and Leuconostoc synthesize various α-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reutera

  3. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.;

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...... ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined...

  4. [The role of angiotensin-converting enzyme gene I/D polymorphism in development of metabolic disorders in patients with cardiovascular pathology].

    Science.gov (United States)

    Vynohradova, S V

    2005-01-01

    The role of angiotensin-converting enzyme (ACE) gene I/D polymorphism in development of cardiovascular pathology (CVP), metabolic syndrom and insulin-independent diabet associated with such metabolic disorders as glucose intolerance and hyperglicemia, intolerance to insulin and hyperinsulinemia, dyslipiproteinemia (DLP) and obesity is discussed. Most of authors consider D-allel and DD genotype to be assosiated with development of DLP and such CVP as ishemic heart disease and myocardial infarction. PMID:16018179

  5. Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

    OpenAIRE

    Klinghammer, Michaela; Tenhaken, Raimund

    2008-01-01

    Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant deve...

  6. Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

    International Nuclear Information System (INIS)

    Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

  7. Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qi-Liang; Luo, Zhi, E-mail: luozhi99@yahoo.com.cn; Pan, Ya-Xiong; Zheng, Jia-Lang; Zhu, Qing-Ling; Sun, Lin-Dan; Zhuo, Mei-Qin; Hu, Wei

    2013-07-15

    Highlights: •Cu downregulates lipogenesis and reduces lipid deposition in liver and adipose tissue. •Mechanism of Cu affecting lipid metabolism is determined at the enzymatic and molecular levels. •Cu exposure differentially influences lipid metabolism between liver and adipose tissue. -- Abstract: The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) μg Cu/l, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu

  8. Identification of ten mevalonate enzyme-encoding genes and their expression in response to juvenile hormone levels in Leptinotarsa decemlineata (Say).

    Science.gov (United States)

    Li, Qian; Meng, Qing-Wei; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2016-06-15

    The mevalonate pathway is responsible for the biosynthesis of many essential molecules important in insect development, reproduction, chemical communication and defense. Based on Leptinotarsa decemlineata transcriptome and genome data, we identified ten genes that encoded acetoacetyl-CoA thiolase (LdAACT1 and LdAACT2), hydroxymethylglutaryl (HMA)-CoA synthase (LdHMGS), HMG-CoA reductase (LdHMGR1 and LdHMGR2), mevalonate kinase (LdMevK), phospho-mevalonate kinase (LdPMK), mevalonate diphosphate decarboxylase (LdMDD), isopentenyl-diphosphate isomerase (LdIDI) and farnesyl pyrophosphate synthetase (LdFPPS). Nine of these genes (except for LdAACT1) were mainly expressed in the larval brain-corpora cardiaca-corpora allata complex, and adult ovary and testis. The 9 genes were transcribed at high levels right after each ecdysis, and at low levels in the mid instar. Therefore, the 9 genes were indicated to be involved in JH biosynthesis. Moreover, knockdown of a JH biosynthesis gene LdJHAMT to lower JH titer significantly downregulated the transcription of the 9 genes. Ingestion of JH to activate JH signaling also significantly suppressed the expression of the 9 genes. It appears that the accumulation of JH precursors in LdJHAMT RNAi larvae and a high JH titer in JH-fed specimens may cause negative feedbacks to repress the expression of the 9 mevalonate enzyme-encoding genes (excluding LdAACT1) to balance the enzyme quantity in L. decemlineata. PMID:26899871

  9. Molecular expression of l-asparaginase gene from Nocardiopsis alba NIOT-VKMA08 in Escherichia coli: A prospective recombinant enzyme for leukaemia chemotherapy.

    Science.gov (United States)

    Meena, Balakrishnan; Anburajan, Lawrance; Vinithkumar, Nambali Valsalan; Shridhar, Divya; Raghavan, Rangamaran Vijaya; Dharani, Gopal; Kirubagaran, Ramalingam

    2016-09-30

    l-Asparaginase is an antineoplastic agent that selectively reduces the level of l-asparagine in blood and diminishes the proliferation of cancerous cells. Studies were carried out on the cloning and heterologous expression of l-asparaginase biosynthesis gene (ansA) from Nocardiopsis alba NIOT-VKMA08 to achieve the stable inducible system that overproduces the glutaminase-free recombinant l-asparaginase. Overexpression of recombinant l-asparaginase was achieved with an optimized final concentration of 1.5mM of isopropyl-β-d-thiogalactoside (IPTG) and the enzyme was expressed as a soluble protein. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and the purified enzyme disclosed an elevated level of asparaginase activity (158.1IU/mL). Optimum pH and temperature of the purified l-asparaginase for the hydrolysis of l-asparagine were 8.0 and 37°C and it was very specific for its natural substrate, l-asparagine. Detailed studies were carried out on the kinetics of enzyme reaction, catalytic activity, temperature and ionic strength and the thermostability of the l-asparaginase enzyme. The functional characterisation of the recombinant l-asparaginase was studied through Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), in silico sequence analysis and protein structural modelling. Glutaminase activity was not detected in the recombinant l-asparaginase, which could reduce the probable side effects during leukaemia therapy. PMID:27155523

  10. Analysis of gene expression changes, caused by exposure to nitrite, in metabolic and antioxidant enzymes in the red claw crayfish, Cherax quadricarinatus.

    Science.gov (United States)

    Jiang, Qichen; Zhang, Wenyi; Tan, Hongyue; Pan, Dongmei; Yang, Yuanhao; Ren, Qian; Yang, Jiaxin

    2014-06-01

    We evaluated the effect of acute exposure to nitrite on expression of antioxidant and metabolic enzyme genes in gill tissue of advanced juvenile Cherax quadricarinatus. A 48h nitrite exposure was conducted, using four test concentrations (NO2-N=0.5, 1, 1.5 and 2mg L(-1)) plus a control group. The relative mRNA expression of mitochondrial manganese superoxide dismutase (mMnSOD), cytosolic MnSOD (cMnSOD), extracellular copper/zinc SOD (exCu/ZnSOD), catalase (CAT), glutathione S-transferase (GST), arginine kinase (AK), glutamate dehydrogenase (GDH), mitochondrial malate dehydrogenase (mMDH), Na(+)/K(+)-ATPase α-subunit and phosphoenolpyruvate carboxykinase (PEPCK) in gill tissue was measured. Significantly increased mRNA expression was observed for all the antioxidant enzymes after 12 and 24h. After 48h, they all decreased at high nitrite concentrations. The gene expression levels of AK, GDH, mMDH and Na(+)/K(+)-ATPase α-subunit showed similar trends as the antioxidant enzymes. Significant depression of gene expression levels of PEPCK occurred throughout the experimental time at high nitrite concentrations. The results indicated that nitrite could induce oxidative and metabolic stress in C. quadricarinatus, in a time dependent manner, which suggests they could be helpful in predicting sublethal nitrite toxicity and useful in environmental monitoring studies. PMID:24680578

  11. Analysis of the metatranscriptome of microbial communities of an alkaline hot sulfur spring revealed different gene encoding pathway enzymes associated with energy metabolism.

    Science.gov (United States)

    Tripathy, Swetaleena; Padhi, Soumesh Kumar; Mohanty, Sriprakash; Samanta, Mrinal; Maiti, Nikhil Kumar

    2016-07-01

    Alkaline sulfur hot springs notable for their specialized and complex ecosystem powered by geothermal energy are abundantly rich in different chemotrophic and phototrophic thermophilic microorganisms. Survival and adaptation of these organisms in the extreme environment is specifically related to energy metabolism. To gain a better understanding of survival mechanism of the organisms in these ecosystems, we determined the different gene encoding enzymes associated with anaerobic pathways of energy metabolism by applying the metatranscriptomics approach. The analysis of the microbial population of hot sulfur spring revealed the presence of both aerobic and anaerobic organisms indicating dual mode of lifestyle of the community members. Proteobacteria (28.1 %) was the most dominant community. A total of 988 reads were associated with energy metabolism, out of which 33.7 % of the reads were assigned to nitrogen, sulfur, and methane metabolism based on KEGG classification. The major lineages of hot spring communities were linked with the anaerobic pathways. Different gene encoding enzymes (hao, nir, nar, cysH, cysI, acs) showed the involvement of microbial members in nitrification, denitrification, dissimilatory sulfate reduction, and methane generation. This study enhances our understanding of important gene encoding enzymes involved in energy metabolism, required for the survival and adaptation of microbial communities in the hot spring. PMID:27290724

  12. DHN melanin biosynthesis in the plant pathogenic fungus Botrytis cinerea is based on two developmentally regulated key enzyme (PKS)-encoding genes.

    Science.gov (United States)

    Schumacher, Julia

    2016-02-01

    Botrytis cinerea is the causal agent of gray mold disease in various plant species and produces grayish macroconidia and/or black sclerotia at the end of the infection cycle. It has been suggested that the pigmentation is due to the accumulation of 1,8-dihydroxynaphthalene (DHN) melanin. To unravel its basis and regulation, the putative melanogenic and regulatory genes were identified and functionally characterized. Unlike other DHN melanin-producing fungi, B. cinerea and other Leotiomycetes contain two key enzyme (PKS)-encoding enzymes. Bcpks12 and bcpks13 are developmentally regulated and are required for melanogenesis in sclerotia and conidia respectively. BcYGH1 converts the BcPKS13 product and contributes thereby to conidial melanogenesis. In contrast, enzymes acting downstream in conversion of the PKS products (BcBRN2, BcSCD1 and BcBRN1) are required for both, sclerotial and conidial melanogenesis, suggesting that DHN melanogenesis in B. cinerea follows a non-linear pathway that is rather unusual for secondary metabolic pathways. Regulation of the melanogenic genes involves three pathway-specific transcription factors (TFs) that are clustered with bcpks12 or bcpks13 and other developmental regulators such as light-responsive TFs. Melanogenic genes are dispensable in vegetative mycelia for proper growth and virulence. However, DHN melanin is considered to contribute to the longevity of the reproduction structures. PMID:26514268

  13. AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco.

    Science.gov (United States)

    Bharti, Poonam; Mahajan, Monika; Vishwakarma, Ajay K; Bhardwaj, Jyoti; Yadav, Sudesh Kumar

    2015-09-01

    In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants. PMID:26116024

  14. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    Science.gov (United States)

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  15. Effects of Cinnamon extract on biochemical enzymes, TNF-α and NF-κB gene expression levels in liver of broiler chickens inoculated with Escherichia coli

    Directory of Open Access Journals (Sweden)

    Seyed Mahmoud Tabatabaei

    2015-09-01

    Full Text Available Abstract: Infection with Escherichia coli (E. coli is a common disease in poultry industry. The use of antibiotics to treat diseases is facing serious criticism and concerns. The medicinal plants may be effective alternatives because of their multiplex activities. The aim of this study was to investigate the effects of cinnamon extract on the levels of liver enzymes, tumor necrosis factor-alpha (TNF-α and nuclear factor-kappa B (NF-κB gene expressions in liver of broiler chickens infected with E. coli. Ninety Ross-308 broilers were divided into healthy or E. coli-infected groups, receiving normal or cinnamon extract (in concentrations of 100 or 200mg/kg of food supplemented diets. E. coli suspension (108cfu was injected subcutaneously after 12 days cinnamon administration. Seventy-two hours after E. coli injection, the blood samples were taken for biochemical analysis of liver enzymes in serum (spectrophotometrically, and liver tissue samples were obtained for detection of gene expression of inflammatory markers TNF-α and NF-κB, using real-time PCR. Infection with E. coli significantly increased the levels of TNF-α and NF-κB gene expressions as well as some liver enzymes including creatine-kinase (CK, lactate-dehydrogenase (LDH, alanine-transferase (ALT and aspartate-transferase (AST as compared with control group (P<0.05. Pre-administration of cinnamon extract in broilers diet (in both concentrations significantly reduced the tissue levels of TNF-α and NF-κB gene expressions and enzymes CK and ALT in serum of broiler chickens inoculated with E. coli in comparison with E. coli group (P<0.05 and P<0.01. The levels of LDH and AST were significantly decreased only by 200mg/kg cinnamon extract in infected broilers. The level of alkaline-phosphatase (ALP was not affected in any groups. Pre-administration of cinnamon extract in diets of broiler chickens inoculated with E. coli could significantly reduce the gene expression levels of pro

  16. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant.

    Science.gov (United States)

    Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

    2016-01-01

    'Hong Anliu' (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of 'Anliu' (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between 'HAL' and 'AL'. Compared with 'AL', the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in 'HAL'. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H(+)-ATPase, vacuolar H(+)-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in 'HAL'. These results implied that 'HAL' has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with 'AL'. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in 'HAL'. PMID:27385485

  17. Exogenous calcium induces tolerance to atrazine stress in Pennisetum seedlings and promotes photosynthetic activity, antioxidant enzymes and psbA gene transcripts.

    Science.gov (United States)

    Erinle, Kehinde Olajide; Jiang, Zhao; Ma, Bingbing; Li, Jinmei; Chen, Yukun; Ur-Rehman, Khalil; Shahla, Andleeb; Zhang, Ying

    2016-10-01

    Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented. PMID:27391035

  18. Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant

    Science.gov (United States)

    Guo, Ling-Xia; Shi, Cai-Yun; Liu, Xiao; Ning, Dong-Yuan; Jing, Long-Fei; Yang, Huan; Liu, Yong-Zhong

    2016-01-01

    ‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’. PMID:27385485

  19. Red meat intake, doneness, polymorphisms in genes that encode carcinogen-metabolizing enzymes and colorectal cancer risk

    OpenAIRE

    Cotterchio, Michelle; Boucher, Beatrice A.; Manno, Michael; Gallinger, Steven; Okey, Allan B; Harper, Patricia A.

    2008-01-01

    Colorectal cancer literature regarding the interaction between polymorphisms in carcinogen-metabolizing enzymes and red meat intake/doneness is inconsistent. A case-control study was conducted to evaluate the interaction between red meat consumption, doneness and polymorphisms in carcinogen-metabolizing enzymes. Colorectal cancer cases diagnosed 1997-2000, aged 20-74 years, were identified through the population-based Ontario Cancer Registry and recruited by the Ontario Family Colorectal Canc...

  20. Bank branches in supermarkets

    OpenAIRE

    Lawrence J. Radecki; John Wenninger; Daniel Orlow

    1996-01-01

    The largest U.S. commercial banks are restructuring their retail operations to reduce the cost disadvantage resulting from a stagnant deposit base and stiffer competition. As part of this effort, some banks are opening "supermarket," or "in-store," branches: a new type of banking office within a large retail outlet. An alternative to the traditional bank office, the supermarket branch enables banks to improve the efficiency of the branch network and offer greater convenience to customers.

  1. The CAZyome of Phytophthora spp.: A comprehensive analysis of the gene complement coding for carbohydrate-active enzymes in species of the genus Phytophthora

    Directory of Open Access Journals (Sweden)

    Laird Emma W

    2010-09-01

    Full Text Available Abstract Background Enzymes involved in carbohydrate metabolism include Carbohydrate esterases (CE, Glycoside hydrolases (GH, Glycosyl transferases (GT, and Polysaccharide lyases (PL, commonly referred to as carbohydrate-active enzymes (CAZymes. The CE, GH, and PL superfamilies are also known as cell wall degrading enzymes (CWDE due to their role in the disintegration of the plant cell wall by bacterial and fungal pathogens. In Phytophthora infestans, penetration of the plant cells occurs through a specialized hyphal structure called appressorium; however, it is likely that members of the genus Phytophthora also use CWDE for invasive growth because hyphal forces are below the level of tensile strength exhibited by the plant cell wall. Because information regarding the frequency and distribution of CAZyme coding genes in Phytophthora is currently unknown, we have scanned the genomes of P. infestans, P. sojae, and P. ramorum for the presence of CAZyme-coding genes using a homology-based approach and compared the gene collinearity in the three genomes. In addition, we have tested the expression of several genes coding for CE in cultures grown in vitro. Results We have found that P. infestans, P. sojae and P. ramorum contain a total of 435, 379, and 310 CAZy homologs; in each genome, most homologs belong to the GH superfamily. Most GH and PL homologs code for enzymes that hydrolyze substances present in the pectin layer forming the middle lamella of the plant cells. In addition, a significant number of CE homologs catalyzing the deacetylation of compounds characteristic of the plant cell cuticle were found. In general, a high degree of gene location conservation was observed, as indicated by the presence of sequential orthologous pairs in the three genomes. Such collinearity was frequently observed among members of the GH superfamily. On the other hand, the CE and PL superfamilies showed less collinearity for some of their putative members

  2. Fat metabolism is regulated by altered gene expression oflipogenic enzymes and regulatory factors in liver and adiposetissue but not in semimembranosus muscle of pigs during thefattening period

    DEFF Research Database (Denmark)

    Duran-Montge, P; Theil, Peter Kappel; Lauridsen, Charlotte;

    2009-01-01

    It has been shown previously that lipid metabolism is regulated by fatty acids (FA) and that thyroid hormones are important regulators of energy metabolism. The effects of weight, dietary fat level and dietary FA profile on thyroid hormone levels and expression of lipogenic genes and tissue FA...... supplemented with approximately 10% fat of different origin and slaughtered at 100 kg BW. The supplemental fats were tallow, high-oleic sunflower oil, sunflower oil (SFO), linseed oil, fat blend (55% tallow, 35% sunflower oil, 10% linseed oil) and fish oil blend (40% fish oil, 60% linseed oil). In general, the...... hormones and genes encoding enzymes of fat synthesis in adipose tissue (acetyl CoA carboxylase (ACACA), fatty acid synthase and stearoyl CoA desaturase (SCD)) and the large differences in expression of lipogenic genes at different weights (60 and 100 kg BW), suggest a role for thyroid hormones and for T3...

  3. Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Highlights: ► We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. ► The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. ► The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. ► The OgUBC1 could protect disruption of plant cells by UV-B radiation. ► OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

  4. Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin; Kim, Hye Jeong [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Shin, Sang Hyun [National Crop Experiment Station, Rural Development Administration, Suwon 441-100 (Korea, Republic of); Lee, Jai Heon; Kim, Doh Hoon; Oh, Ju Sung [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Oh, Boung-Jun [BioControl Center, Jeonnam 516-942 (Korea, Republic of); Jung, Ho Won, E-mail: hwjung@dau.ac.kr [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young Soo, E-mail: chungys@dau.ac.kr [Department of Genetic Engineering, Dong-A University, Busan 604-714 (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

  5. The engineered thymidylate kinase (TMPK/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

    Directory of Open Access Journals (Sweden)

    Takeya Sato

    Full Text Available We previously described a novel suicide (or 'cell fate control' gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK that potentiates azidothymidine (AZT activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs. Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43 and Pannexin1 (Panx1, but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

  6. alpha. -Amylase of Clostridium thermosulfurogenes EM1: Nucleotide sequence of the gene, processing of the enzyme, and comparison to other. alpha. -amylases

    Energy Technology Data Exchange (ETDEWEB)

    Bahl, H.; Burchhardt, G.; Spreinat, A.; Haeckel, K.; Wienecke, A.; Antranikian, G.; Schmidt, B. (Georg-August-Univ., Gottingen (Germany))

    1991-05-01

    The nucleotide sequence of the {alpha}-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes Em1 suggested that the {alpha}-amylase is translated form mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature {alpha}-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 {alpha}-amylase with those from other bacterial and eukaryotic {alpha}-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca{sup 2+}-binding site (consensus region I) of this Ca{sub 2+}-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the {alpha}-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the {beta}-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

  7. Human nuclear NAD+ ADP-ribosyltransferase: Localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli

    International Nuclear Information System (INIS)

    The gene for human nuclear NAD+ ADP-ribosyltransferase was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pAD-PRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at interval translational start signals located within the pADPRT cDNA

  8. The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy

    International Nuclear Information System (INIS)

    Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the “gold standard” nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT

  9. The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Green, Laura K.; Storey, Mathew A. [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Williams, Elsie M. [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Victoria University Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington 6140 (New Zealand); Patterson, Adam V.; Smaill, Jeff B. [Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1142 (New Zealand); Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland 1142 (New Zealand); Copp, Janine N.; Ackerley, David F., E-mail: david.ackerley@vuw.ac.nz [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Victoria University Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington 6140 (New Zealand); Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1142 (New Zealand)

    2013-08-08

    Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the “gold standard” nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

  10. Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring

    Directory of Open Access Journals (Sweden)

    Miroslav Fojta

    2008-01-01

    Full Text Available Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE, followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

  11. Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

    Science.gov (United States)

    Gotoh, Takeshi; Awa, Hirono; Kikuchi, Ken-Ichi; Nirasawa, Satoru; Takahashi, Saori

    2010-01-01

    In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells. PMID:20139610

  12. Identification of Target Genes Involved in the Antiproliferative Effect of Enzyme-Modified Ginseng Extract in HepG2 Hepatocarcinoma Cell

    Directory of Open Access Journals (Sweden)

    Sung-Il Jang

    2013-01-01

    Full Text Available Ginsenosides are ginseng saponins, which are the major biologically active components of Panax ginseng, often metabolized by intestinal bacteria into more effective forms. In this study, we found that the antiproliferative activity of ginseng increased after enzymatic processing of ginseng saponin (50% inhibitory concentration [IC50], >30 μg/mL, which may be the result of the accumulation of minor saponins, such as Rh1, Rg3, compound K, and PPT constituents in ginseng saponin. Using the Agilent PrimeView Human Gene Expression Array, we found that the expression of several genes involved in apoptosis (caspase-4, Annexin A2, HSPA9, AIFM1, UQCRC2, and caspase-7 were increased in HepG2 human hepatocarcinoma cells after their treatment with enzyme-modified ginseng extract (EMGE. Furthermore, several genes implicated in cell cycle progression (CDCA3, CDCA8, CABLES2, CDC25B, CNNM3, and CCNK showed decreased expression in HepG2 cells treated with EMGE. Finally, from flow cytometric analysis, we found that EMGE-treated HepG2 cells showed increased apoptotic sub-G1 population (24%, compared with that observed in DMSO-treated control cells (1.6%. Taken together, our results suggest that EMGE induces anticancer activity through the induction of apoptosis-related genes and cell cycle arrest via decreased expression of cell cycle regulatory genes.

  13. Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

    OpenAIRE

    Weichenhan, D; Wackernagel, W

    1988-01-01

    We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.

  14. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.; Gilbert, Lawrence I.

    2006-01-01

    hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this......-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the...

  15. CLONING AND SEQUENCING OF THE GENE FOR A LACTOCOCCAL ENDOPEPTIDASE, AN ENZYME WITH SEQUENCE SIMILARITY TO MAMMALIAN ENKEPHALINASE

    NARCIS (Netherlands)

    Mierau, Igor; Tan, Paris S.T.; Haandrikman, Alfred J.; Kok, Jan; Leenhouts, Kees J.; Konings, Wil N.; Venema, Gerard

    1993-01-01

    The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-247 in lambdaEMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport syste

  16. Deubiquitylating enzymes and disease

    OpenAIRE

    Baker Rohan T; Taylor Matthew C; Singhal Shweta

    2008-01-01

    Abstract Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been charac...

  17. Branching processes and neutral evolution

    CERN Document Server

    Taïb, Ziad

    1992-01-01

    The Galton-Watson branching process has its roots in the problem of extinction of family names which was given a precise formulation by F. Galton as problem 4001 in the Educational Times (17, 1873). In 1875, an attempt to solve this problem was made by H. W. Watson but as it turned out, his conclusion was incorrect. Half a century later, R. A. Fisher made use of the Galton-Watson process to determine the extinction probability of the progeny of a mutant gene. However, it was J. B. S. Haldane who finally gave the first sketch of the correct conclusion. J. B. S. Haldane also predicted that mathematical genetics might some day develop into a "respectable branch of applied mathematics" (quoted in M. Kimura & T. Ohta, Theoretical Aspects of Population Genetics. Princeton, 1971). Since the time of Fisher and Haldane, the two fields of branching processes and mathematical genetics have attained a high degree of sophistication but in different directions. This monograph is a first attempt to apply the current sta...

  18. Interaction and Relationship Between Angiotensin Converting Enzyme Gene and Environmental Factors Predisposing to Essential Hypertension in Mongolian Population of China

    Institute of Scientific and Technical Information of China (English)

    QUN XU; HUA FENG; SHUANG-LIAN BAI; HAI-HUA PANG; GUI-RONG HUANG; MING-WU FANG; YONG-HONG ZHANG; ZHENG-LAI WU; CHANG-CHUN QIU; YAN-HUA WANG; WEI-JUN TONG; MING-LIANG GU; GANG WU; BATU BUREN; YONG-YUE LIU; JIAN WANG; YONG-SHAN LI

    2004-01-01

    Objective To investigate the association of specific functional gene ACE (I/D) variants of the renin-angiotensin system with essential hypertension (EH) and interaction between ACE (I/D) gene and risk factors for EH in a genetically homogenous Mongolia rural population of China. Methods Individuals (n=1099) were recruited from general population of Kezuohouqi Banner in Inner Mongolian Autonomous Region. Results The association was found between ACE genotype DD plus ID and EH, with an interaction between ACE genotype DD plus ID and cigarette smoking in an additive model. Cigarette smoking index and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 7.10 to 1.16. Interaction between ACE genotype DD plus ID and alcohol drinking on EH appeared an additive model. Alcohol drinking index and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 1.66 to 1.09. BMI and ACE gene showed a low exposure-gene (LEG) effect on EH, with interaction indices from 6.15 to 2.49. Interactions between ACE genotype and WHR on EH showed a multiplicative model. In a short, there was an interaction between ACE gene and cigarette smoking, alcohol drinking and BMI on EH, especially in a low dose-exposure effect. Conclusion It is important for individuals who carry ACE D allele gene to prevent EH, and furthermore, to prevent and control coronary heart disease, in a view of population-based prevention.

  19. Materials Test Branch

    Science.gov (United States)

    Gordon, Gail

    2012-01-01

    The Materials Test Branch resides at Marshall Space Flight Center's Materials and Processing laboratory and has a long history of supporting NASA programs from Mercury to the recently retired Space Shuttle. The Materials Test Branch supports its customers by supplying materials testing expertise in a wide range of applications. The Materials Test Branch is divided into three Teams, The Chemistry Team, The Tribology Team and the Mechanical Test Team. Our mission and goal is to provide world-class engineering excellence in materials testing with a special emphasis on customer service.

  20. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  1. Enhancement of flowering and branching phenotype in chrysanthemum by expression of ipt under the control of a 0.821 kb fragment of the LEACO1 gene promoter

    Czech Academy of Sciences Publication Activity Database

    Khodakovskaya, M.; Vaňková, Radomíra; Malbeck, Jiří; Li, A.; Li, Y.; McAvoy, R.

    2009-01-01

    Roč. 28, č. 9 (2009), s. 1351-1362. ISSN 0721-7714 Institutional research plan: CEZ:AV0Z50380511 Keywords : ipt Gene * Cytokinins * Transgenic chrysanthemum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.301, year: 2009

  2. Elucidating the interplay between DNA-condensing and free polycations in gene transfection through a mechanistic study of linear and branched PEI

    DEFF Research Database (Denmark)

    Dai, Zhuojun; Gjetting, Torben; Mattebjerg, Maria Ahlm;

    2011-01-01

    In the present study we compare LPEI and BPEI characteristics related to DNA condensation and their role as free polycation chains in gene transfection. Using radioactive 32P labeled DNA, we investigated the effect of free PEI chains on the cellular uptake of polyplexes. Our investigations show d...

  3. Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count.

    Science.gov (United States)

    Loureiro, Bárbara; Ereno, Ronaldo L; Favoreto, Mauricio G; Barros, Ciro M

    2016-07-15

    Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus animals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concentration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concentration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers. PMID:26948295

  4. Implication of Xenobiotic Metabolizing Enzyme gene (CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 Polymorphisms in Breast Carcinoma

    Directory of Open Access Journals (Sweden)

    Gabbouj Sallouha

    2008-04-01

    Full Text Available Abstract Background Xenobiotic Metabolizing Enzymes (XMEs contribute to the detoxification of numerous cancer therapy-induced products. This study investigated the susceptibility and prognostic implications of the CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 gene polymorphisms in breast carcinoma patients. Methods The authors used polymerase chain reaction and restriction enzyme digestion to characterize the variation of the CYP2E1, CYP2C19, CYP2D6, mEH and NAT2 gene in a total of 560 unrelated subjects (246 controls and 314 patients. Results The mEH (C/C mutant and the NAT2 slow acetylator genotypes were significantly associated with breast carcinoma risk (p = 0.02; p = 0.01, respectively. For NAT2 the association was more pronounced among postmenopausal patients (p = 0.006. A significant association was found between CYP2D6 (G/G wild type and breast carcinoma risk only in postmenopausal patients (p = 0.04. Association studies of genetic markers with the rates of breast carcinoma specific overall survival (OVS and the disease-free survival (DFS revealed among all breast carcinoma patients no association to DFS but significant differences in OVS only with the mEH gene polymorphisms (p = 0.02. In addition, the mEH wild genotype showed a significant association with decreased OVS in patients with axillary lymph node-negative patients (p = 0.03 and with decreasesd DFS in patients with axillary lymph node-positive patients (p = 0.001. However, the NAT2 intermediate acetylator genotype was associated with decreased DFS in axillary lymph node-negative patients. Conclusion The present study may prove that polymorphisms of some XME genes may predict the onset of breast carcinoma as well as survival after treatment.

  5. Acute intermittent porphyria: A single-base deletion and a nonsense mutation in the human hydroxymethylbilane synthase gene, predicting truncations of the enzyme polypeptide

    Energy Technology Data Exchange (ETDEWEB)

    Lee, G.L.; Astrin, K.H.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States)

    1995-08-28

    Acute intermittent porphyria (AIP) is an autosomal-dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase (HMB-synthase). AIP is an ecogenetic condition, since the life-threatening acute attacks are precipitated by various factors, including drugs, alcohol, fasting, and certain hormones. Biochemical diagnosis is problematic, and the identification of mutations in the HMB-synthase gene provides accurate detection of presymptomatic heterozygotes, permitting avoidance of the acute precipitating factors. By direct solid-phase sequencing, two mutations causing AIP were identified, an adenine deletion at position 629 in exon 11(629delA), which alters the reading frame and predicts premature truncation of the enzyme protein after amino acid 255, and a nonsense mutation in exon 12 (R225X). These mutations were confirmed by either restriction enzyme analysis or family studies of symptomatic patients, permitting accurate presymptomatic diagnosis of affected relatives. 29 refs., 2 figs.

  6. PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

    OpenAIRE

    Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

    2014-01-01

    Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by enviro...

  7. Dietary probiotic live yeast modulates antioxidant enzyme activities and gene expression of sea bass (Dicentrarchus labrax) larvae

    OpenAIRE

    Tovar-ramirez, D.; Mazurais, David; Gatesoupe, J. F.; Quazuguel, Patrick; Cahu, Chantal; Zambonino-infante, J. L.

    2010-01-01

    The main goal of this work was to determine the effect of dietary live yeast Debaryomyces hansenii on the enzymatic antioxidative status of sea bass Dicentrarchus labrax larvae. Growth, activity and expression of the main antioxidative enzymes: catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD), and heat shock protein (HSP70) were measured in sea bass larvae at 23 and 48 days after hatching. Larvae were fed on two microdiets: group one, fed microdiet containing live y...

  8. Branching processes in biology

    CERN Document Server

    Kimmel, Marek

    2015-01-01

    This book provides a theoretical background of branching processes and discusses their biological applications. Branching processes are a well-developed and powerful set of tools in the field of applied probability. The range of applications considered includes molecular biology, cellular biology, human evolution and medicine. The branching processes discussed include Galton-Watson, Markov, Bellman-Harris, Multitype, and General Processes. As an aid to understanding specific examples, two introductory chapters, and two glossaries are included that provide background material in mathematics and in biology. The book will be of interest to scientists who work in quantitative modeling of biological systems, particularly probabilists, mathematical biologists, biostatisticians, cell biologists, molecular biologists, and bioinformaticians. The authors are a mathematician and cell biologist who have collaborated for more than a decade in the field of branching processes in biology for this new edition. This second ex...

  9. The Olive Branch Awards.

    Science.gov (United States)

    Harnack, William

    1984-01-01

    The first annual Olive Branch Awards, sponsored by the Writers' and Publishers Alliance and the Editors' Organizing Committee, were given to ten magazines, out of 60 that submitted entries. Winning entries are described briefly. (IM)

  10. Koenigs function and branching processes

    OpenAIRE

    Tchikilev, O. G.

    2001-01-01

    An explicit solution of time-homogeneous pure birth branching processes is described. It gives alternative extensions for the negative binomial distribution (branching processes with immigration) and for the Furry-Yule distribution (branching processes without immigration).

  11. PTM-Switchboard—a database of posttranslational modifications of transcription factors, the mediating enzymes and target genes

    OpenAIRE

    Everett, Logan; Vo, Antony; Hannenhalli, Sridhar

    2008-01-01

    Gene transcription is largely regulated by sequence-specific transcription factors (TFs). The TF activity is significantly regulated by its posttranslational modifications (PTMs). TF-PTMs serve as ‘molecular switchboards’ that map multiple upstream signaling events, in response to various environmental perturbations, to the downstream transcriptional events. While many instances of TF-PTMs and their effect on gene regulation have been experimentally determined, a systematic meta-analysis or a...

  12. Genetic variation in angiotensin-converting enzyme 2 gene is associated with extent of left ventricular hypertrophy in hypertrophic cardiomyopathy

    OpenAIRE

    van der Merwe, Lize; Cloete, Ruben; Revera, Miriam; Heradien, Marshall; Goosen, Althea; Corfield, Valerie A.; Paul A Brink; Moolman-Smook, Johanna C

    2008-01-01

    Hypertrophic cardiomyopathy, a common, inherited cardiac muscle disease, is primarily caused by mutations in sarcomeric protein-encoding genes and is characterized by overgrowth of ventricular muscle that is highly variable in extent and location. This variability has been partially attributed to locus and allelic heterogeneity of the disease-causing gene, but other factors, including unknown genetic factors, also modulate the extent of hypertrophy that develops in response to the defective s...

  13. The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

    OpenAIRE

    Jaramillo-Gutierrez, Giovanna; Molina-Cruz, Alvaro; Kumar, Sanjeev; Barillas-Mury, Carolina

    2010-01-01

    Background OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. Methodology/Principal Findings OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OX...

  14. In situ hybridization chain reaction mediated ultrasensitive enzyme-free and conjugation-free electrochemcial genosensor for BRCA-1 gene in complex matrices.

    Science.gov (United States)

    Yang, Hui; Gao, Yang; Wang, Siqi; Qin, You; Xu, Lu; Jin, Dan; Yang, Fan; Zhang, Guo-Jun

    2016-06-15

    In this study, we report an enzyme-free and conjugation-free electrochemical genosensor enabling an ultrasensitive readout of BRCA-1, a breast cancer susceptibility gene. The sensor employs a target-responsive hybridization chain reaction (HCR) to significantly amplify the detectable current signals. By means of a functional auxiliary probe pair and a versatile initiator sequence, a linear DNA concatemer structure can be formed via spontaneous and continuous polymerization of DNA oligomers in the presence of target sequence. Such a DNA nanoassembly endows the genosensor an ultrahigh sensitivity up to 1 aM, which is higher than that of the nanomaterials-based or enzyme mediated amplification approaches by several orders of magnitude. More importantly, the sensor's responsive peak current exhibits a favorable linear correlation to the logarithm of the concentrations of target sequence ranging from 1aM to 10pM. In addition, the sensor is highly selective, and can discriminate a single mismatched sequence. This HCR-based genosensor is also capable of probing low-abundance BRCA-1 gene sequence directly in complex matrices, such as 50% human serum, with minimal interference. These advantages will make our tailor-engineered HCR-based electrochemical genosensor appealing to genetic analysis and clinical diagnostics. PMID:26875018

  15. Ameliorating effect of wheat bran, Beta-carotene and Curcumin on K-ras gene mutations and expression of ntioxidant enzymes in rat colon cancer

    International Nuclear Information System (INIS)

    In Egypt, colon cancer has unique characterises differ than other countries, more than third cases happen in people under 40 years, with advanced stage, high grade tumors that carry more mutations . This may be return to increase pollution in food and water. The aim of the present study, is the investigation of the role of some natural products approaches for colorectal carcinoma including curcumin, wheat bran and β-Carotene. Accordingly, animals were injected with 1,2-dimethylhydrazine hydrochloride (DMH) and/or dually exposed to ionizing radiation to induce colorectal cancer. The frequency of mutation of K-ras gene, the level activity of SOD, GpX antioxidant enzymes and expression of SOD1, SOD2 and GpX1 in tissue of 120 colon rats from 10 different treated groups were studied. Curcumin, wheat bran and D-carotene have inhibition effect on formation of colon cancer and decrease the mutations in K-ras gene. Moreover, they have ameliorating effect on antioxidants enzymes activities and expressions. The present study revealed that wheat bran and D-carotene have better effect than curcumin.

  16. EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF); Scientific Opinion on Flavouring Group Evaluation 3, Revision 2 (FGE.03Rev2): Acetals of branched- and straight-chain aliphatic saturated primary alcohols and branched- and straight-chain saturated or unsaturated

    DEFF Research Database (Denmark)

    Larsen, John Christian; Nørby, Karin Kristiane; Beltoft, Vibe Meister;

    The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to evaluate one flavouring substance, acetaldehyde ethyl isopropyl acetal [FL-no: 06.137], structurally related to the 58 flavouring substances in the Flavouring Group...... structure-activity relationships, intake from current uses, toxicological threshold of concern, and available data on metabolism and toxicity. The Panel concluded as for the other already evaluated substances that the substance [FL-no: 06.137] do not give rise to safety concern at its level of dietary...

  17. Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis

    Directory of Open Access Journals (Sweden)

    McLendon Roger E

    2010-11-01

    Full Text Available Abstract Background Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. Results B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. Conclusions These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

  18. Studying the organization of genes encoding plant cell wall degrading enzymes in Chrysomela tremula provides insights into a leaf beetle genome.

    Science.gov (United States)

    Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G

    2014-06-01

    The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. PMID:24456018

  19. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

    Science.gov (United States)

    Meneghini, Vasco; Lattanzi, Annalisa; Tiradani, Luigi; Bravo, Gabriele; Morena, Francesco; Sanvito, Francesca; Calabria, Andrea; Bringas, John; Fisher-Perkins, Jeanne M; Dufour, Jason P; Baker, Kate C; Doglioni, Claudio; Montini, Eugenio; Bunnell, Bruce A; Bankiewicz, Krystof; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2016-01-01

    Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD. PMID

  20. Kunstige Enzymer

    DEFF Research Database (Denmark)

    Bols, Mikael; Bjerre, Jeannette; Marinescu, Lavinia

    2007-01-01

    Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin.......Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin....

  1. Evaluation of the gene encoding the enzyme βHPMEH for the bacterial wilt inhibition caused by Ralstonia solanacearum

    Directory of Open Access Journals (Sweden)

    Elizabeth Fernandez

    2015-10-01

    Full Text Available Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density. R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

  2. Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides.

    Science.gov (United States)

    Schwintner, C; Sabaty, M; Berna, B; Cahors, S; Richaud, P

    1998-08-15

    Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1. PMID:9742704

  3. Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme

    Directory of Open Access Journals (Sweden)

    Buonocore Vincenzo

    2006-10-01

    Full Text Available Abstract Background Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts. Results In order to obtain a simple and efficient source of laccase, the lcc1 cDNA isolated from the white-rot fungus Trametes trogii has been successfully expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol induced alcohol oxidase promoter PAOX1. The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass. The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by T. trogii. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid. Conclusion Heterologous expression of T. trogii laccase lcc1 in the methylotrophic yeast P. pastoris was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.

  4. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report. [In vitro cultivation of Nicotiana tissues and enzymological studies of gene expression at the cell level

    Energy Technology Data Exchange (ETDEWEB)

    None

    1979-01-01

    Progress over the first 9 months of the project has been substantial along several avenues. We have focussed on Nicotiana sylvestris for intensive study for the reasons specified. The individual characteristics of this organism dictate the need to adapt cell culture techniques to the particular requirements of this species. We have devoted considerable effort to optimization of our system through largely empirical experimentation. Methodological advances have been made to improve techniques for isolating enzyme substrates (mainly pretyrosine) that are not commercially available and for refining analytical techniques for the qualitative assay of the new enzyme activities of aromatic biosynthesis recently found by our group. Enzymological studies have been carried out in organismal plant material as a part of the ultimate goal of defining gene expression at the organismal level in relationship to expression at the cell culture level.

  5. Distinct Regulatory Changes Underlying Differential Expression of TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR Genes Associated with Petal Variations in Zygomorphic Flowers of Petrocosmea spp. of the Family Gesneriaceae.

    Science.gov (United States)

    Yang, Xia; Zhao, Xiao-Ge; Li, Chao-Qun; Liu, Jing; Qiu, Zhi-Jing; Dong, Yang; Wang, Yin-Zheng

    2015-11-01

    CYCLOIDEA (CYC)-like genes, belonging to the plant-specific TCP transcription factor family that is named after TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYC from Antirrhinum majus, and the PROLIFERATING CELL FACTORS (PCF) from rice (Oryza sativa), have conserved dorsal identity function in patterning floral zygomorphy mainly through specific expression in dorsal petals of a flower. Their expression changes are usually related to morphological diversity of zygomorphic flowers. However, it is still a challenge to elucidate the molecular mechanism underlying their expression differentiation. It is also unknown whether CINCINNATA (CIN)-like TCP genes, locally controlling cell growth and proliferation, are involved in the evolution of floral zygomorphy. To address these questions, we selected two closely related species, i.e. Petrocosmea glabristoma and Petrocosmea sinensis, with distinct petal morphology to conduct expression, hybridization, mutant, and allele-specific expression analyses. The results show that the size change of the dorsal petals between the two species is mainly mediated by the expression differentiation of CYC1C and CYC1D, while the shape variation of all petals is related to the expression change of CIN1. In reciprocal F1 hybrids, the expression of CYC1C, CYC1D, and CIN1 conforms to an additive inheritance mode, consistent with the petal phenotypes of hybrids. Through allele-specific expression analyses, we find that the expression differentiation of these TCP genes is underlain by distinctly different types of regulatory changes. We suggest that highly redundant paralogs with identical expression patterns and interspecific expression differentiation may be controlled by remarkably different regulatory pathways because natural selection may favor different regulatory modifications rather than coding sequence changes of key developmental genes in generating morphological diversity. PMID:26351309

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

    Science.gov (United States)

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

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism. PMID:26882658

  7. Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

    Science.gov (United States)

    Lei, B; Liu, M; Huang, S; Tu, S C

    1994-01-01

    NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism. Images PMID:8206832

  8. Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efifciency During the Conversion of Lycopene-to-Provitamin A

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-cheng; ZHOU Wen-jing; XU Qiang; TAO Neng-guo; YE Jun-li; GUO Fei; XU Juan; DENG Xiu-xin

    2013-01-01

    Citrus fruits are rich in carotenoids. In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYb, EC:1.14.-.-) is a key regulatory enzyme in the catalysis of lycopene to β-carotene, an important dietary precursor of vitamin A for human nutrition. Two closely related lycopeneβ-cyclase cDNAs, designated CsLCYb1 and CsLCYb2, were isolated from the pulp of orange fruits (Citrus sinensis). The expression level of CsLCYb genes is lower in the lfavedo and juice sacs of a lycopene-accumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efifciently converted lycopene into the bicyclicβ-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into theβ-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the caulilfower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene intoβ-carotene, and the ripe fruits displayed a bright orange colour. However, the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation. In fruits of the CsLCYb1 transgenic plants, most of the lycopene was converted intoβ-carotene with provitamin A levels reaching about 700 µg g-1 DW. Unexpectedly, most transgenic tomatoes showed a reduction in total carotenoid accumulation, and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits. Collectively, these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efifciency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

  9. Comparative genomics of the protocatechuate branch of the β-ketoadipate pathway in the Roseobacter lineage.

    Science.gov (United States)

    Alejandro-Marín, Catalina Maria; Bosch, Rafael; Nogales, Balbina

    2014-10-01

    The protocatechuate branch of the β-ketoadipate pathway is the most common pathway for degradation of monoaromatic compounds in the Roseobacter lineage. We analyzed 43 Roseobacter genomes in order to determine if they possessed all genetic elements for this pathway and if there were common patterns in gene organization. The eight genes of the pathway (pcaG, -H, -B, -C, -D, -I, -J, and -F), possible regulators, and genes encoding for proteins with related function (i.e. catabolism of 4-hydroxybenzoate, catechol, and meta-cleavage of protocatechuate) were predicted by sequence homology analysis. Most of the Roseobacters studied had putatively a complete protocatechuate branch of the β-ketoadipate pathway while 11 of them would probably have an incomplete pathway. Thirty-one Roseobacters would be potentially able of transforming 4-hydroxybenzoate to protocatechuate, and 13 of them might transform catechol via ortho-cleavage, the starting reaction of the catechol branch of the β-ketoadipate pathway. We observed variability in gene organization, with no clear relationship between gene order and Roseobacter taxonomy. Genes were usually organized in several gene clusters. One of the clusters (pcaRIJF) was not reported previously in Roseobacters. The presence of the putative regulator pcaR in these bacteria was also a novel finding. The conserved ORF (chp), encoding for a protein of family DUF849 whose functional role has been proven recently, was detected in 34 genomes. Sequence homology confirmed that proteins encoded by chp corresponded to putative BKACE G4 proteins, which are able to transform β-ketoadipate. Therefore, most Roseobacters seemed to possess two different enzymes for transforming β-ketoadipate. We also report two possible regulation mechanisms of gene pobA (encoding for the enzyme transforming 4-hydroxybenzoate to protocatechuate): via PcaQ, the regulator commonly found with pca genes, and via an independent regulator (PobR). The results of this study

  10. Cloning of the beta-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme.

    OpenAIRE

    Nanmori, T; M. Nagai; Shimizu, Y.; Shinke, R; Mikami, B

    1993-01-01

    The gene encoding the beta-amylase of Bacillus cereus BQ10-S1 (SpoII) was cloned into Escherichia coli JM 109. A sequenced DNA fragment of 2,001 bp contains the beta-amylase gene. The N-terminal sequences (AVNGKG MNPDYKAYLMAPLKKI), the C-terminal sequences (SHTSSW), and the amino acid sequences of the five regions in the beta-amylase molecules were determined. The mature beta-amylase contains 514 amino acid residues with a molecular mass of 57,885 Da. The amino acid sequence homology with tho...

  11. Fragrance Release from the Surface of Branched Poly (Amide S

    Directory of Open Access Journals (Sweden)

    T. Youngs

    2005-01-01

    Full Text Available Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased.

  12. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo;

    2014-01-01

    hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas...

  13. Cloning and expression of lipoxygenase genes and enzyme activity in ripening persimmon fruit in response to GA and ABA treatment

    Science.gov (United States)

    Two genes of the lipoxygenase (LOX) family, DkLox1 and DkLox3 (GenBank accession No. JF436951 and JF436950), were cloned from persimmon fruit (Diospyros kaki L. ‘Fuping Jianshi’). Sequence analysis indicated that they belong to the 9-LOX sub-group. Heterologous expression of DkLox1 in E. coli produc...

  14. Silencing C19-GA 2-oxidases induces parthenocarpic development and inhibits lateral branching in tomato plants.

    Science.gov (United States)

    Martínez-Bello, Liliam; Moritz, Thomas; López-Díaz, Isabel

    2015-09-01

    Gibberellins (GAs) are phytohormones that regulate a wide range of developmental processes in plants. Levels of active GAs are regulated by biosynthetic and catabolic enzymes like the GA 2-oxidases (GA2oxs). In tomato (Solanum lycopersicum L.) C19 GA2oxs are encoded by a small multigenic family of five members with some degree of redundancy. In order to investigate their roles in tomato, the silencing of all five genes in transgenic plants was induced. A significant increase in active GA4 content was found in the ovaries of transgenic plants. In addition, the transgenic unfertilized ovaries were much bigger than wild-type ovaries (about 30 times) and a certain proportion (5-37%) were able to develop parthenocarpically. Among the GA2ox family, genes GA2ox1 and -2 seem to be the most relevant for this phenotype since their expression was induced in unfertilized ovaries and repressed in developing fruits, inversely correlating with ovary growth. Interestingly, transgenic lines exhibited a significant inhibition of branching and a higher content of active GA4 in axillary buds. This phenotype was reverted, in transgenic plants, by the application of paclobutrazol, a GA biosynthesis inhibitor, suggesting a role for GAs as repressors of branching. In summary, this work demonstrates that GA 2-oxidases regulate gibberellin levels in ovaries and axillary buds of tomato plants and their silencing is responsible for parthenocarpic fruit growth and branching inhibition. PMID:26093022

  15. Right bundle branch block

    DEFF Research Database (Denmark)

    Bussink, Barbara E; Holst, Anders Gaarsdal; Jespersen, Lasse;

    2013-01-01

    AimsTo determine the prevalence, predictors of newly acquired, and the prognostic value of right bundle branch block (RBBB) and incomplete RBBB (IRBBB) on a resting 12-lead electrocardiogram in men and women from the general population.Methods and resultsWe followed 18 441 participants included in...... men vs. 0.5%/2.3% in women, P <0.001). Significant predictors of newly acquired RBBB were male gender, increasing age, high systolic blood pressure, and presence of IRBBB, whereas predictors of newly acquired IRBBB were male gender, increasing age, and low BMI. Right bundle branch block was associated...... with significantly increased all-cause and cardiovascular mortality in both genders with age-adjusted hazard ratios (HR) of 1.31 [95% confidence interval (CI), 1.11-1.54] and 1.87 (95% CI, 1.48-2.36) in the gender pooled analysis with little attenuation after multiple adjustment. Right bundle branch...

  16. Comparison between ultrafine and fine particulate matter collected in Lebanon: Chemical characterization, in vitro cytotoxic effects and metabolizing enzymes gene expression in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    During the last few years, the induction of toxicological mechanisms by atmospheric ultrafine particles (UFP) has become one of the most studied topics in toxicology and a subject of huge debates. Fine particles (FP) and UFP collected at urban and rural sites in Lebanon were studied for their chemical composition and toxicological effects. UFP were found more enriched in trace elements, secondary inorganic ions, total carbon and organic compounds than FP. For toxicological analysis, BEAS-2B cells were exposed for 24, 48 and 72 h to increasing concentrations of FP, water-UFP suspension (UFPw) and UFP organic extract (UFPorg). Our findings showed that UFP caused earlier alterations of mitochondrial metabolism and membrane integrity from the lowest concentrations. Moreover, a significant induction of CYP1A1, CYP1B1 and AhRR genes expression was showed after cells exposure to UFPorg and to a lesser extent to UFPw and FP samples. - Highlights: • The main source of air pollution in Beirut is road traffic and generator sets. • More trace elements, secondary inorganic ions and organic compounds are found in UFP. • UFP cause early alterations of mitochondrial metabolism and membrane integrity. • PM induce XME gene expression leading to the formation of PAHs-reactive metabolites. • All the results evidenced a higher toxicity of UFP compared to FP. - Ultrafine particles collected in Lebanon: higher organic compounds contents than in fine particles and therefore higher induction of xenobiotic-metabolizing enzymes genes expression leading to more DNA damages

  17. Clinically relevant genetic variants of drug-metabolizing enzyme and transporter genes detected in Thai children and adolescents with autism spectrum disorder

    Science.gov (United States)

    Medhasi, Sadeep; Pasomsub, Ekawat; Vanwong, Natchaya; Ngamsamut, Nattawat; Puangpetch, Apichaya; Chamnanphon, Montri; Hongkaew, Yaowaluck; Limsila, Penkhae; Pinthong, Darawan; Sukasem, Chonlaphat

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) among drug-metabolizing enzymes and transporters (DMETs) influence the pharmacokinetic profile of drugs and exhibit intra- and interethnic variations in drug response in terms of efficacy and safety profile. The main objective of this study was to assess the frequency of allelic variants of drug absorption, distribution, metabolism, and elimination-related genes in Thai children and adolescents with autism spectrum disorder. Blood samples were drawn from 119 patients, and DNA was extracted. Genotyping was performed using the DMET Plus microarray platform. The allele frequencies of the DMET markers were generated using the DMET Console software. Thereafter, the genetic variations of significant DMET genes were assessed. The frequencies of SNPs across the genes coding for DMETs were determined. After filtering the SNPs, 489 of the 1,931 SNPs passed quality control. Many clinically relevant SNPs, including CYP2C19*2, CYP2D6*10, CYP3A5*3, and SLCO1B1*5, were found to have frequencies similar to those in the Chinese population. These data are important for further research to investigate the interpatient variability in pharmacokinetics and pharmacodynamics of drugs in clinical practice. PMID:27110117

  18. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia

    Energy Technology Data Exchange (ETDEWEB)

    Vulliamy, T.J.; D' Urso, M.; Battistuzzi, G.; Estrada, M.; Foulkes, N.S.; Martini, G.; Calabro, V.; Poggi, V.; Giordano, R.; Town, M.; Luzzatto, L.; Persico, M.G. (Royal Postgraduate Medical School, London (England))

    1988-07-01

    Glucose-6-phosphate dehydrogenase deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. The authors have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A they have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. The mutations observed show a striking predominance of C {yields} T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

  19. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia

    International Nuclear Information System (INIS)

    Glucose-6-phosphate dehydrogenase deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. The authors have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A they have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. The mutations observed show a striking predominance of C → T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency

  20. A Nitrogen-Regulated Glutamine Amidotransferase (GAT1_2.1) Represses Shoot Branching in Arabidopsis[C][W

    Science.gov (United States)

    Zhu, Huifen; Kranz, Robert G.

    2012-01-01

    Shoot branching in plants is regulated by many environmental cues and by specific hormones such as strigolactone (SL). We show that the GAT1_2.1 gene (At1g15040) is repressed over 50-fold by nitrogen stress, and is also involved in branching control. At1g15040 is predicted to encode a class I glutamine amidotransferase (GAT1), a superfamily for which Arabidopsis (Arabidopsis thaliana) has 30 potential members. Most members can be categorized into known biosynthetic pathways, for the amidation of known acceptor molecules (e.g. CTP synthesis). Some members, like GAT1_2.1, are of unknown function, likely involved in amidation of unknown acceptors. A gat1_2.1 mutant exhibits a significant increase in shoot branching, similar to mutants in SL biosynthesis. The results suggest that GAT1_2.1 is not involved in SL biosynthesis since exogenously applied GR24 (a synthetic SL) does not correct the mutant phenotype. The subfamily of GATs (GATase1_2), with At1g15040 as the founding member, appears to be present in all plants (including mosses), but not other organisms. This suggests a plant-specific function such as branching control. We discuss the possibility that the GAT1_2.1 enzyme may activate SLs (e.g. GR24) by amidation, or more likely could embody a new pathway for repression of branching. PMID:22885937

  1. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  2. Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case

    DEFF Research Database (Denmark)

    Hansen, Bjarne Gram; Salomonsen, Bo; Nielsen, Morten Thrane;

    2011-01-01

    Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamen...

  3. Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli.

    OpenAIRE

    Berry, A M; Lock, R A; Thomas, S M; Rajan, D P; Hansman, D.; Paton, J C

    1994-01-01

    A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert,...

  4. Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved From Different Invertases. Expression of Fructan Genes Throughout Chicory Development

    OpenAIRE

    Wim Van den Ende; An Michiels; Joke De Roover; Andrea Van Laere

    2002-01-01

    Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The biochemistry of fructan biosynthesis in dicots has been resolved, and the respective cDNAs have been cloned. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar-type invertase...

  5. A CHROMATIN MODIFYING ENZYME, SDG8, IS REQUIRED FOR MORPHOLOGICAL, GENE EXPRESSION, AND EPIGENETIC RESPONSES TO MECHANICAL STIMULATION

    OpenAIRE

    Christopher Ian Cazzonelli; Nazia eNisar; Roberts, Andrea C.; Kevin eMurray; Borevitz, Justin O; Barry James Pogson

    2014-01-01

    Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzy...

  6. Pulmonary Embolism in a Sarcoidosis Patient Double Heterozygous for Methylenetetrahydrofolate Reductase Gene Polymorphisms and Factor V Leiden and Homozygous for the D-Allele of Angiotensin Converting Enzyme Gene

    Directory of Open Access Journals (Sweden)

    Nadim El-Majzoub

    2015-01-01

    Full Text Available Sarcoidosis is a multisystem granulomatous disease of unknown etiology and pathogenesis. It presents in patients younger than 40 years of age. The lungs are the most commonly affected organ. Till the present day, there is no single specific test that will accurately diagnose sarcoidosis; as a result, the diagnosis of sarcoidosis relies on a combination of clinical, radiologic, and histologic findings. Patients with sarcoidosis have been found to have an increased risk of pulmonary embolism compared to the normal population. MTHFR and factor V Leiden mutations have been reported to increase the risk of thrombosis in patients. We hereby present a case of a middle aged man with sarcoidosis who developed a right main pulmonary embolism and was found to be double heterozygous for methylenetetrahydrofolate reductase gene polymorphisms and factor V Leiden and homozygous for the D-allele of the angiotensin converting enzyme gene.

  7. Gene expression of drug metabolizing enzymes in adult and aged mouse liver: A modulation by immobilization stress

    International Nuclear Information System (INIS)

    The role of stress in the regulation of enzymatic systems involved in the biotransformation of xenobiotics, as well as endogenous substrates in the liver was investigated using single immobilization stress as a model. Adult (3 months of age) and aged (26 months) C3H/a male mice were used. Cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2), glutathione S-transferase M1 (GSTM1), aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and catechol-O-methyltransferase (COMT) mRNA levels in the mouse liver were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Excluding CYP1A1, experiments revealed significant differences in the expression of these genes between adult- and aged-control animals. The influence of stress on the expression of genes studied was shown to be higher in adult mice than in aged ones. Our results clearly demonstrate the lack of response or even the attenuation of gene expression in aged animals that may play an important role in age-related pathologies and diseases

  8. Cloning and Characterization of the Phytoene Desaturase(pds) Gene-a Key Enzyme for Carotenoids Synthesis in Dunaliella (Chlorophyta)

    Institute of Scientific and Technical Information of China (English)

    SUN Guohua; SUI Zhenghong; ZHANG Xuecheng

    2008-01-01

    The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga, phytoene desaturase (pals) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp (GenBank Accession No. DQ243892) was de- duced from RACE results, including untranslated 21 bp 5'- and 520 bp 3'- flanking regions and an open reading frame of 582 amino acids, coding a protein of 64.196 kDa. The DNA sequence of 2908 bp (GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex, including two bases' perfect repeats (GT)10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species, and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance ofpds was discussed.

  9. Metabolic enzyme activities, metabolism-related genes expression and bioaccumulation in juvenile white shrimp Litopenaeus vannamei exposed to benzo[a]pyrene.

    Science.gov (United States)

    Ren, Xianyun; Pan, Luqing; Wang, Lin

    2014-06-01

    The purpose of this study was to investigate the impact of benzo[a]pyrene (BaP) on metabolic detoxification system and bioaccumulation of white shrimp Litopenaeus vannamei. In this study, juvenile white shrimp L. vannamei were exposed for 21 days at four different concentrations of 0, 0.03, 0.3 and 3μg/L. Detoxification enzyme activities of phase I (aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (EROD), epoxide hydrolase (EH)) and phase II (glutathione-S-transferase (GST), sulfotransferase (SULT), uridine diphosphate glucuronyl transferase (UGT)) were determined, and results showed that all the detoxification enzyme activities increased in a dose-dependent manner except for the low BaP exposure. Transcription of genes was detected and measured by real-time RT-PCR. It showed that at day six BaP increased cytochrome P450 (CYP) 1A1, GST, SULT visa aryl hydrocarbon receptor (AhR) mRNA expression in a dose-dependent manner, which suggests that they could be potential targets of BaP that disrupt the detoxification system. The consistency of their responses to BaP exposure implies that AhR action may be involved in invertebrate CYP regulation. Additionally, BaP bioaccumulation increased rapidly first and showed an incoming plateau. Besides, the enzyme activities and bioaccumulation in the hepatopancreas were higher than those in the gills. These results will not only provide information on BaP metabolic mechanism for this species, but also scientific data for pollution monitoring. PMID:24636950

  10. Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.

  11. DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

    International Nuclear Information System (INIS)

    DNA samples prepared from human SP3 cells, which had not been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3±0.7) x 10-3/Mbp/Gy was deduced for 80 kV X-rays. (Author)

  12. Expression analysis of ROS producing and scavenging enzyme-encoding genes in rubber tree infected by Pseudocercospora ulei.

    Science.gov (United States)

    Koop, Daniela Martins; Rio, Maryannick; Sabau, Xavier; Almeida Cardoso, Saulo Emilio; Cazevieille, Chantal; Leclercq, Julie; Garcia, Dominique

    2016-07-01

    South American Leaf Blight (SALB), caused by the ascomycete Pseudocercospora ulei, is responsible for the low productivity of rubber trees in Latin America and is a serious threat to rubber plantations in Asia and Africa, where the rubber trees are derived from highly susceptible clones. Three contrasted genotypes were chosen for their levels of resistance to the pathogen: FX2784 (totally resistant), MDF180 (partially resistant) and PB314 (susceptible). Array analyses were previously performed to identify genes differentially expressed in resistant and susceptible genotypes. Twenty-one genes were selected for further gene expression analysis in non-inoculated and inoculated genotypes from 24 to 216 h post infection (hpi). These genes are involved in ROS production (HbRBOHA, HbRBOHB, HbRBOHC, HbRBOHD), ROS-scavenging systems (cytoplasmic and chloroplastic HbCuZnSOD, HbMnSOD, HbCAT, HbAPX1, HbAPX2, HbMDHAR, HbGCL1, HbGCL2, HbOASTL, HbGPX, HbDHAR), and leaf senescence (HbCASP, HbPCYST, HbWRKY2, HbPLY, HbKAT2). First, a genotype-dependent level of expression was observed. The genes HbRBOHA, HbCuZnSOD cyto, HbCAT, HbGCL and HbWRKY2 were constitutively expressed at lower levels in the MDF180 genotype than in the FX2784 and PB314 genotypes. Conversely, the levels of expression of HbDHAR, HbGPX and HbPCYST were higher in the older, non-inoculated leaves of MDF180. Lower production of ROS and efficient regeneration of reduced ascorbate ensure a balanced redox intracellular state in this genotype. Second, inoculation of the leaves induced few modifications in the expression level of the studied genes. In the MDF180 partially resistant genotype, an increase in the expression level of HbRBOHB, HbRBOHD 48 hpi and a decrease in the expression level of HbDHAR 216 hpi were observed. In the FX2784 totally resistant genotype, an increase in the expression level of HbRBOHD and HbCuZnSOD cyto and a decrease in HbCAT were observed 48 hpi. This transitory variation could be associated

  13. Association between insertion/deletion polymorphism in angiotensin-converting enzyme gene and acute lung injury/acute respiratory distress syndrome: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Matsuda Akihisa

    2012-08-01

    Full Text Available Abstract Background A previous meta-analysis reported a positive association between an insertion/deletion (I/D polymorphism in the angiotensin-converting enzyme gene (ACE and the risk of acute lung injury (ALI/acute respiratory distress syndrome (ARDS. Here, we updated this meta-analysis and additionally assessed the association of this polymorphism with ALI/ARDS mortality. Methods We searched electronic databases through October 2011 for the terms “angiotensin-converting enzyme gene”, “acute lung injury”, and “acute respiratory distress syndrome,” and reviewed all studies that reported the relationship of the I/D polymorphism in ACE with ALI/ARDS in humans. Seven studies met the inclusion criteria, comprising 532 ALI/ARDS patients, 3032 healthy controls, and 1432 patients without ALI/ARDS. We used three genetic models: the allele, dominant, and recessive models. Results The ACE I/D polymorphism was not associated with susceptibility to ALI/ARDS for any genetic model. However, the ACE I/D polymorphism was associated with the mortality risk of ALI/ARDS in Asian subjects ( Pallele Pdominant = 0.001, Precessive = 0.002. This finding remained significant after correction for multiple comparisons. Conclusions There is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.

  14. Reduction in Activity/Gene Expression of Anthocyanin Degradation Enzymes in Lychee Pericarp is Responsible for the Color Protection of the Fruit by Heat and Acid Treatment

    Institute of Scientific and Technical Information of China (English)

    FANG Fang; ZHANG Zhao-qi; ZHANG Xue-lian; WU Zhen-xian; YIN Hui-fang; PANG Xue-qun

    2013-01-01

    Heat and acid treatments were reported to be a promising substitute for SO2 fumigation in color protection of postharvest lychee (Litchi chinensis Sonn.) fruits, but the mechanism was not clear. In the present study, hot water (70°C) dipping followed by immersion in 2%HCl (heat-acid) substantially protected the red color of the fruit during storage at 25°C and inhibited anthocyanin degradation while hot water dipping alone (heat) led to rapidly browning and about 90%loss in anthocyanin content. The pH values in the pericarp of the heat-acid treated fruit dropped to 3.2, while the values maintained around 5.0 in the heat-treated and control fruit. No significantly different pH values were detected among the arils of heat-acid, heat treated and control fruit. Heat-acid treatment dramatically reduced the activities of anthocyanin degradation enzyme (ADE), peroxidase (POD) and polyphenol oxidase in the pericarp. A marked reduction in LcPOD gene expression was also detected in heat-acid treated fruit, in contrast, induction was found in heat treated fruit. The pericarp of heat-acid treated fruit exhibited significantly lower respiration rate but faster water loss than that of the untreated or heat treated fruit. Taken together, heat treatment triggered quick browning and anthocyanin loss in lychee fruit, while heat-acid treatment protected the fruit color by a great reduction in the activities/gene expression of anthocyanin degradation enzymes and acidification of lychee pericarp.

  15. Tobacco smoking, polymorphisms in carcinogen metabolism enzyme genes, and risk of localized and advanced prostate cancer: results from the California Collaborative Prostate Cancer Study

    International Nuclear Information System (INIS)

    The relationship between tobacco smoking and prostate cancer (PCa) remains inconclusive. This study examined the association between tobacco smoking and PCa risk taking into account polymorphisms in carcinogen metabolism enzyme genes as possible effect modifiers (9 polymorphisms and 1 predicted phenotype from metabolism enzyme genes). The study included cases (n = 761 localized; n = 1199 advanced) and controls (n = 1139) from the multiethnic California Collaborative Case–Control Study of Prostate Cancer. Multivariable conditional logistic regression was performed to evaluate the association between tobacco smoking variables and risk of localized and advanced PCa risk. Being a former smoker, regardless of time of quit smoking, was associated with an increased risk of localized PCa (odds ratio [OR] = 1.3; 95% confidence interval [CI] = 1.0–1.6). Among non-Hispanic Whites, ever smoking was associated with an increased risk of localized PCa (OR = 1.5; 95% CI = 1.1–2.1), whereas current smoking was associated with risk of advanced PCa (OR = 1.4; 95% CI = 1.0–1.9). However, no associations were observed between smoking intensity, duration or pack-year variables, and advanced PCa. No statistically significant trends were seen among Hispanics or African-Americans. The relationship between smoking status and PCa risk was modified by the CYP1A2 rs7662551 polymorphism (P-interaction = 0.008). In conclusion, tobacco smoking was associated with risk of PCa, primarily localized disease among non-Hispanic Whites. This association was modified by a genetic variant in CYP1A2, thus supporting a role for tobacco carcinogens in PCa risk

  16. Tracheobronchial Branching Anomalies

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Min Ji; Kim, Young Tong; Jou, Sung Shick [Soonchunhyang University, Cheonan Hospital, Cheonan (Korea, Republic of); Park, A Young [Soonchunhyang University College of Medicine, Asan (Korea, Republic of)

    2010-04-15

    There are various congenital anomalies with respect to the number, length, diameter, and location of tracheobronchial branching patterns. The tracheobronchial anomalies are classified into two groups. The first one, anomalies of division, includes tracheal bronchus, cardiac bronchus, tracheal diverticulum, pulmonary isomerism, and minor variations. The second one, dysmorphic lung, includes lung agenesis-hypoplasia complex and lobar agenesis-aplasia complex

  17. H2O2-Activated Up-Regulation of Glutathione in Arabidopsis Involves Induction of Genes Encoding Enzymes Involved in Cysteine Synthesis in the Chloroplast

    Institute of Scientific and Technical Information of China (English)

    Guillaume Queval; Dorothée Thominet; Hélène Vanacker; Myroslawa Miginiac-Maslow; Bertrand Gakière; Graham Noctor

    2009-01-01

    Glutathione is a key player in cellular redox homeostasis and, therefore, in the response to H2O2, but the factors regulating oxidation-activated glutathione synthesis are still unclear. We investigated H2O2-induced glutathione synthesis in a conditional Arabidopsis catalase-deficient mutant (cat2). Plants were grown from seed at elevated CO2 for 5 weeks, then transferred to air in either short-day or long-day conditions. Compared to cat2 at elevated CO2 or wild-type plants in any condition, transfer of cat2 to air in both photoperiods caused measurable oxidation of the leaf glutathione pool within hours. Oxidation continued on subsequent days and was accompanied by accumulation of glutathione. This effect was stronger in cat2 transferred to air in short days, and was not linked to appreciable increases in the extractable activities of or transcripts encoding enzymes involved in the committed pathway of glutathione synthesis. In contrast, it was accompanied by increases in serine, O-acetylserine, and cysteine. These changes in metabolites were accompanied by induction of genes encoding adenosine phosphosulfate reductase (APR), particularly APR3, as well as a specific serine acetyltransferase gene (SAT2.1) encoding a chloroplastic SAT. Marked induction of these genes was only observed in cat2 transferred to air in short-day conditions, where cysteine and glutathione accumulation was most dramatic. Unlike other SAT genes, which showed negligible induction in cat2, the relative abundance of APR and SAT2.1 transcripts was closely correlated with marker transcripts for H2O2 signaling. Together, the data underline the importance of cysteine synthesis in oxidant-induced up-regulation of glutathione synthesis and suggest that the chloroplast makes an important contribution to cysteine production under these circumstances.

  18. Prolonged expression of the BX1 signature enzyme is associated with a recombination hotspot in the benzoxazinoid gene cluster in Zea mays.

    Science.gov (United States)

    Zheng, Linlin; McMullen, Michael D; Bauer, Eva; Schön, Chris-Carolin; Gierl, Alfons; Frey, Monika

    2015-07-01

    Benzoxazinoids represent preformed protective and allelopathic compounds. The main benzoxazinoid in maize (Zea mays L.) is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to herbivores and microbes. Protective concentrations are found predominantly in young plantlets. We made use of the genetic diversity present in the maize nested association mapping (NAM) panel to identify lines with significant benzoxazinoid concentrations at later developmental stages. At 24 d after imbibition (dai), only three lines, including Mo17, showed effective DIMBOA concentrations of 1.5mM or more; B73, by contrast, had low a DIMBOA content. Mapping studies based on Mo17 and B73 were performed to reveal mechanisms that influence the DIMBOA level in 24 dai plants. A major quantitative trait locus mapped to the Bx gene cluster located on the short arm of chromosome 4, which encodes the DIMBOA biosynthetic genes. Mo17 was distinguished from all other NAM lines by high transcriptional expression of the Bx1 gene at later developmental stages. Bx1 encodes the signature enzyme of the pathway. In Mo17×B73 hybrids at 24 dai, only the Mo17 Bx1 allele transcript was detected. A 3.9kb cis-element, termed DICE (distal cis-element), that is located in the Bx gene cluster approximately 140 kb upstream of Bx1, was required for high Bx1 transcript levels during later developmental stages in Mo17. The DICE region was a hotspot of meiotic recombination. Genetic analysis revealed that high 24 dai DIMBOA concentrations were not strictly dependent on high Bx1 transcript levels. However, constitutive expression of Bx1 in transgenics increased DIMBOA levels at 24 dai, corroborating a correlation between DIMBOA content and Bx1 transcription. PMID:25969552

  19. Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

    International Nuclear Information System (INIS)

    Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 'factory' cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products

  20. The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

    OpenAIRE

    Puri, Sumant; O'Brian, Mark R.

    2006-01-01

    Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ (bll7075) and hmuD (bll7423) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staph...

  1. Angiotensin Converting Enzyme Gene I/D Polymorphism in Pakistani Rheumatic Heart Disease Patients and Healthy Controls

    Directory of Open Access Journals (Sweden)

    Sadia Rehman

    2015-09-01

    Full Text Available Background: Valve scarring and collagen deposition are crucial in pathogenesis of Rheumatic Heart Disease (RHD, an autoimmune disorder of the heart. Angiotensin I-Converting Enzyme (ACE plays a major role in fibrous tissue formation. Objectives: The present research work aimed to assess the role of ACE Insertion/Deletion (I/D polymorphism in progress of RHD. Patients and Methods: DNA was pre pared from blood samples from 156 RHD patients (156 and 204 healthy ethnically-matched controls. Then, it was screened using sequence-specific Primers. Polymerase chain reaction and Agarose gel electrophoresis. The data were analyzed using Vassar stats (http://faculty.vassar.edu/lowry/VassarStats.html. Results: I allele (P = 0.024, OR = 1.42 and II genotype (P = 0.001, OR = 3.07 were significantly higher in Pakistani RHD patients compared to the healthy controls. Also, a significant difference was found between the female, but not male, patients and the controls regarding I allele and II genotype. Conclusions: The study results provided information about involvement of ACE I/D polymorphism in molecular mechanism of RHD. Thus, it can become one of the useful tools in risk assessment and help with designing strategies to combat the disease.

  2. Gene Expression of Desaturase (FADS1 and FADS2) and Elongase (ELOVL5) Enzymes in Peripheral Blood: Association with Polyunsaturated Fatty Acid Levels and Atopic Eczema in 4-Year-Old Children

    OpenAIRE

    Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Àngels; López-Sabater, M Carmen

    2013-01-01

    Abstract Background It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. Methods AE (n=20) and non-AE (n=104) children participating in the ...

  3. Gene expression of desaturase (FADS1 and FADS2) and elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children

    OpenAIRE

    Chisaguano, Aida M.; Montes, Rosa; P??rez Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante Pineda, Mariona; Morales, Eva; Garc??a Esteban, Raquel; Sunyer Deu, Jordi; Franch, ??ngels; L??pez Sabater, M. Carmen

    2013-01-01

    Background: It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. Methods: AE (n=20) and non-AE (n=104) children participating in the Sabadel...

  4. Impact of the interaction between 3’-UTR SNPs and microRNA on the expression of human xenobiotic metabolism enzyme and transporter (XMETs genes

    Directory of Open Access Journals (Sweden)

    Rongrong eWei

    2012-11-01

    Full Text Available Genetic variation in the expression of human XMETs leads to inter-individual variability in metabolism of therapeutic agents as well as differed susceptibility to various diseases. Recent eQTL (expression quantitative traits loci mapping in a few human cells/tissues have identified a number of SNPs significantly associated with mRNA expression of many XMET genes. These eQTLs are therefore important candidate markers for pharmacogenetic studies. However, questions remain about whether these SNPs are causative and in what mechanism these SNPs may function. Given the important role of microRNAs in gene transcription regulation, we hypothesize that those eQTLs or their proxies in strong linkage disequilibrium (LD altering microRNA targeting are likely causative SNPs affecting gene expression. The aim of this study is to identify eQTLs potentially regulating major XMETs via interference with microRNA targeting. To this end, we performed a genome-wide screening for eQTLs for 409 genes encoding major drug metabolism enzymes transporters and transcription factors, in publically available eQTL datasets generated from the HapMap lymphoblastoid cell lines (LCLs and human liver and brain tissue. As a result, 308 eQTLs significantly (p<10-5 associated with mRNA expression of 101 genes were identified. We further identified 7,869 SNPs in strong LD (r2≥0.8 with these eQTLs using the 1000 Genome SNP data. Among these 8,177 SNPs, 27 are located in the 3’-UTR of 14 genes. Using two algorithms predicting microRNA-SNP interaction, we found that almost all these SNPs (26 out of 27 were predicted to create, abolish or change the target site for microRNAs in both algorithms. Many of these microRNAs were also expressed in the same tissue that the eQTL were identified. Our study provides a strong rationale for continued investigation for the functions of these eQTLs in pharmacogenetic settings.

  5. Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

    Directory of Open Access Journals (Sweden)

    Facincani Agda Paula

    2003-01-01

    Full Text Available The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor, Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

  6. Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

    Science.gov (United States)

    Miyazawa, Ken; Yoshimi, Akira; Zhang, Silai; Sano, Motoaki; Nakayama, Mayumi; Gomi, Katsuya; Abe, Keietsu

    2016-09-01

    Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions. PMID:27442340

  7. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

    Science.gov (United States)

    Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

    2016-07-01

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. PMID:26975754

  8. Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides

    DEFF Research Database (Denmark)

    Lübbehüsen, Tina Louise; Polo, V.G.; Rossi, S.; Nielsen, Jens; Moreno, S.; Mcintyre, Mhairi; Arnau, J.

    2004-01-01

    The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift....... An inverse relationship between the pkaR expression level in KFA121 and the hyphal growth unit length was observed in KFA121, suggesting a central role for PKAR in branching. A biochemical analysis of PKAR using antibodies and enzyme assay demonstrated that the level of PKAR is higher in KFA121 under...

  9. Evolutionary rate patterns of the Gibberellin pathway genes

    Directory of Open Access Journals (Sweden)

    Zhang Fu-min

    2009-08-01

    Full Text Available Abstract Background Analysis of molecular evolutionary patterns of different genes within metabolic pathways allows us to determine whether these genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution of proteins in an interacting system. Although previous studies found that upstream genes in the pathway evolved more slowly than downstream genes, the correlation between evolutionary rate and position of the genes in metabolic pathways as well as its implications in molecular evolution are still less understood. Results We sequenced and characterized 7 core structural genes of the gibberellin biosynthetic pathway from 8 representative species of the rice tribe (Oryzeae to address alternative hypotheses regarding evolutionary rates and patterns of metabolic pathway genes. We have detected significant rate heterogeneity among 7 GA pathway genes for both synonymous and nonsynonymous sites. Such rate variation is mostly likely attributed to differences of selection intensity rather than differential mutation pressures on the genes. Unlike previous argument that downstream genes in metabolic pathways would evolve more slowly than upstream genes, the downstream genes in the GA pathway did not exhibited the elevated substitution rate and instead, the genes that encode either the enzyme at the branch point (GA20ox or enzymes catalyzing multiple steps (KO, KAO and GA3ox in the pathway had the lowest evolutionary rates due to strong purifying selection. Our branch and codon models failed to detect signature of positive selection for any lineage and codon of the GA pathway genes. Conclusion This study suggests that significant heterogeneity of evolutionary rate of the GA pathway genes is mainly ascribed to differential constraint relaxation rather than the positive selection and supports the pathway flux theory that predicts that natural selection primarily targets enzymes that have the greatest control on fluxes.

  10. Survival and Predictive Factors of Lethality in Hemodyalisis: D/I Polymorphism of The Angiotensin I-Converting Enzyme and of the Angiotensinogen M235T Genes

    International Nuclear Information System (INIS)

    End-stage kidney disease patients continue to have markedly increased cardiovascular disease morbidity and mortality. Analysis of genetic factors connected with the renin-angiotensin system that influences the survival of the patients with end-stage kidney disease supports the ongoing search for improved outcomes. To assess survival and its association with the polymorphism of renin-angiotensin system genes: angiotensin I-converting enzyme insertion/deletion and angiotensinogen M235T in patients undergoing hemodialysis. Our study was designed to examine the role of renin-angiotensin system genes. It was an observational study. We analyzed 473 chronic hemodialysis patients in four dialysis units in the state of Rio de Janeiro. Survival rates were calculated by the Kaplan-Meier method and the differences between the curves were evaluated by Tarone-Ware, Peto-Prentice, and log rank tests. We also used logistic regression analysis and the multinomial model. A p value ≤ 0.05 was considered to be statistically significant. The local medical ethics committee gave their approval to this study. The mean age of patients was 45.8 years old. The overall survival rate was 48% at 11 years. The major causes of death were cardiovascular diseases (34%) and infections (15%). Logistic regression analysis found statistical significance for the following variables: age (p = 0.000038), TT angiotensinogen (p = 0.08261), and family income greater than five times the minimum wage (p = 0.03089), the latter being a protective factor. The survival of hemodialysis patients is likely to be influenced by the TT of the angiotensinogen M235T gene

  11. Survival and Predictive Factors of Lethality in Hemodyalisis: D/I Polymorphism of The Angiotensin I-Converting Enzyme and of the Angiotensinogen M235T Genes

    Directory of Open Access Journals (Sweden)

    Mauro Alves

    2014-09-01

    Full Text Available Background: End-stage kidney disease patients continue to have markedly increased cardiovascular disease morbidity and mortality. Analysis of genetic factors connected with the renin-angiotensin system that influences the survival of the patients with end-stage kidney disease supports the ongoing search for improved outcomes. Objective: To assess survival and its association with the polymorphism of renin-angiotensin system genes: angiotensin I-converting enzyme insertion/deletion and angiotensinogen M235T in patients undergoing hemodialysis. Methods: Our study was designed to examine the role of renin-angiotensin system genes. It was an observational study. We analyzed 473 chronic hemodialysis patients in four dialysis units in the state of Rio de Janeiro. Survival rates were calculated by the Kaplan-Meier method and the differences between the curves were evaluated by Tarone-Ware, Peto-Prentice, and log rank tests. We also used logistic regression analysis and the multinomial model. A p value ≤ 0.05 was considered to be statistically significant. The local medical ethics committee gave their approval to this study. Results: The mean age of patients was 45.8 years old. The overall survival rate was 48% at 11 years. The major causes of death were cardiovascular diseases (34% and infections (15%. Logistic regression analysis found statistical significance for the following variables: age (p = 0.000038, TT angiotensinogen (p = 0.08261, and family income greater than five times the minimum wage (p = 0.03089, the latter being a protective factor. Conclusions: The survival of hemodialysis patients is likely to be influenced by the TT of the angiotensinogen M235T gene.

  12. Survival and Predictive Factors of Lethality in Hemodyalisis: D/I Polymorphism of The Angiotensin I-Converting Enzyme and of the Angiotensinogen M235T Genes

    Energy Technology Data Exchange (ETDEWEB)

    Alves, Mauro, E-mail: malves@cardiol.br; Silva, Nelson Albuquerque de Souza e; Salis, Lucia Helena Alvares; Pereira, Basilio de Bragança; Godoy, Paulo Henrique; Nascimento, Emília Matos do [Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Oliveira, Jose Mario Franco [Universidade Federal Fluminense, Niterói, RJ (Brazil)

    2014-09-15

    End-stage kidney disease patients continue to have markedly increased cardiovascular disease morbidity and mortality. Analysis of genetic factors connected with the renin-angiotensin system that influences the survival of the patients with end-stage kidney disease supports the ongoing search for improved outcomes. To assess survival and its association with the polymorphism of renin-angiotensin system genes: angiotensin I-converting enzyme insertion/deletion and angiotensinogen M235T in patients undergoing hemodialysis. Our study was designed to examine the role of renin-angiotensin system genes. It was an observational study. We analyzed 473 chronic hemodialysis patients in four dialysis units in the state of Rio de Janeiro. Survival rates were calculated by the Kaplan-Meier method and the differences between the curves were evaluated by Tarone-Ware, Peto-Prentice, and log rank tests. We also used logistic regression analysis and the multinomial model. A p value ≤ 0.05 was considered to be statistically significant. The local medical ethics committee gave their approval to this study. The mean age of patients was 45.8 years old. The overall survival rate was 48% at 11 years. The major causes of death were cardiovascular diseases (34%) and infections (15%). Logistic regression analysis found statistical significance for the following variables: age (p = 0.000038), TT angiotensinogen (p = 0.08261), and family income greater than five times the minimum wage (p = 0.03089), the latter being a protective factor. The survival of hemodialysis patients is likely to be influenced by the TT of the angiotensinogen M235T gene.

  13. The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluster

    Directory of Open Access Journals (Sweden)

    Scott Christopher J

    2010-04-01

    Full Text Available Abstract Background The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A. Subsequently we have identified a number of human family members and shown that one of these (DUB-3 is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1. Results Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable beta-defensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating. Conclusions The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

  14. Tau leptonic branching ratios

    CERN Document Server

    Buskulic, Damir; De Bonis, I; Décamp, D; Ghez, P; Goy, C; Lees, J P; Lucotte, A; Minard, M N; Odier, P; Pietrzyk, B; Ariztizabal, F; Chmeissani, M; Crespo, J M; Efthymiopoulos, I; Fernández, E; Fernández-Bosman, M; Gaitan, V; Garrido, L; Martínez, M; Orteu, S; Pacheco, A; Padilla, C; Palla, Fabrizio; Pascual, A; Perlas, J A; Sánchez, F; Teubert, F; Colaleo, A; Creanza, D; De Palma, M; Farilla, A; Gelao, G; Girone, M; Iaselli, Giuseppe; Maggi, G; Maggi, M; Marinelli, N; Natali, S; Nuzzo, S; Ranieri, A; Raso, G; Romano, F; Ruggieri, F; Selvaggi, G; Silvestris, L; Tempesta, P; Zito, G; Huang, X; Lin, J; Ouyang, Q; Wang, T; Xie, Y; Xu, R; Xue, S; Zhang, J; Zhang, L; Zhao, W; Bonvicini, G; Cattaneo, M; Comas, P; Coyle, P; Drevermann, H; Engelhardt, A; Forty, Roger W; Frank, M; Hagelberg, R; Harvey, J; Jacobsen, R; Janot, P; Jost, B; Kneringer, E; Knobloch, J; Lehraus, Ivan; Markou, C; Martin, E B; Mato, P; Minten, Adolf G; Miquel, R; Oest, T; Palazzi, P; Pater, J R; Pusztaszeri, J F; Ranjard, F; Rensing, P E; Rolandi, Luigi; Schlatter, W D; Schmelling, M; Schneider, O; Tejessy, W; Tomalin, I R; Venturi, A; Wachsmuth, H W; Wiedenmann, W; Wildish, T; Witzeling, W; Wotschack, J; Ajaltouni, Ziad J; Bardadin-Otwinowska, Maria; Barrès, A; Boyer, C; Falvard, A; Gay, P; Guicheney, C; Henrard, P; Jousset, J; Michel, B; Monteil, S; Montret, J C; Pallin, D; Perret, P; Podlyski, F; Proriol, J; Rossignol, J M; Saadi, F; Fearnley, Tom; Hansen, J B; Hansen, J D; Hansen, J R; Hansen, P H; Nilsson, B S; Kyriakis, A; Simopoulou, Errietta; Siotis, I; Vayaki, Anna; Zachariadou, K; Blondel, A; Bonneaud, G R; Brient, J C; Bourdon, P; Passalacqua, L; Rougé, A; Rumpf, M; Tanaka, R; Valassi, Andrea; Verderi, M; Videau, H L; Candlin, D J; Parsons, M I; Focardi, E; Parrini, G; Corden, M; Delfino, M C; Georgiopoulos, C H; Jaffe, D E; Antonelli, A; Bencivenni, G; Bologna, G; Bossi, F; Campana, P; Capon, G; Chiarella, V; Felici, G; Laurelli, P; Mannocchi, G; Murtas, F; Murtas, G P; Pepé-Altarelli, M; Dorris, S J; Halley, A W; ten Have, I; Knowles, I G; Lynch, J G; Morton, W T; O'Shea, V; Raine, C; Reeves, P; Scarr, J M; Smith, K; Smith, M G; Thompson, A S; Thomson, F; Thorn, S; Turnbull, R M; Becker, U; Braun, O; Geweniger, C; Graefe, G; Hanke, P; Hepp, V; Kluge, E E; Putzer, A; Rensch, B; Schmidt, M; Sommer, J; Stenzel, H; Tittel, K; Werner, S; Wunsch, M; Beuselinck, R; Binnie, David M; Cameron, W; Colling, D J; Dornan, Peter J; Konstantinidis, N P; Moneta, L; Moutoussi, A; Nash, J; San Martin, G; Sedgbeer, J K; Stacey, A M; Dissertori, G; Girtler, P; Kuhn, D; Rudolph, G; Bowdery, C K; Brodbeck, T J; Colrain, P; Crawford, G; Finch, A J; Foster, F; Hughes, G; Sloan, Terence; Whelan, E P; Williams, M I; Galla, A; Greene, A M; Kleinknecht, K; Quast, G; Raab, J; Renk, B; Sander, H G; Wanke, R; Van Gemmeren, P; Zeitnitz, C; Aubert, Jean-Jacques; Bencheikh, A M; Benchouk, C; Bonissent, A; Bujosa, G; Calvet, D; Carr, J; Diaconu, C A; Etienne, F; Thulasidas, M; Nicod, D; Payre, P; Rousseau, D; Talby, M; Abt, I; Assmann, R W; Bauer, C; Blum, Walter; Brown, D; Dietl, H; Dydak, Friedrich; Ganis, G; Gotzhein, C; Jakobs, K; Kroha, H; Lütjens, G; Lutz, Gerhard; Männer, W; Moser, H G; Richter, R H; Rosado-Schlosser, A; Schael, S; Settles, Ronald; Seywerd, H C J; Saint-Denis, R; Wolf, G; Alemany, R; Boucrot, J; Callot, O; Cordier, A; Courault, F; Davier, M; Duflot, L; Grivaz, J F; Heusse, P; Jacquet, M; Kim, D W; Le Diberder, F R; Lefrançois, J; Lutz, A M; Musolino, G; Nikolic, I A; Park, H J; Park, I C; Schune, M H; Simion, S; Veillet, J J; Videau, I; Abbaneo, D; Azzurri, P; Bagliesi, G; Batignani, G; Bettarini, S; Bozzi, C; Calderini, G; Carpinelli, M; Ciocci, M A; Ciulli, V; Dell'Orso, R; Fantechi, R; Ferrante, I; Foà, L; Forti, F; Giassi, A; Giorgi, M A; Gregorio, A; Ligabue, F; Lusiani, A; Marrocchesi, P S; Messineo, A; Rizzo, G; Sanguinetti, G; Sciabà, A; Spagnolo, P; Steinberger, Jack; Tenchini, Roberto; Tonelli, G; Triggiani, G; Vannini, C; Verdini, P G; Walsh, J; Betteridge, A P; Blair, G A; Bryant, L M; Cerutti, F; Gao, Y; Green, M G; Johnson, D L; Medcalf, T; Mir, L M; Perrodo, P; Strong, J A; Bertin, V; Botterill, David R; Clifft, R W; Edgecock, T R; Haywood, S; Edwards, M; Maley, P; Norton, P R; Thompson, J C; Bloch-Devaux, B; Colas, P; Emery, S; Kozanecki, Witold; Lançon, E; Lemaire, M C; Locci, E; Marx, B; Pérez, P; Rander, J; Renardy, J F; Roussarie, A; Schuller, J P; Schwindling, J; Trabelsi, A; Vallage, B; Johnson, R P; Kim, H Y; Litke, A M; McNeil, M A; Taylor, G; Beddall, A; Booth, C N; Boswell, R; Cartwright, S L; Combley, F; Dawson, I; Köksal, A; Letho, M; Newton, W M; Rankin, C; Thompson, L F; Böhrer, A; Brandt, S; Cowan, G D; Feigl, E; Grupen, Claus; Lutters, G; Minguet-Rodríguez, J A; Rivera, F; Saraiva, P; Smolik, L; Stephan, F; Apollonio, M; Bosisio, L; Della Marina, R; Giannini, G; Gobbo, B; Ragusa, F; Rothberg, J E; Wasserbaech, S R; Armstrong, S R; Bellantoni, L; Elmer, P; Feng, Z; Ferguson, D P S; Gao, Y S; González, S; Grahl, J; Harton, J L; Hayes, O J; Hu, H; McNamara, P A; Nachtman, J M; Orejudos, W; Pan, Y B; Saadi, Y; Schmitt, M; Scott, I J; Sharma, V; Turk, J; Walsh, A M; Wu Sau Lan; Wu, X; Yamartino, J M; Zheng, M; Zobernig, G

    1996-01-01

    A sample of 62249 \\tau-pair events is selected from data taken with the ALEPH detector in 1991, 1992 and 1993. The measurement of the branching fractions for \\tau decays into electrons and muons is presented with emphasis on the study of systematic effects from selection, particle identification and decay classification. Combined with the most recent ALEPH determination of the \\tau lifetime, these results provide a relative measurement of the leptonic couplings in the weak charged current for transverse W bosons.

  15. Holographic Coulomb branch vevs

    CERN Document Server

    Skenderis, K; Skenderis, Kostas; Taylor, Marika

    2006-01-01

    We compute holographically the vevs of all chiral primary operators for supergravity solutions corresponding to the Coulomb branch of N=4 SYM and find exact agreement with the corresponding field theory computation. Using the dictionary between 10d geometries and field theory developed to extract these vevs, we propose a gravity dual of a half supersymmetric deformation of N=4 SYM by certain irrelevant operators.

  16. Modeling branching in cereals

    OpenAIRE

    Jochem B. Evers; Vos, Jan

    2013-01-01

    Cereals and grasses adapt their structural development to environmental conditions and the resources available. The primary adaptive response is a variable degree of branching, called tillering in cereals. Especially for heterogeneous plant configurations the degree of tillering varies per plant. Functional–structural plant modeling (FSPM) is a modeling approach allowing simulation of the architectural development of individual plants, culminating in the emergent behavior at the canopy level....

  17. Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes.

    OpenAIRE

    Labedan Bernard; Glansdorff Nicolas; Xu Ying

    2006-01-01

    Abstract Background The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i) the classical N-acetylglutamate synthase (NAGS, gene argA) first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii) the bifunctional version of ornithine acetyltransferase (OAT, gene argJ)...

  18. Angiotensin-converting enzyme gene deletion polymorphism determines an increase in frequency of migraine attacks in patients suffering from migraine without aura.

    Science.gov (United States)

    Paterna, S; Di Pasquale, P; D'Angelo, A; Seidita, G; Tuttolomondo, A; Cardinale, A; Maniscalchi, T; Follone, G; Giubilato, A; Tarantello, M; Licata, G

    2000-01-01

    Many authors have reported an association between the angiotensin-converting enzyme (ACE)-D allele and coronary heart disease and other cardiovascular diseases. The mechanism underlying the positive associations between the ACE-D alleles and diseases are not yet clear. Previous reports showed an association between migraine without aura and ACE-D allele polymorphism. The study is aimed to evaluate if the DD genotype could also be associated with the frequency and duration of migraine without aura. In 302 patients suffering from migraine without aura (at least for 1 year), with no history of cardiovascular diseases and major risk factors for ischemic events, the genotypes of the ACE gene, plasma ACE activity, and the frequency (weekly) and duration of migraine attacks were evaluated. No drugs were given before (4 weeks) and during the study. The same evaluations were performed in 201 subjects without migraine. The molecular biologist and the physician evaluating the patient data were blinded to the clinical history and ACE-DD gene determination. Genotypes were determined by polymerase chain reaction amplification. Plasma ACE activity was performed by the HPLC method. The groups were similar for sex, age and smoking habit (migraines: 302 patients (200 F/102 M), mean age 37.8 +/- 8.2 years; control: 201 subjects (127 F/74 M), mean age 37.5 +/- 9.3 years). Patients with migraine without aura showed higher incidence of the ACE-DD gene (48.34%) than control subjects (37.32%), p < 0.05. The frequency of migraine (average attacks per week) was higher in patients with DD (2.11 +/- 1.9) than in patients with ID (1.54 +/- 1. 44), p < 0.05. No difference in duration of migraine attacks (hours per week) was observed. Plasma ACE activity was increased in patients with the ACE-DD gene. Our data suggest that ACE-DD gene polymorphism could have an important role in determining migraine attacks and the frequency of these attacks. Further data are needed through further studies

  19. Formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr) from the hyperthermophilic Methanopyrus kandleri. Cloning, sequencing and functional expression of the ftr gene and one-step purification of the enzyme overproduced in Escherichia coli.

    Science.gov (United States)

    Shima, S; Weiss, D S; Thauer, R K

    1995-06-15

    Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 degrees C. The gene ftr encoding the formylmethanofuran:tetrahydromethanopterin formyltransferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90 degrees C in 1.5 M K2HPO4 pH 8.0 for 30 min. From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained. The purified enzyme showed essentially the same catalytic properties as that purified from M. kandleri cells. The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 degrees C) and Methanothermus fervidus (83 degrees C). Of the three enzymes that from M. kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition. The enzyme from M. kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine. These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M. kandleri is adapted to both hyperthermophilic and halophilic conditions. PMID:7601152

  20. The making of a branching annelid

    OpenAIRE

    Aguado, M. Teresa; Glasby, Christopher J.; Schroeder, Paul C.; Weigert, Anne; Bleidorn, Christoph

    2015-01-01

    Ramisyllis multicaudata is a member of Syllidae (Annelida, Errantia, Phyllodocida) with a remarkable branching body plan. Using a next-generation sequencing approach, the complete mitochondrial genomes of R. multicaudata and Trypanobia sp. are sequenced and analysed, representing the first ones from Syllidae. The gene order in these two syllids does not follow the order proposed as the putative ground pattern in Errantia. The phylogenetic relationships of R. multicaudata are discerned usin...

  1. Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi

    Directory of Open Access Journals (Sweden)

    Agrawal Parul

    2010-07-01

    Full Text Available Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR for GyrA gene followed by restriction fragment length polymorphism (RFLP with restriction enzyme (RE SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18% were susceptible to nalidixic acid (NASST and the remaining 48 (82% were resistant to nalidixic acid (NARST. Of these 48 NARST strains, 46 (97.5% had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL, whereas 2 strains (2.75% were resistant to ciprofloxacin (MIC 4.0 μg/mL. In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46 had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe, whereas double mutations (Ser 83→Phe and Asp 87→Asn were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12 revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further

  2. Method for altering starch using a microbial branching enzyme

    NARCIS (Netherlands)

    van der Maarel, Marc; Palomo Reixach, Marta; Booiman, Thijs; Dijkhuizen, Lubbert

    2010-01-01

    The present invention relates to methods for construction of pharmamers i.e. vaccine components characterized by their multimerization domain and the attached biologically active molecules, and their use in preparation of vaccines that contains the pharmamers alone or in combination with other molec

  3. Thermal Energy Conversion Branch

    Science.gov (United States)

    Bielozer, Matthew C.; Schreiber, Jeffrey, G.; Wilson, Scott D.

    2004-01-01

    The Thermal Energy Conversion Branch (5490) leads the way in designing, conducting, and implementing research for the newest thermal systems used in space applications at the NASA Glenn Research Center. Specifically some of the most advanced technologies developed in this branch can be broken down into four main areas: Dynamic Power Systems, Primary Solar Concentrators, Secondary Solar Concentrators, and Thermal Management. Work was performed in the Dynamic Power Systems area, specifically the Stirling Engine subdivision. Today, the main focus of the 5490 branch is free-piston Stirling cycle converters, Brayton cycle nuclear reactors, and heat rejection systems for long duration mission spacecraft. All space exploring devices need electricity to operate. In most space applications, heat energy from radioisotopes is converted to electrical power. The Radioisotope Thermoelectric Generator (RTG) already supplies electricity for missions such as the Cassini Spacecraft. The focus of today's Stirling research at GRC is aimed at creating an engine that can replace the RTG. The primary appeal of the Stirling engine is its high system efficiency. Because it is so efficient, the Stirling engine will significantly reduce the plutonium fuel mission requirements compared to the RTG. Stirling is also being considered for missions such as the lunar/Mars bases and rovers. This project has focused largely on Stirling Engines of all types, particularly the fluidyne liquid piston engine. The fluidyne was developed by Colin D. West. This engine uses the same concepts found in any type of Stirling engine, with the exception of missing mechanical components. All the working components are fluid. One goal was to develop and demonstrate a working Stirling Fluidyne Engine at the 2nd Annual International Energy Conversion Engineering Conference in Providence, Rhode Island.

  4. Combustion Branch Website Development

    Science.gov (United States)

    Bishop, Eric

    2004-01-01

    The NASA combustion branch is a leader in developing and applying combustion science to focused aerospace propulsion systems concepts. It is widely recognized for unique facilities, analytical tools, and personnel. In order to better communicate the outstanding research being done in this Branch to the public and other research organization, a more substantial website was desired. The objective of this project was to build an up-to-date site that reflects current research in a usable and attractive manner. In order to accomplish this, information was requested from all researchers in the Combustion branch, on their professional skills and on the current projects. This information was used to fill in the Personnel and Research sections of the website. A digital camera was used to photograph all personnel and these photographs were included in the personnel section as well. The design of the site was implemented using the latest web standards: xhtml and external css stylesheets. This implementation conforms to the guidelines recommended by the w3c. It also helps to ensure that the web site is accessible by disabled users, and complies with Section 508 Federal legislation (which mandates that all Federal websites be accessible). Graphics for the new site were generated using the gimp (www.gimp.org) an open-source graphics program similar to Adobe Photoshop. Also, all graphics on the site were of a reasonable size (less than 20k, most less than 2k) so that the page would load quickly. Technologies such as Macromedia Flash and Javascript were avoided, as these only function on some clients which have the proper software installed or enabled. The website was tested on different platforms with many different browsers to ensure there were no compatibility issues. The website was tested on windows with MS IE 6, MSIE 5 , Netscape 7, Mozilla and Opera. On a Mac, the site was tested with MS IE 5 , Netscape 7 and Safari.

  5. Analysis of the apo E/apo C-I, angiotensin converting enzyme and methylenetetrahydrofolate reductase genes as candidates affecting human longevity.

    Science.gov (United States)

    Galinsky, D; Tysoe, C; Brayne, C E; Easton, D F; Huppert, F A; Dening, T R; Paykel, E S; Rubinsztein, D C

    1997-03-21

    Genetic factors are likely to affect human survival, since twin studies have shown greater concordance for age of death in monozygotic compared to dizygotic twins. Coronary artery disease is an important contributor to premature mortality in the UK. Accordingly, we have chosen genes associated with cardiovascular risk, apo E/apo C-I, angiotensin converting enzyme (ACE) and methylenetetrahydrofolate reductase (MTHFR), as candidates which may affect longevity/survival into old age. An association study was performed by comparing allele and genotype frequencies at polymorphic loci associated with these genes in 182 women and 100 men aged 84 years and older with 100 boys and 100 girls younger than 17 years. MTHFR allele and genotype frequencies were similar in the elderly and young populations. Apo C-I allele and genotype frequencies were significantly different in the elderly women compared to the younger sample (P Hardy-Weinberg equilibrium and compared to observed genotypes in elderly men and women. In contrast to previous studies, apo E2 was not overrepresented in the elderly men or women. Thus, the proposition that apo E2, E3 and E4 protein isoforms are themselves functionally associated with increasing risks for early death, may be too simplistic. The I/I ACE was depleted in the elderly males but not the elderly females. Furthermore, significant differences were observed between ACE genotypes in elderly men and elderly women. These data suggest that the penetrance of loci which influence survival may vary according to sex. The depletion of the ACE I/I genotype in elderly men is generally consistent with a previous study which found decreased frequencies of the I allele in French centenarians compared to younger controls. However, these results are apparently paradoxical, since others have suggested that the I allele is associated with increased cardiovascular risk. Clarification of the overall effect of a genotype on survival will be vital if therapies are to be

  6. Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Amos O. Abolaji

    2015-08-01

    Full Text Available The compounds 4-vinylcyclohexene 1,2-monoepoxide (VCM and 4-Vinylcyclohexene diepoxide (VCD are the two downstream metabolites of 4-vinylcyclohexene (VCH, an ovotoxic agent in mammals. In addition, VCM and VCD may be found as by-products of VCH oxidation in the environment. Recently, we reported the involvement of oxidative stress in the toxicity of VCH in Drosophila melanogaster. However, it was not possible to determine the individual contributions of VCM and VCD in VCH toxicity. Hence, we investigated the toxicity of VCM and VCD (10–1000 µM in flies after 5 days of exposure via the diet. Our results indicated impairments in climbing behaviour and disruptions in antioxidant balance and redox status evidenced by an increase in DCFH oxidation, decreases in total thiol content and glutathione-S-transferase (GST activity in the flies exposed to VCM and VCD (p<0.05. These effects were accompanied by disruptions in the transcription of the genes encoding the proteins superoxide dismutase (SOD1, kelch-like erythroid-derived cap-n-collar (CNC homology (ECH-associated protein 1 (Keap-1, mitogen activated protein kinase 2 (MAPK-2, catalase, Cyp18a1, JAFRAC 1 (thioredoxin peroxidase 1 and thioredoxin reductase 1 (TrxR-1 (p<0.05. VCM and VCD inhibited acetylcholinesterase (AChE and delta aminolevulinic acid dehydratase (δ-ALA D activities in the flies (p<0.05. Indeed, here, we demonstrated that different target enzymes and genes were modified by the electrophiles VCM and VCD in the flies. Thus, D. melanogaster has provided further lessons on the toxicity of VCM and VCD which suggest that the reported toxicity of VCH may be mediated by its transformation to VCM and VCD.

  7. SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato.

    NARCIS (Netherlands)

    Vogel, J.T.; Walter, M.H.; Giavalisco, P.; Lytovchenko, A.; Kohlen, W.; Charnikhova, T.; Simkin, A.J.; Goulet, C.; Strack, D.; Bouwmeester, H.J.; Fernie, A.R.; Klee, H.J.

    2010-01-01

    The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8,

  8. A gene controlling the number of primary rachis branches also controls the vascular bundle formation and hence is responsible to increase the harvest index and grain yield in rice.

    Science.gov (United States)

    Terao, Tomio; Nagata, Kenji; Morino, Kazuko; Hirose, Tatsuro

    2010-03-01

    The quantitative trait locus controlling the number of primary rachis branches (PRBs) in rice was identified using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. The resultant gene was ABERRANT PANICLE ORGANIZATION 1 (APO1). Habataki-genotype segregated reciprocal recombinant lines for the APO1 locus increased both the number of PRB (12-13%) and the number of grains per panicle (9-12%), which increased the grain yield per plant (5-7%). Further recombination dividing this region revealed that different alleles regulated the number of PRB and the number of grains per panicle. The PRB1 allele, which includes the APO1 open reading frame (ORF) and the proximal promoter region, controlled only the number of PRB but not the number of grains per panicle. In contrast, the HI1 allele, which includes only the distal promoter region, increased the grain yield and harvest index in Habataki-genotype plants, nevertheless, the ORF expressed was Sasanishiki type. It also increased the number of large vascular bundles in the peduncle. APO1 expression occurred not only in developing panicles but also in the developing vascular bundle systems. In addition, Habataki plants displayed increased APO1 expression in comparison to Sasanishiki plants. It suggests that APO1 enhances the formation of vascular bundle systems which, consequently, promote carbohydrate translocation to panicles. The HI1 allele is suggested to regulate the amount of APO1 expression, and thereby control the development of vascular bundle systems. These findings may be useful to improve grain yield as well as quality through the improvement of translocation efficiency. PMID:20151298

  9. Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Garcia, Lucia; Alonso-Sanz, Mercedes; Rebollo, Maria J.; Tercero, Juan C.; Chaves, Fernando

    2001-01-01

    Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rifr) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rifr isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rifr allele within each strain, 10 of the 11 Rifr genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rifr organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods. PMID:11325996

  10. Association between angiotensin converting enzyme gene insertion/deletion polymorphism and renal scar risk in children vesicoureteral reflex: a reappraise meta-analysis

    Science.gov (United States)

    Ai, Jin-Wei; Zeng, Xian-Tao; Liu, Ying; Fu, Yu; Liu, Tong-Zu; Pei, Bin

    2016-01-01

    Vesicoureteral reflex(VUR) is a common disease in children. Some studies indicated that the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism associated with the renal scar in VUR, but not all researchers agreed with it. To clarify the effect of ACE I/D polymorphism on renal scar risk in children with VUR, we performed the present meta-analysis. PubMed, CNKI, CBM, and Embase databases were searched for studies that examined the relationship between ACE I/D polymorphism and renal scar risk in children with VUR. The Stata 12.0 software was used for statistical analyses. 11 case-control studies with 1,032 VUR patients were analyzed. The results showed that the DD genotype and D allele were associated with renal scar risk in overall VUR patients, DD vs. DI + II: OR = 1.61, 95% CI = 1.04–2.49, P = 0.03; DD vs. II: OR = 1.78, 95% CI = 1.20–2.65, P < 0.01; D vs. I: OR = 1.38, 95% CI = 1.02–1.86, P = 0.04. Similar results were revealed in Turks, but not in Caucasians and Asians. Our meta-analysis indicated that the ACE DD genotype may increase the risk of renal scar in children with VUR. PMID:27506878

  11. Role Of Shark Cartilage In Reducing Changes In Gene Expression Of Some Enzymes Induced By N-Nitroso-N-Methyl Urea In Prostate Of Irradiated Rats

    International Nuclear Information System (INIS)

    There is overwhelming evidence to indicate that free radicals cause oxidative damage to lipids, proteins and nucleic acids and are involved in the pathogenesis of several diseases. Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of these diseases. Recent studies demonstrated the role of shark cartilage in protecting cells against reactive oxygen species induced DNA damage and mutagenesis. Reactive oxygen species and other free radicals are known to be the mediators of phenotypic and genotypic changes that lead from mutation to neoplasia. There are some primary antioxidants such as glutathione peroxidase (GSHPx), glutathione-S-transferase (GST-π) and super oxide dismutase (SOD), which protects against cellular and molecular damage caused by the reactive oxygen metabolites (ROMs).In this study, the effect of shark cartilage against the N-nitroso-N-methyl urea + testosterone and/or gamma radiation-induced mutagens and carcinogens in rat prostate were investigated.The data showed significant decrease in gene expression of manganese superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GSHPx1) , enzyme activities of total superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) and non-significant increase in glutathione-S-transferase (GST-π) in N-nitroso-N-methyl urea + testosterone, N-nitroso-N-methyl urea + testosterone + gamma radiation groups as compared to control group.The results revealed that shark cartilage administration afford a significant protective effect against N-nitroso-N-methyl urea + testosterone and/or gamma radiation- induced oxidative injury.

  12. Clinically relevant genetic variants of drug-metabolizing enzyme and transporter genes detected in Thai children and adolescents with autism spectrum disorder

    Directory of Open Access Journals (Sweden)

    Medhasi S

    2016-04-01

    Full Text Available Sadeep Medhasi,1–3 Ekawat Pasomsub,4 Natchaya Vanwong,1,2 Nattawat Ngamsamut,5 Apichaya Puangpetch,1,2 Montri Chamnanphon,1,2 Yaowaluck Hongkaew,1,2 Penkhae Limsila,5 Darawan Pinthong,3 Chonlaphat Sukasem1,2 1Division of Pharmacogenomics and Personalized Medicine, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 2Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 3Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand; 4Division of Virology, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 5Yuwaprasart Waithayopathum Child and Adolescent Psychiatric Hospital, Department of Mental Health Services, Ministry of Public Health, Samut Prakarn, Thailand Abstract: Single-nucleotide polymorphisms (SNPs among drug-metabolizing enzymes and transporters (DMETs influence the pharmacokinetic profile of drugs and exhibit intra- and interethnic variations in drug response in terms of efficacy and safety profile. The main objective of this study was to assess the frequency of allelic variants of drug absorption, distribution, metabolism, and elimination-related genes in Thai children and adolescents with autism spectrum disorder. Blood samples were drawn from 119 patients, and DNA was extracted. Genotyping was performed using the DMET Plus microarray platform. The allele frequencies of the DMET markers were generated using the DMET Console software. Thereafter, the genetic variations of significant DMET genes were assessed. The frequencies of SNPs across the genes coding for DMETs were determined. After filtering the SNPs, 489 of the 1,931 SNPs passed quality control. Many clinically relevant SNPs, including CYP2C19*2, CYP2D6*10, CYP3A5*3, and SLCO1B1*5, were found to have frequencies similar to those in the Chinese

  13. Path-valued branching processes and nonlocal branching superprocesses

    CERN Document Server

    Li, Zenghu

    2012-01-01

    A family of continuous-state branching processes with immigration are constructed as the solution flow of a stochastic equation system driven by time-space noises. The family can be regarded as an inhomogeneous increasing path-valued branching process with immigration. Two nonlocal branching immigration superprocesses can be defined from the flow. We identify explicitly the branching and immigration mechanisms of those processes. The results provide new perspectives into the tree-valued Markov processes of Aldous and Pitman [Ann. Inst. H. Poincare Probab. Statist. 34 (1998), 637--686] and Abraham and Delmas [Ann. Probab. To appear].

  14. Maritime Branch Analysis

    DEFF Research Database (Denmark)

    Mougaard, Krestine; Neugebauer, Line Maria; Garcia i Mateu, Adrià;

    after-sales service and have therefore embarked on business development activities that tightly combine product and service offerings in their portfolios. Closer customer contact, commoditisation of goods, total cost of ownership, and product liability are just some of the reasons for this transition......The vast majority of countries in the developed world are now dependent on their service sectors for between 70-80% of their gross domestic product. Even companies with decades of expertise in producing manufactured products are experiencing an increased need to understand before-, during- and...... and influencing a move towards intensified service integration into business- and product development activities. The workbook has been created by condensing the transcripts and the insights we have gained from a wealth of studies across the maritime branch, and it serves to provide a unique insight...

  15. Quiver Varieties and Branching

    Directory of Open Access Journals (Sweden)

    Hiraku Nakajima

    2009-01-01

    Full Text Available Braverman and Finkelberg recently proposed the geometric Satake correspondence for the affine Kac-Moody group Gaff [Braverman A., Finkelberg M., arXiv:0711.2083]. They conjecture that intersection cohomology sheaves on the Uhlenbeck compactification of the framed moduli space of Gcpt-instantons on $R^4/Z_r$ correspond to weight spaces of representations of the Langlands dual group $G_{aff}^{vee}$ at level $r$. When $G = SL(l$, the Uhlenbeck compactification is the quiver variety of type $sl(r_{aff}$, and their conjecture follows from the author's earlier result and I. Frenkel's level-rank duality. They further introduce a convolution diagram which conjecturally gives the tensor product multiplicity [Braverman A., Finkelberg M., Private communication, 2008]. In this paper, we develop the theory for the branching in quiver varieties and check this conjecture for $G = SL(l$.

  16. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

    Science.gov (United States)

    Jung, W; Yu, O; Lau, S M; O'Keefe, D P; Odell, J; Fader, G; McGonigle, B

    2000-02-01

    Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes. PMID:10657130

  17. Metabolic evolution of a deep-branching hyperthermophilic chemoautotrophic bacterium.

    Science.gov (United States)

    Braakman, Rogier; Smith, Eric

    2014-01-01

    Aquifex aeolicus is a deep-branching hyperthermophilic chemoautotrophic bacterium restricted to hydrothermal vents and hot springs. These characteristics make it an excellent model system for studying the early evolution of metabolism. Here we present the whole-genome metabolic network of this organism and examine in detail the driving forces that have shaped it. We make extensive use of phylometabolic analysis, a method we recently introduced that generates trees of metabolic phenotypes by integrating phylogenetic and metabolic constraints. We reconstruct the evolution of a range of metabolic sub-systems, including the reductive citric acid (rTCA) cycle, as well as the biosynthesis and functional roles of several amino acids and cofactors. We show that A. aeolicus uses the reconstructed ancestral pathways within many of these sub-systems, and highlight how the evolutionary interconnections between sub-systems facilitated several key innovations. Our analyses further highlight three general classes of driving forces in metabolic evolution. One is the duplication and divergence of genes for enzymes as these progress from lower to higher substrate specificity, improving the kinetics of certain sub-systems. A second is the kinetic optimization of established pathways through fusion of enzymes, or their organization into larger complexes. The third is the minimization of the ATP unit cost to synthesize biomass, improving thermodynamic efficiency. Quantifying the distribution of these classes of innovations across metabolic sub-systems and across the tree of life will allow us to assess how a tradeoff between maximizing growth rate and growth efficiency has shaped the long-term metabolic evolution of the biosphere. PMID:24516572

  18. Metabolic evolution of a deep-branching hyperthermophilic chemoautotrophic bacterium.

    Directory of Open Access Journals (Sweden)

    Rogier Braakman

    Full Text Available Aquifex aeolicus is a deep-branching hyperthermophilic chemoautotrophic bacterium restricted to hydrothermal vents and hot springs. These characteristics make it an excellent model system for studying the early evolution of metabolism. Here we present the whole-genome metabolic network of this organism and examine in detail the driving forces that have shaped it. We make extensive use of phylometabolic analysis, a method we recently introduced that generates trees of metabolic phenotypes by integrating phylogenetic and metabolic constraints. We reconstruct the evolution of a range of metabolic sub-systems, including the reductive citric acid (rTCA cycle, as well as the biosynthesis and functional roles of several amino acids and cofactors. We show that A. aeolicus uses the reconstructed ancestral pathways within many of these sub-systems, and highlight how the evolutionary interconnections between sub-systems facilitated several key innovations. Our analyses further highlight three general classes of driving forces in metabolic evolution. One is the duplication and divergence of genes for enzymes as these progress from lower to higher substrate specificity, improving the kinetics of certain sub-systems. A second is the kinetic optimization of established pathways through fusion of enzymes, or their organization into larger complexes. The third is the minimization of the ATP unit cost to synthesize biomass, improving thermodynamic efficiency. Quantifying the distribution of these classes of innovations across metabolic sub-systems and across the tree of life will allow us to assess how a tradeoff between maximizing growth rate and growth efficiency has shaped the long-term metabolic evolution of the biosphere.

  19. Branched-Chain Amino Acids

    Directory of Open Access Journals (Sweden)

    Matteo Ghiringhelli

    2015-07-01

    Full Text Available Our study is focused on evaluation and use of the most effective and correct nutrients. In particular, our attention is directed to the role of certain amino acids in cachectic patients.During parenteral nutrition in humans, physician already associates in the PN-bags different formulations including amino acids, lipids and glucose solutions or essential amino acids solution alone or exclusively branched-chain amino acids (BCAA. Studies investigated the effects of dietary BCAA ingestion on different diseases and conditions such as obesity and metabolic disorders, liver disease, muscle atrophy, cancer, impaired immunity or injuries (surgery, trauma, burns, and sepsis. BCAAs have been shown to affect gene expression, protein metabolism, apoptosis and regeneration of hepatocytes, and insulin resistance. They have also been shown to inhibit the proliferation of liver cancer cells in vitro, and are essential for lymphocyte proliferation and dendritic cell maturation. Oral or parenteral administration of these three amino acids will allow us to evaluate the real efficacy of these compounds during a therapy to treat malnutrition in subjects unable to feed themselves.

  20. Methods and Technologies Branch (MTB)

    Science.gov (United States)

    The Methods and Technologies Branch focuses on methods to address epidemiologic data collection, study design and analysis, and to modify technological approaches to better understand cancer susceptibility.

  1. Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN.

    Science.gov (United States)

    Miyanaga, Akimasa; Hayakawa, Yuki; Numakura, Mario; Hashimoto, Junko; Teruya, Kuniko; Hirano, Takashi; Shin-Ya, Kazuo; Kudo, Fumitaka; Eguchi, Tadashi

    2016-05-01

    Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2. PMID:26818633

  2. Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells

    Institute of Scientific and Technical Information of China (English)

    Suqin Gao; Lin Sun; Enji Han; Hongshun Qi; Jinbo Feng; Shunliang Xu; Wen Xia

    2009-01-01

    BACKGROUND:Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level.In addition,the piperlonguminine (A) and dihydropiperlonguminine (B) components (1:0.8),which can be separated from Futokadsura stem,selectively inhibit expression of the APP at mRNA and protein levels.OBJECTIVE:Based on previous findings,the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem,respectively.DESIGN,TIME AND SETTING:A gene interference-based randomized,controlled,in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research,Ministries of Education and Public Health,and Institute of Pharmacologic Research,School of Pharmaceutical Science & Department of Biochemistry,School of Medicine,Shandong University between July 2006 and December 2007.MATERIALS:SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences,Chinese Academy of Sciences,Shanghai,China;mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems,USA;mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology,USA;and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma,USA.METHODS:The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez).Three pairs of siRNAs,specific to human BACE1 gene,were synthesized through the use of Silencer? pre-designed siRNA specification,and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10-50 nmol/L) on SK-N-SH cells.Futokadsura stem was separated and purified with chemical methods,and the crystal was composed of

  3. Gene expression pattern of some classes of cytochrome P-450 and glutathione S-transferase enzymes in differentiated hepatocytes-like cells from menstrual blood stem cells.

    Science.gov (United States)

    Esmaeili-Rad, Aida; Khanjani, Sayeh; Vaziri, Hamidreza; Kazemnejad, Somaieh

    2015-05-01

    Recently, valuable characteristics of menstrual blood stem cells (MenSCs) have impelled scientists to take its advantages for cell therapy of different diseases including liver disorders. In this study, we examined messenger RNA (mRNA) expression levels of phases I and II drug metabolizing enzymes including glutathione S-transferase (GST) and cytochrome P-450 (CYP) in differentiated hepatocyte-like cells from MenSCs. The isolated MenSCs were characterized and differentiated into hepatocyte-like cells using hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum-free culture media. After primary characterization of hepatocyte markers, mRNA expression of GSTA1, GSTA2, GSTP1, CYP3A4, and CYP7A1 was assessed in differentiated cells in reference to undifferentiated cells using real-time PCR. Based on immunofluorescent staining and real-time PCR data, the differentiated MenSCs could express functional hepatocyte markers at mRNA and/or protein levels suggesting development of hepatocyte-like cells from MenSCs. Moreover, the expression levels of GSTA1, GSTA2, and CYP3A4 mRNA were upregulated in differentiated cells compared to undifferentiated cells. The expression of CYP7A1 gene was also remarkable on the last day of differentiation process. However, the expression level of GSTP1 did not exhibit statistically significant change during differentiation (P = 0.6). Based on accumulative data, MenSCs could be viewed as an accessible population of stem cells with differentiation ability into drug-metabolizing hepatocyte-like cells. PMID:25614436

  4. Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli.

    Directory of Open Access Journals (Sweden)

    Moushree Pal Roy

    Full Text Available The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1, respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

  5. Can the branching exponent reliably relate the branching indexes?

    Czech Academy of Sciences Publication Activity Database

    Netopilík, Miloš

    2015-01-01

    Roč. 24, č. 2 (2015), s. 80-84. ISSN 1022-1344 R&D Projects: GA ČR(CZ) GA13-02938S Institutional support: RVO:61389013 Keywords : branching exponent * branching indexes * intrinsic viscosity Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.667, year: 2014

  6. 酵母菌合成酯类化合物关键酶基因的研究进展%Study on key enzyme genes of yeast for esters synthesis

    Institute of Scientific and Technical Information of China (English)

    庄世文; 付俊淑; 黄金海

    2012-01-01

    酯类化合物是发酵食品的重要香气成分之一,酵母发酵是酯类化合物产生的主要来源。在分析酵母菌酯类生物合成途径的基础上,对合成途径中的关键酶基因,包括脂肪酶/酯酶基因、醇酰基转移酶基因和醇脱氢酶基因及其酶作用进展作了综述。%Esters are one of the most important flavor compounds in fermented food and mainly synthesized by yeasts when fermenting.The key enzyme genes of yeast for esters synthesis, including lipase/esterase genes, alcohol acetyltransferase genes and alcohol dehydrogenase genes, based on the biosynthetic pathways of esters production were introduced.

  7. Effect of Potato virusY on activities of antioxidant and anaplerotic enzymes in Nicotina tabacum L. transgenic plants with the gene for p3 protein

    Czech Academy of Sciences Publication Activity Database

    Doubnerová, V.; Janoušková, M.; Synková, Helena; Šubr, Z.; Čeřovská, Noemi; Ryšlavá, H.

    2007-01-01

    Roč. 33, 3-4 (2007), s. 123-140. ISSN 1312-8183 Institutional research plan: CEZ:AV0Z50380511 Keywords : antioxidant enzymes * NADP-malic enzyme * PEPC Subject RIV: EB - Genetics ; Molecular Biology www.bio21.bas.bg/ipp/gapbfiles/v-33/07_3-4_123-140.pdf

  8. Branched-chain 2-keto acid decarboxylases derived from Psychrobacter.

    Science.gov (United States)

    Wei, Jiashi; Timler, Jacobe G; Knutson, Carolann M; Barney, Brett M

    2013-09-01

    The conversion of branched-chain amino acids to branched-chain acids or alcohols is an important aspect of flavor in the food industry and is dependent on the Ehrlich pathway found in certain lactic acid bacteria. A key enzyme in the pathway, the 2-keto acid decarboxylase (KDC), is also of interest in biotechnology applications to produce small branched-chain alcohols that might serve as improved biofuels or other commodity feedstocks. This enzyme has been extensively studied in the model bacterium Lactococcus lactis, but is also found in other bacteria and higher organisms. In this report, distinct homologs of the L. lactis KDC originally annotated as pyruvate decarboxylases from Psychrobacter cryohalolentis K5 and P. arcticus 273-4 were cloned and characterized, confirming a related activity toward specific branched-chain 2-keto acids derived from branched-chain amino acids. Further, KDC activity was confirmed in intact cells and cell-free extracts of P. cryohalolentis K5 grown on both rich and defined media, indicating that the Ehrlich pathway may also be utilized in some psychrotrophs and psychrophiles. A comparison of the similarities and differences in the P. cryohalolentis K5 and P. arcticus 273-4 KDC activities to other bacterial KDCs is presented. PMID:23826991

  9. Value of Angiotensin-Converting Enzyme and Monoxide Nitrogen in Pathogenesis of Myocardium Remodeling Depending on Genes' Polymorphism of Асе (I/D) and eNOS (894T>G) in Patients with Arterial Hypertension

    OpenAIRE

    Sydorchuk L.P.; Gaborec I.Y.; Sydorchuk A.R.; Bukach O.P.; Sokolenko A.A.; Ursuliak J.V.; Ivaschuk S.I.; Antoniuk M.V.; Yarynych J.M.

    2013-01-01

    Introduction: Humans genes mutations in altered social conditions through interaction with environmental factors and harmful habits become an individual risk factor. Objectives: To evaluate Angiotensin-Converting Enzyme (ACE) and nitrogen monoxide metabolites (NO/NO2-/NO3-) blood levels depending on I/D polymorphism of ACE gene (dbSNP id:rs4646994), 894T>G of Endothelial Nitric Oxide Synthase (eNOS, dbSNP id:rs1799983) in pathogenesis of left ventricular hypertrophy (LVH) in patients with Ess...

  10. Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver

    OpenAIRE

    Tydén, Eva; Tjälve, Hans; Larsson, Pia

    2014-01-01

    Background Among the cytochrome P450 enzymes (CYP), families 1–3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to ...

  11. An arterial-specific enhancer of the human Endothelin-converting enzyme 1 (ECE1) gene is synergistically activated by Sox17, FoxC2, and Etv2

    OpenAIRE

    Robinson, Ashley S.; Materna, Stefan C.; Barnes, Ralston M.; De Val, Sarah; Xu, Shan-Mei; Black, Brian L.

    2014-01-01

    Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on ...

  12. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar.

    OpenAIRE

    Mirelman, D; Nuchamowitz, Y; Stolarsky, T

    1997-01-01

    A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

  13. Functional polymorphism of genes inactivating biogenic amines and cognitive deficits in paranoid schizophrenia [Funkcjonalny polimorfizm genów enzymów inaktywujących aminy biogenne a deficyty procesów poznawczych w schizofrenii paranoidalnej

    OpenAIRE

    Tylec, Aneta; Kucharska-Pietura, Katarzyna; Jeleniewicz, Witold; Cybulski, Marek; Czernikiewicz, Andrzej

    2013-01-01

    Aim. The aim of the work was to assess relationship between gene polymorphism of enzymes influencing dopaminergic-, serotoninergic, and noradrenergic transfer and cognitive functioning of paranoid schizophrenic inpatients (ICD-10). Method. The following methods have been used in the study: The Test of Everyday Attention (TEA) and The Visual Object and Space Perception Battery (VOSP ), psychiatric scales (SAPS, SANS, BDI) and techniques of genetic engineering (PCR reaction, RFLP and VNTR techn...

  14. Tau hadronic branching ratios

    CERN Document Server

    Buskulic, Damir; De Bonis, I; Décamp, D; Ghez, P; Goy, C; Lees, J P; Lucotte, A; Minard, M N; Odier, P; Pietrzyk, B; Ariztizabal, F; Chmeissani, M; Crespo, J M; Efthymiopoulos, I; Fernández, E; Fernández-Bosman, M; Gaitan, V; Martínez, M; Orteu, S; Pacheco, A; Padilla, C; Palla, Fabrizio; Pascual, A; Perlas, J A; Sánchez, F; Teubert, F; Colaleo, A; Creanza, D; De Palma, M; Farilla, A; Gelao, G; Girone, M; Iaselli, Giuseppe; Maggi, G; Maggi, M; Marinelli, N; Natali, S; Nuzzo, S; Ranieri, A; Raso, G; Romano, F; Ruggieri, F; Selvaggi, G; Silvestris, L; Tempesta, P; Zito, G; Huang, X; Lin, J; Ouyang, Q; Wang, T; Xie, Y; Xu, R; Xue, S; Zhang, J; Zhang, L; Zhao, W; Bonvicini, G; Cattaneo, M; Comas, P; Coyle, P; Drevermann, H; Engelhardt, A; Forty, Roger W; Frank, M; Hagelberg, R; Harvey, J; Jacobsen, R; Janot, P; Jost, B; Kneringer, E; Knobloch, J; Lehraus, Ivan; Markou, C; Martin, E B; Mato, P; Minten, Adolf G; Miquel, R; Oest, T; Palazzi, P; Pater, J R; Pusztaszeri, J F; Ranjard, F; Rensing, P E; Rolandi, Luigi; Schlatter, W D; Schmelling, M; Schneider, O; Tejessy, W; Tomalin, I R; Venturi, A; Wachsmuth, H W; Wiedenmann, W; Wildish, T; Witzeling, W; Wotschack, J; Ajaltouni, Ziad J; Bardadin-Otwinowska, Maria; Barrès, A; Boyer, C; Falvard, A; Gay, P; Guicheney, C; Henrard, P; Jousset, J; Michel, B; Monteil, S; Pallin, D; Perret, P; Podlyski, F; Proriol, J; Rossignol, J M; Saadi, F; Fearnley, Tom; Hansen, J B; Hansen, J D; Hansen, J R; Hansen, P H; Nilsson, B S; Kyriakis, A; Simopoulou, Errietta; Siotis, I; Vayaki, Anna; Zachariadou, K; Blondel, A; Bonneaud, G R; Brient, J C; Bourdon, P; Passalacqua, L; Rougé, A; Rumpf, M; Tanaka, R; Valassi, Andrea; Verderi, M; Videau, H L; Candlin, D J; Parsons, M I; Focardi, E; Parrini, G; Corden, M; Delfino, M C; Georgiopoulos, C H; Jaffe, D E; Antonelli, A; Bencivenni, G; Bologna, G; Bossi, F; Campana, P; Capon, G; Chiarella, V; Felici, G; Laurelli, P; Mannocchi, G; Murtas, F; Murtas, G P; Pepé-Altarelli, M; Dorris, S J; Halley, A W; ten Have, I; Knowles, I G; Lynch, J G; Morton, W T; O'Shea, V; Raine, C; Reeves, P; Scarr, J M; Smith, K; Smith, M G; Thompson, A S; Thomson, F; Thorn, S; Turnbull, R M; Becker, U; Braun, O; Geweniger, C; Graefe, G; Hanke, P; Hepp, V; Kluge, E E; Putzer, A; Rensch, B; Schmidt, M; Sommer, J; Stenzel, H; Tittel, K; Werner, S; Wunsch, M; Beuselinck, R; Binnie, David M; Cameron, W; Colling, D J; Dornan, Peter J; Konstantinidis, N P; Moneta, L; Moutoussi, A; Nash, J; San Martin, G; Sedgbeer, J K; Stacey, A M; Dissertori, G; Girtler, P; Kuhn, D; Rudolph, G; Bowdery, C K; Brodbeck, T J; Colrain, P; Crawford, G; Finch, A J; Foster, F; Hughes, G; Sloan, Terence; Whelan, E P; Williams, M I; Galla, A; Greene, A M; Kleinknecht, K; Quast, G; Raab, J; Renk, B; Sander, H G; Wanke, R; Van Gemmeren, P; Zeitnitz, C; Aubert, Jean-Jacques; Bencheikh, A M; Benchouk, C; Bonissent, A; Bujosa, G; Calvet, D; Carr, J; Diaconu, C A; Etienne, F; Thulasidas, M; Nicod, D; Payre, P; Rousseau, D; Talby, M; Abt, I; Assmann, R W; Bauer, C; Blum, Walter; Brown, D; Dietl, H; Dydak, Friedrich; Ganis, G; Gotzhein, C; Jakobs, K; Kroha, H; Lütjens, G; Lutz, Gerhard; Männer, W; Moser, H G; Richter, R H; Rosado-Schlosser, A; Schael, S; Settles, Ronald; Seywerd, H C J; Saint-Denis, R; Wolf, G; Alemany, R; Boucrot, J; Callot, O; Cordier, A; Courault, F; Davier, M; Duflot, L; Grivaz, J F; Heusse, P; Jacquet, M; Kim, D W; Le Diberder, F R; Lefrançois, J; Lutz, A M; Musolino, G; Nikolic, I A; Park, H J; Park, I C; Schune, M H; Simion, S; Veillet, J J; Videau, I; Abbaneo, D; Azzurri, P; Bagliesi, G; Batignani, G; Bettarini, S; Bozzi, C; Calderini, G; Carpinelli, M; Ciocci, M A; Ciulli, V; Dell'Orso, R; Fantechi, R; Ferrante, I; Foà, L; Forti, F; Giassi, A; Giorgi, M A; Gregorio, A; Ligabue, F; Lusiani, A; Marrocchesi, P S; Messineo, A; Rizzo, G; Sanguinetti, G; Sciabà, A; Spagnolo, P; Steinberger, Jack; Tenchini, Roberto; Tonelli, G; Triggiani, G; Vannini, C; Verdini, P G; Walsh, J; Betteridge, A P; Blair, G A; Bryant, L M; Cerutti, F; Gao, Y; Green, M G; Johnson, D L; Medcalf, T; Mir, L M; Perrodo, P; Strong, J A; Bertin, V; Botterill, David R; Clifft, R W; Edgecock, T R; Haywood, S; Edwards, M; Maley, P; Norton, P R; Thompson, J C; Bloch-Devaux, B; Colas, P; Emery, S; Kozanecki, Witold; Lançon, E; Lemaire, M C; Locci, E; Marx, B; Pérez, P; Rander, J; Renardy, J F; Roussarie, A; Schuller, J P; Schwindling, J; Trabelsi, A; Vallage, B; Johnson, R P; Kim, H Y; Litke, A M; McNeil, M A; Taylor, G; Beddall, A; Booth, C N; Boswell, R; Cartwright, S L; Combley, F; Dawson, I; Köksal, A; Letho, M; Newton, W M; Rankin, C; Thompson, L F; Böhrer, A; Brandt, S; Cowan, G D; Feigl, E; Grupen, Claus; Lutters, G; Minguet-Rodríguez, J A; Rivera, F; Saraiva, P; Smolik, L; Stephan, F; Apollonio, M; Bosisio, L; Della Marina, R; Giannini, G; Gobbo, B; Ragusa, F; Rothberg, J E; Wasserbaech, S R; Armstrong, S R; Bellantoni, L; Elmer, P; Feng, Z; Ferguson, D P S; Gao, Y S; González, S; Grahl, J; Harton, J L; Hayes, O J; Hu, H; McNamara, P A; Nachtman, J M; Orejudos, W; Pan, Y B; Saadi, Y; Schmitt, M; Scott, I J; Sharma, V; Turk, J; Walsh, A M; Wu Sau Lan; Wu, X; Yamartino, J M; Zheng, M; Zobernig, G

    1996-01-01

    From 64492 selected \\tau-pair events, produced at the Z^0 resonance, the measurement of the tau decays into hadrons from a global analysis using 1991, 1992 and 1993 ALEPH data is presented. Special emphasis is given to the reconstruction of photons and \\pi^0's, and the removal of fake photons. A detailed study of the systematics entering the \\pi^0 reconstruction is also given. A complete and consistent set of tau hadronic branching ratios is presented for 18 exclusive modes. Most measurements are more precise than the present world average. The new level of precision reached allows a stringent test of \\tau-\\mu universality in hadronic decays, g_\\tau/g_\\mu \\ = \\ 1.0013 \\ \\pm \\ 0.0095, and the first measurement of the vector and axial-vector contributions to the non-strange hadronic \\tau decay width: R_{\\tau ,V} \\ = \\ 1.788 \\ \\pm \\ 0.025 and R_{\\tau ,A} \\ = \\ 1.694 \\ \\pm \\ 0.027. The ratio (R_{\\tau ,V} - R_{\\tau ,A}) / (R_{\\tau ,V} + R_{\\tau ,A}), equal to (2.7 \\pm 1.3) \\ \\%, is a measure of the importance of Q...

  15. Molecular and phenotypic characteristics of seven novel mutations causing branched-chain organic acidurias.

    Science.gov (United States)

    Stojiljkovic, M; Klaassen, K; Djordjevic, M; Sarajlija, A; Brasil, S; Kecman, B; Grkovic, S; Kostic, J; Rodriguez-Pombo, P; Desviat, L R; Pavlovic, S; Perez, B

    2016-09-01

    Specific mitochondrial enzymatic deficiencies in the catabolism of branched-chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease-causing mutations were identified in nine unrelated branched-chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non-responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South-Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype-phenotype correlation for these rare diseases. PMID:26830710

  16. Bioinformatic and Expression Analysis of Rice Ubiquitin-conjugating Enzyme Gene Family%水稻泛素结合酶基因家族的生物信息学与表达分析

    Institute of Scientific and Technical Information of China (English)

    刘鑫; 张恒; 阚虎飞; 周立帅; 黄昊; 宋林林; 翟焕趁; 张君; 鲁国东

    2016-01-01

    The ubiquitin /proteasome system plays an important role in plant growth and development ,morphogenesis and disease resistance .Recent studies have shown that some pathogens can mimic the host plant ubiquitin /proteasome system components to achieve their own purposes .Ubiquitin‐conjugating enzyme is the second enzyme in the ubiquitination process and is indispensable for the plant ubiquitin/proteasome system .Previous studies showed that there are 48 predicted ubiquitin‐conjugating enzyme genes in rice genome .In order to preliminarily elucidate the functions of rice ubiquitin‐conjugating enzyme genes in plant disease resistance ,bioinformatic ,RNA‐seq and qRT‐PCR methods were used to analyze characteristics and expression patterns of rice ubiquitin‐conjugating enzyme gene family . Phylogenetic tree analyses indicate that the 48 rice ubiquitin‐conjugating enzyme genes can be divided into 3 groups ,7 sub‐groups in total .Protein domain analysis showed that ubiquitin‐conjugating enzyme genes mainly consist of a big ubiquitin‐conjugating enzyme catalytic domain .Expression analysis in silico suggested that most of the rice ubiquitin‐conjugating enzymes can be induced by blast fungus infection .Plant cis‐acting elements analysis indicated that four pathogen resistance cis‐acting elements and one hypersensitivity reaction cis‐acting element have high distribution in the promoter region of the 48 rice ubiquitin‐conjugating enzyme genes .RNA‐seq data from compatible and incompatible monogenic rice after rice blast fungus infection showed that 44 rice ubiquitin‐conjugating enzyme genes were expressed at 36 hours after treatment ,among which more than 50% were highly expressed genes .qRT‐PCR analysis showed that expression of some ubiquitin‐conjugating enzyme genes can be induced by the inoculation of rice blast fungus both in compatible and incompatible monogenic rice .However ,in incompatible rice the expression of rice ubiquitin

  17. Uptake of Branched-Chain Amino Acids by Streptococcus thermophilus

    OpenAIRE

    Akpemado, K. M.; Bracquart, P. A.

    1983-01-01

    The transport of branched-chain amino acids in Streptococcus thermophilus was energy dependent. The metabolic inhibitors of glycolysis and ATPase enzymes were active, but the proton-conducting uncouplers were not. Transport was optimal at temperatures of between 30 and 45°C and at pH 7.0 for the three amino acids leucine, valine, and isoleucine; a second peak existed at pH 5.0 with valine and isoleucine. By competition and kinetics studies, the branched-chain amino acids were found to share a...

  18. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990

    Energy Technology Data Exchange (ETDEWEB)

    Okita, T.W.

    1990-12-31

    The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of the gene that encodes for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. During the last two years we have obtained information on the structure of this enzyme from both potato tuber and rice endosperm, using a combination of biochemical and molecular biological approaches. Moreover, we present evidence that this enzyme may be localized at discrete regions of the starch grain within the amyloplast, and plays a role in controlling overall starch biosynthesis in potato tubers.

  19. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants

    KAUST Repository

    Chen, Hao

    2010-08-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed. © 2010 Blackwell Publishing Ltd.

  20. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  1. Food Enzymes

    Science.gov (United States)

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  2. The psychrophilic bacterium Pseudoalteromonas halosplanktis TAC125 possesses a gene coding for a cold-adapted feruloyl esterase activity that shares homology with esterase enzymes from gamma-proteobacteria and yeast.

    Science.gov (United States)

    Aurilia, Vincenzo; Parracino, Antonietta; Saviano, Michele; Rossi, Mose'; D'Auria, Sabato

    2007-08-01

    The complete genome of the psychrophilic bacteria Pseudoalteromonas haloplanktis TAC 125, recently published, owns a gene coding for a putative esterase activity corresponding to the ORF PSHAa1385, also classified in the Carbohydrate Active Enzymes database (CAZY) belonging to family 1 of carbohydrate esterase proteins. This ORF is 843 bp in length and codes for a protein of 280 amino acid residues. In this study we characterized and cloned the PSHAa1385 gene in Escherichia coli. We also characterized the recombinant protein by biochemical and biophysical methodologies. The PSHAa1385 gene sequence showed a significant homology with several carboxyl-esterase and acetyl-esterase genes from gamma-proteobacteria genera and yeast. The recombinant protein exhibited a significant activity towards pNP-acetate, alpha-and beta-naphthyl acetate as generic substrates, and 4-methylumbelliferyl p-trimethylammonio cinnamate chloride (MUTMAC) as a specific substrate, indicating that the protein exhibits a feruloyl esterase activity that it is displayed by similar enzymes present in other organisms. Finally, a three-dimensional model of the protein was built and the amino acid residues involved in the catalytic function of the protein were identified. PMID:17543477

  3. Interception of Cocaine by Enzyme or Antibody Delivered with Viral Gene Transfer: A Novel Strategy for Preventing Relapse in Recovering Drug Users

    OpenAIRE

    Brimijoin, Stephen

    2011-01-01

    Recent progress in enzyme engineering has led to versions of human butyrylcholinesterase (BChE) that hydrolyze cocaine efficiently in plasma, reduce concentrations reaching reward neurocircuity in the brain, and weaken behavioral responses to this drug. Along with enzyme advances, increasingly avid anti-cocaine antibodies and potent anti-cocaine vaccines have also been developed. Here we review these developments and consider the potential advantages along with the risks of delivering drug-in...

  4. Continuous-state branching processes

    CERN Document Server

    Li, Zenghu

    2012-01-01

    These notes were used in a short graduate course on branching processes the author gave in Beijing Normal University. The following main topics are covered: scaling limits of Galton--Watson processes, continuous-state branching processes, extinction probabilities, conditional limit theorems, decompositions of sample paths, martingale problems, stochastic equations, Lamperti's transformations, independent and dependent immigration processes. Some of the results are simplified versions of those in the author's book "Measure-valued branching Markov processes" (Springer, 2011). We hope these simplified results will set out the main ideas in an easy way and lead the reader to a quick access of the subject.

  5. Photoreactivating enzymes

    International Nuclear Information System (INIS)

    Photoreactivating enzymes (PRE) also called photolyases (EC 4.1.99.3) catalyze the light 300 to 600 nm)-dependent monomerization of cyclobutyl pyrimidine dimers, formed between adjacent pyrimidines on the same DNA strand, upon exposure to ultraviolet (uv) irradiation (220 to 320 nm). Although much is known about the substrate and product of these unusual enzymes, their identification required the development and synthesis of such fields as photochemistry, biochemistry, and microbiology. Photoreactivation was first known as a biological recovery phenomenon: cells exposed to visible light following uv irradiation showed higher survival than those kept in the dark. Early investigators examined the photoreactivability of an enormous range of cellular damage in both prokaryotes and eukaryotes. This review article discusses the purification and properties of PRE, the kinetics of photoreactivation and the biological role of this repair process

  6. Engineering enzymes

    OpenAIRE

    Dutton, P. Leslie; Moser, Christopher C.

    2011-01-01

    Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of str...

  7. Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae

    International Nuclear Information System (INIS)

    Highlights: •Novel identification of DNA repair-related genes in fish. •Investigation of whole expression profiling of DNA repair genes upon gamma radiation. •Analysis of effects of gamma radiation on antioxidant system and cell stress proteins. •Usefulness of verification of pathway-based profiling for mechanistic understanding. -- Abstract: To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4 Gy of radiation, and biochemical and molecular damage became substantial from 8 Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6 Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae

  8. Left bundle-branch block

    DEFF Research Database (Denmark)

    Risum, Niels; Strauss, David; Sogaard, Peter;

    2013-01-01

    The relationship between myocardial electrical activation by electrocardiogram (ECG) and mechanical contraction by echocardiography in left bundle-branch block (LBBB) has never been clearly demonstrated. New strict criteria for LBBB based on a fundamental understanding of physiology have recently...

  9. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.

    Science.gov (United States)

    Chiu, Hsien-Tai; Weng, Chien-Pao; Lin, Yu-Chin; Chen, Kuan-Hung

    2016-02-14

    Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis. PMID:26754528

  10. X-ray structure of MalY from Escherichia coli: a pyridoxal 5′-phosphate-dependent enzyme acting as a modulator in mal gene expression

    OpenAIRE

    Clausen, Tim; Schlegel, Anja; Peist, Ralf; Schneider, Eva; Steegborn, Clemens; Chang, Yuh-Shin; Haase, Andrea; Bourenkov, Gleb P.; Bartunik, Hans D.; Boos, Winfried

    2000-01-01

    MalY represents a bifunctional pyridoxal 5′-phosphate-dependent enzyme acting as a β–cystathionase and as a repressor of the maltose regulon. Here we present the crystal structures of wild-type and A221V mutant protein. Each subunit of the MalY dimer is composed of a large pyridoxal 5′-phosphate-binding domain and a small domain similar to aminotransferases. The structural alignment with related enzymes identifies residues that are generally responsible for β–lyase activity and depicts a uniq...

  11. Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis

    OpenAIRE

    Baguma, Yona; Sun, Chuanxin; Borén, Mats; Olsson, Helena; Rosenqvist, Sara; Mutisya, Joel; Patrick R. Rubaihayo; Jansson, Christer

    2008-01-01

    Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expr...

  12. Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase.

    OpenAIRE

    Walfridsson, M; Hallborn, J; Penttilä, M.; Keränen, S; Hahn-Hägerdal, B

    1995-01-01

    Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-con...

  13. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    Science.gov (United States)

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  14. Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes

    Directory of Open Access Journals (Sweden)

    Labedan Bernard

    2006-01-01

    Full Text Available Abstract Background The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i the classical N-acetylglutamate synthase (NAGS, gene argA first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii the bifunctional version of ornithine acetyltransferase (OAT, gene argJ present in Bacteria, Archaea and many Eukaryotes. This paper focuses on a new and surprising aspect of glutamate acetylation. We recently showed that in Moritella abyssi and M. profunda, two marine gamma proteobacteria, the gene for the last enzyme in arginine biosynthesis (argH is fused to a short sequence that corresponds to the C-terminal, N-acetyltransferase-encoding domain of NAGS and is able to complement an argA mutant of E. coli. Very recently, other authors identified in Mycobacterium tuberculosis an independent gene corresponding to this short C-terminal domain and coding for a new type of NAGS. We have investigated the two prokaryotic Domains for patterns of gene-enzyme relationships in the first committed step of arginine biosynthesis. Results The argH-A fusion, designated argH(A, and discovered in Moritella was found to be present in (and confined to marine gamma proteobacteria of the Alteromonas- and Vibrio-like group. Most of them have a classical NAGS with the exception of Idiomarina loihiensis and Pseudoalteromonas haloplanktis which nevertheless can grow in the absence of arginine and therefore appear to rely on the arg(A sequence for arginine biosynthesis. Screening prokaryotic genomes for virtual argH-X 'fusions' where X stands for a homologue of arg(A, we retrieved a large number of Bacteria and several Archaea, all of them devoid of a classical NAGS. In the case of Thermus thermophilus and Deinococcus radiodurans, the arg(A-like sequence

  15. Fragrance release from the surface of branched poly (amide)s.

    Science.gov (United States)

    Aulenta, Francesca; Drew, Michael G B; Foster, Alison; Hayes, Wayne; Rannard, Steven; Thornthwaite, David W; Youngs, Tristan G A

    2005-01-01

    Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased. PMID:18007278

  16. The Effects of HBx Gene on the Expression of DNA Repair Enzymes hOGG1 and hMYHα mRNA in HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    Bin CHENG; Xiaorong GUO; Yaochu ZHENG; Ying WANG; Chunyan LIU; Peiyuan LI

    2009-01-01

    To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHα and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma,the gene-transfected cells HepG2/HBx which stably expressed HBx was established,and the effect of HBx on the cell cycle and proliferation of HepG2 was examined.By using the β-actin as the interior control,real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx,the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1).The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1.Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx (0.021±0.007) was significantly lower than that of HepG2 (0.099±0.041) (P0.05).The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05).It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage,to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function,thus participate in the occurrence and development of hepatocellular carcinoma.

  17. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  18. Roles for auxin, cytokinin, and strigolactone in regulating shoot branching.

    Science.gov (United States)

    Ferguson, Brett J; Beveridge, Christine A

    2009-04-01

    Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth. PMID:19218361

  19. A pre- and co-knockdown of RNAseT enzyme, Eri-1, enhances the efficiency of RNAi induced gene silencing in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Pooja Jadiya

    Full Text Available BACKGROUND: The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown. METHODOLOGY/PRINCIPAL FINDINGS: Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans. CONCLUSIONS/SIGNIFICANCE: Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains.

  20. Multiple pathways regulate shoot branching

    Directory of Open Access Journals (Sweden)

    Catherine eRameau

    2015-01-01

    Full Text Available Shoot branching patterns result from the spatio-temporal regulation of axillary bud outgrowth. Numerous endogenous, developmental and environmental factors are integrated at the bud and plant levels to determine numbers of growing shoots. Multiple pathways that converge to common integrators are most probably involved. We propose several pathways involving not only the classical hormones auxin, cytokinins and strigolactones, but also other signals with a strong influence on shoot branching such as gibberellins, sugars or molecular actors of plant phase transition. We also deal with recent findings about the molecular mechanisms and the pathway involved in the response to shade as an example of an environmental signal controlling branching. We propose the TCP transcription factor TB1/BRC1 and the polar auxin transport stream in the stem as possible integrators of these pathways. We finally discuss how modeling can help to represent this highly dynamic system by articulating knowledges and hypothesis and calculating the phenotype properties they imply.

  1. Computer simulation of long-chain branching and branching indexes

    Czech Academy of Sciences Publication Activity Database

    Netopilík, Miloš

    Vienna : University of Vienna, 2014. s. 22. [International Conference on Polymer Behaviour /6./. 22.09.2014-26.09.2014, Vienna] R&D Projects: GA ČR(CZ) GA13-02938S Institutional support: RVO:61389013 Keywords : branching indexes * intrinsic viscosity * radius of gyration Subject RIV: CD - Macromolecular Chemistry

  2. Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

    Directory of Open Access Journals (Sweden)

    Spatz Linus

    2000-01-01

    Full Text Available The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

  3. Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

    OpenAIRE

    Sá-Correia, I.; Darzins, A; Wang, S K; Berry, A.; Chakrabarty, A M

    1987-01-01

    The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide prec...

  4. Evolution of Arginine Biosynthesis in the Bacterial Domain: Novel Gene-Enzyme Relationships from Psychrophilic Moritella Strains (Vibrionaceae) and Evolutionary Significance of N-α-Acetyl Ornithinase

    OpenAIRE

    Xu, Ying; Liang, Ziyuan; Legrain, Christianne; Rüger, Hans J.; Glansdorff, Nicolas

    2000-01-01

    In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of N-α-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (argJ encoded). Only two exceptions had been reported—the Enterobacteriaceae and Myxococcus xanthus (members of the γ and δ groups of the class Proteobacteria, respectively)—in which ornithine is produced from N-α-acetylornithine by a deacylase, acetylornithinase (...

  5. Similar Therapeutic Efficacy Between a Single Administration of Gene Therapy and Multiple Administrations of Recombinant Enzyme in a Mouse Model of Lysosomal Storage Disease

    OpenAIRE

    Ferla, Rita; Claudiani, Pamela; Cotugno, Gabriella; Saccone, Paola; De Leonibus, Elvira; Auricchio, Alberto

    2014-01-01

    Enzyme replacement therapy (ERT) has become the standard of care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSD manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI, we compared the efficacy of a single intravascular administration of an adeno-associated viral vector targeting liver to weekly infusions o...

  6. The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

    OpenAIRE

    P. Marini(GANIL); Li, S J; Gardiol, D; Cronan, J E; De Mendoza, D

    1995-01-01

    The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

  7. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

    OpenAIRE

    Anjum, Awais; Kelly, Brathwaite; Aidley, Jack B; Connerton, Phillippa L.; Cummings, Nicola J; Parkhill, Julian; Ian F Connerton; Bayliss, Christopher D

    2016-01-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system ...

  8. Enzyme-free and sensitive electrochemical determination of the FLT3 gene based on a dual signal amplified strategy: Controlled nanomaterial multilayers and a target-catalyzed hairpin assembly.

    Science.gov (United States)

    Sun, Yingying; Ren, Qunxiang; Liu, Bo; Qin, Yan; Zhao, Shuang

    2016-04-15

    An isothermal, enzyme-free and sensitive electrochemical DNA sensor was developed for the detection of the FLT3 gene in acute myeloid leukemia (AML). First, aminated multi-walled carbon nanotubes (AMWNTs) and gold nanoparticles (AuNPs) were alternately self-assembled on a gold electrode using a layer-by-layer strategy. Then, the hairpin DNA probe 1 (H1), with a thiol group at the 3' end and a ferrocenyl moiety (Fc) at the 5' end, was immobilized on the AMWNTs/AuNPs multilayer films through Au-S bonding. When the target DNA (TD) appeared, it hybridized with and opened the hairpin structure of H1, and Fc was forced away from the electrode surface, leading to a significant decrease in the current peak of square wave voltammetry. Subsequently, the hairpin DNA probe 2 (H2) bound to H1, freeing the TD to trigger another reaction cycle. The combination of this target-catalyzed hairpin assembly and the LBL assembly of nanomaterials achieved a detection limit of 0.1 pM with a wide linear range of 0.1-1000 pM. The sensor discriminated between mismatched DNA and the target DNA with high selectivity. This dual signal amplification strategy is relatively simple and inexpensive because it does not need any enzymes or sophisticated equipment and successfully assayed the FLT3 gene from real samples. PMID:26584077

  9. Environmental enrichment attenuates the age-related decline in the mRNA expression of steroidogenic enzymes and reduces the methylation state of the steroid 5α-reductase type 1 gene in the rat hippocampus.

    Science.gov (United States)

    Rossetti, María F; Varayoud, Jorgelina; Moreno-Piovano, Guillermo S; Luque, Enrique H; Ramos, Jorge G

    2015-09-01

    We analyzed the effects of aging and environmental enrichment on the mRNA expression and DNA methylation state of steroidogenic enzymes in the hippocampus. The effects of aging were evaluated by comparing young adult (90-day-old) and middle-aged (450-day-old) female Wistar rats. To elucidate the effects of environmental enrichment, a subgroup of middle-aged rats exposed to sensory and social stimulation for 105 days was compared to rats housed under standard laboratory conditions. Aging decreased the transcription of neurosteroidogenic-related genes and increased the promoter methylation state of cytochrome P450 side chain cleavage, 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5α-reductase-1. Exposure of middle-aged rats to environmental enrichment increased mRNA levels of 5α-reductase-1, 3α-HSD and cytochrome P450 17α-hydroxylase/c17,20-lyase and decreased the methylation state of the 5α-reductase-1 gene. Thus, sensory and social stimulation attenuate the age-related decline in the mRNA expression of hippocampal steroidogenic enzymes. Epigenetic mechanisms associated with differential promoter methylation could be involved. PMID:26021641

  10. Association of Angiotensin-Converting Enzyme Intron 16 Insertion/Deletion and Angiotensin II Type 1 Receptor A1166C Gene Polymorphisms with Preeclampsia in South East of Iran

    Directory of Open Access Journals (Sweden)

    Saeedeh Salimi

    2011-01-01

    Full Text Available Some evidence suggests that a variety of genetic factors contributed in pathogenesis of the preeclampsia. The aim of this study was to assess the association between the angiotensin-converting enzyme (ACE I/D and angiotensin II type1 receptor A1166C polymorphisms with preeclampsia. This study was performed in 125 preeclamptic pregnant women and 132 controls. The I/D Polymorphism of the ACE gene was assessed by polymerase chain reaction and the A1166C Polymorphism of the AT1R gene was determined by restriction fragment length polymorphism. The genotype and allele frequencies of I/D polymorphism differed between two groups. The risk of preeclampsia was 3.2-fold in pregnant women with D allele (OR, 3.2 [95% CI, 1.1 to 3.8]; P=0.01. The distribution of the AT1R gene A1166C polymorphism was similar in affected and control groups. Our results supported that presence of the I/D polymorphism of ACE gene is a marker for the increased risk of preeclampsia.

  11. NCI: DCTD: Biometric Research Branch

    Science.gov (United States)

    The Biometric Research Branch (BRB) is the statistical and biomathematical component of the Division of Cancer Treatment, Diagnosis and Centers (DCTDC). Its members provide statistical leadership for the national and international research programs of the division in developmental therapeutics, developmental diagnostics, diagnostic imaging and clinical trials.

  12. Branching of keratin intermediate filaments.

    Science.gov (United States)

    Nafeey, Soufi; Martin, Ines; Felder, Tatiana; Walther, Paul; Felder, Edward

    2016-06-01

    Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation. PMID:27039023

  13. Multi-Level Flow Branching

    Czech Academy of Sciences Publication Activity Database

    Tesař, Václav

    Praha: UTAM AV ČR, 2006 - (Náprstek, J.; Fischer, C.), s. 380-381 ISBN 80-86246-27-2. [Engineering mechanics 2006 : national conference with international participation. Svratka (CZ), 15.05.2006-18.05.2006] Institutional research plan: CEZ:AV0Z20760514 Keywords : branching * respiratory system * aortic system * microfluidics Subject RIV: BK - Fluid Dynamics

  14. Cash efficiency for bank branches.

    Science.gov (United States)

    Cabello, Julia García

    2013-01-01

    Bank liquidity management has become a major issue during the financial crisis as liquidity shortages have intensified and have put pressure on banks to diversity and improve their liquidity sources. While a significant strand of the literature concentrates on wholesale liquidity generation and on the alternative to deposit funding, the management of an inventory of cash holdings within the banks' branches is also a relevant issue as any significant improvement in cash management at the bank distribution channels may have a positive effect in reducing liquidity tensions. In this paper, we propose a simple programme of cash efficiency for the banks' branches, very easy to implement, which conform to a set of instructions to be imposed from the bank to their branches. This model proves to significantly reduce cash holdings at branches thereby providing efficiency improvements in liquidity management. The methodology we propose is based on the definition of some stochastic processes combined with renewal processes, which capture the random elements of the cash flow, before applying suitable optimization programmes to all the costs involved in cash movements. The classical issue of the Transaction Demand for the Cash and some aspects of Inventory Theory are also present. Mathematics Subject Classification (2000) C02, C60, E50. PMID:24010026

  15. Fluid Flow at Branching Junctions

    OpenAIRE

    Sochi, Taha

    2013-01-01

    The flow of fluids at branching junctions plays important kinematic and dynamic roles in most biological and industrial flow systems. The present paper highlights some key issues related to the flow of fluids at these junctions with special emphasis on the biological flow networks particularly blood transportation vasculature.

  16. Risk Factor Assessment Branch (RFAB)

    Science.gov (United States)

    The Risk Factor Assessment Branch (RFAB) focuses on the development, evaluation, and dissemination of high-quality risk factor metrics, methods, tools, technologies, and resources for use across the cancer research continuum, and the assessment of cancer-related risk factors in the population.

  17. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase genes. Progress report, [April 15, 1990--April 14, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Okita, T.W.

    1990-12-31

    The long term goal of this project is to assess the feasibility of increasing the conversion of photosynthate a key regulatory enzyme in starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is primarily regulated by the gene activation, expression, and allosteric regulation of ADPglucose pyrophosphorylase, as well as starch synthase, and branching enzyme. During the last year we have elucidated the structure of both subunits which compose this tetrameric enzyme and determined the temporal and spatial expression of the genes encoding each subunit as well as their correlation to starch biosynthesis. Genomic clones to both subunits have also been isolated and the gene structure of the small subunit determined. Transgenic potato plants have been produced containing deletions of the small subunit promoter. Currently, cis acting elements and their involvement in spatial and temporal expression are under investigation.

  18. Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

    International Nuclear Information System (INIS)

    A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids

  19. Registration of durum wheat germplasm lines with combined mutations in SBEIIa and SBEIIb genes conferring increased amylose and resistant starch

    OpenAIRE

    Hazard, B; Zhang, X.; Naemeh, M; Dubcovsky, J.

    2014-01-01

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations i...

  20. Interactions between auxin and strigolactone in shoot branching control.

    Science.gov (United States)

    Hayward, Alice; Stirnberg, Petra; Beveridge, Christine; Leyser, Ottoline

    2009-09-01

    In Arabidopsis (Arabidopsis thaliana), the carotenoid cleavage dioxygenases MORE AXILLARY GROWTH3 (MAX3) and MAX4 act together with MAX1 to produce a strigolactone signaling molecule required for the inhibition of axillary bud outgrowth. We show that both MAX3 and MAX4 transcripts are positively auxin regulated in a manner similar to the orthologous genes from pea (Pisum sativum) and rice (Oryza sativa), supporting evolutionary conservation of this regulation in plants. This regulation is important for branching control because large auxin-related reductions in these transcripts are associated with increased axillary branching. Both transcripts are up-regulated in max mutants, and consistent with max mutants having increased auxin in the polar auxin transport stream, this feedback regulation involves auxin signaling. We suggest that both auxin and strigolactone have the capacity to modulate each other's levels and distribution in a dynamic feedback loop required for the coordinated control of axillary branching. PMID:19641034

  1. Deubiquitylating enzymes and disease

    Directory of Open Access Journals (Sweden)

    Baker Rohan T

    2008-10-01

    Full Text Available Abstract Deubiquitylating enzymes (DUBs can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin, including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development. Publication history Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  2. Starch granules size distribution in superior and inferior grains of wheat is related to enzyme activities and their gene expressions during grain filling

    DEFF Research Database (Denmark)

    Zhang, Chuanhui; Jiang, Dong; Liu, Fulai; Cai, Jian; Dai, Tingbo; Cao, Weixing

    2010-01-01

    Mature wheat endosperm contains A-, B-, C-type starch granules, and each class has unique physiochemical properties which determine the quality of starch. The dynamics of the starch granule size distribution, activities of starch synthases and expression of starch synthase encoding genes were...... studied in superior and inferior grains during grain filling. Compared with inferior grains, superior grains showed higher grain weight, contents of starch, amylose and amylopectin. The formation of A-, B-, C-type starch granules initiated at 4, 8, 20 DAF, respectively, and was well consistent with the...... temporally change patterns of starch synthase activities and relative gene expression levels. For instance, activities of soluble and granule-bound starch synthases (designated SSS and GBSS) peaked at 20 and 24 DAF. Genes encoding isoforms of starch synthases expressed at different grain filling periods. In...

  3. UPLC/Q-TOF MS-based metabolomics and qRT-PCR in enzyme gene screening with key role in triterpenoid saponin biosynthesis of Polygala tenuifolia.

    Directory of Open Access Journals (Sweden)

    Fusheng Zhang

    Full Text Available The dried root of Polygala tenuifolia, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in P. tenuifolia is unclear.In this study, ultra-performance liquid chromatography (UPLC coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of P. tenuifolia. A total of 22 marker compounds (VIP>1 were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of P. tenuifolia. The expression levels of squalene synthase (SQS, squalene monooxygenase (SQE, and beta-amyrin synthase (β-AS were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis.This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of P. tenuifolia.

  4. Accessory enzymes from Aspergillus involved in xylan and pectin degradation

    OpenAIRE

    Vries, de, G.

    1999-01-01

    The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding genes. This thesis describes the purification and characterisation of two accessory enzymes from Aspergillus , feruloyl esterase A (FaeA) andα-glucuronidase A (AguA), and the activities of these enz...

  5. Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine-β-Hydroxylase- and CRH-Knockout Mice Exposed to Stress

    OpenAIRE

    Richard, Kvetnansky; Olga, Krizanova; Andrej, Tillinger; Sabban Esther, L.; Thomas Steven, A; Lucia, Kubovcakova

    2008-01-01

    Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-β-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, sin...

  6. Branching processes in disease epidemics

    Science.gov (United States)

    Singh, Sarabjeet

    Branching processes have served as a model for chemical reactions, biological growth processes and contagion (of disease, information or fads). Through this connection, these seemingly different physical processes share some common universalities that can be elucidated by analyzing the underlying branching process. In this thesis, we focus on branching processes as a model for infectious diseases spreading between individuals belonging to different populations. The distinction between populations can arise from species separation (as in the case of diseases which jump across species) or spatial separation (as in the case of disease spreading between farms, cities, urban centers, etc). A prominent example of the former is zoonoses -- infectious diseases that spill from animals to humans -- whose specific examples include Nipah virus, monkeypox, HIV and avian influenza. A prominent example of the latter is infectious diseases of animals such as foot and mouth disease and bovine tuberculosis that spread between farms or cattle herds. Another example of the latter is infectious diseases of humans such as H1N1 that spread from one city to another through migration of infectious hosts. This thesis consists of three main chapters, an introduction and an appendix. The introduction gives a brief history of mathematics in modeling the spread of infectious diseases along with a detailed description of the most commonly used disease model -- the Susceptible-Infectious-Recovered (SIR) model. The introduction also describes how the stochastic formulation of the model reduces to a branching process in the limit of large population which is analyzed in detail. The second chapter describes a two species model of zoonoses with coupled SIR processes and proceeds into the calculation of statistics pertinent to cross species infection using multitype branching processes. The third chapter describes an SIR process driven by a Poisson process of infection spillovers. This is posed as a

  7. Effects of dietary probiotic supplementation on LXRα and CYP7α1 gene expression, liver enzyme activities and fat metabolism in ducks.

    Science.gov (United States)

    Huang, Z; Mu, C; Chen, Y; Zhu, Z; Chen, C; Lan, L; Xu, Q; Zhao, W; Chen, G

    2015-04-01

    1. The objective of this study was to investigate the effects of dietary probiotic supplementation on liver X receptor alpha (LXRα) and cholesterol 7α-hydroxylase (CYP7α1) mRNA levels, protein enzymatic activities and fat metabolism in Cherry Valley Pekin ducks. 2. A total of 750 one-day-old Cherry Valley Pekin ducks were randomly divided into 5 groups with three replicates of 50 ducks each in a completely randomised experiment. Each group was fed on a basal diet supplemented with 0, 500, 1000, 1500 or 2000 mg probiotics/kg. 3. Body rate and feed conversion ratio were highest and abdominal subcutaneous fat % was lowest at 1000 mg probiotic/kg. 4. The mRNA levels of LXRα and CYP7α1 in liver tissue was estimated by RT-PCR; serum triglyceride (TG) and total cholesterol (TC) concentrations were measured by ELISA. 5. The expression levels and enzyme activity of LXRα and CYP7α1 increased in conjunction with decreases in TG and TC concentrations following probiotic supplementation to a maximum at 1000 mg probiotics/kg and decreased thereafter. 6. It is concluded that dietary probiotics can enhance LXRα and CYP7α1 enzyme activities in the liver and reduce lipid concentrations and fat deposition in ducks. PMID:25559164

  8. Prediction of Wild-type Enzyme Characteristics

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus

    article presents a sequence-based approach to discovery of proteolytic enzymes in metagenomes obtained from the Polar oceans. We show that microorganisms living in these extreme environments of constant low temperature harbour genes encoding novel proteolytic enzymes with potential industrial relevance...

  9. Cytochrome P450 enzyme systems in fungi

    NARCIS (Netherlands)

    Brink, H.M. van den; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    1998-01-01

    The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have

  10. Stereoselective interactions of warfarin enantiomers with the pregnane X nuclear receptor in gene regulation of major drug-metabolizing cytochrome P450 enzymes

    Czech Academy of Sciences Publication Activity Database

    Rulcová, A.; Prokopová, I.; Krausová, L.; Bitman, M.; Vrzal, R.; Dvořák, Z.; Blahoš, Jaroslav; Pávek, P.

    2010-01-01

    Roč. 8, č. 12 (2010), s. 2708-2717. ISSN 1538-7933 R&D Projects: GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50520514 Keywords : CYP3A4 * gene regulation * warfarin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.439, year: 2010

  11. Metabolic and chaperone gene loss marks the origin of animals: evidence for Hsp104 and Hsp78 chaperones sharing mitochondrial enzymes as clients.

    Directory of Open Access Journals (Sweden)

    Albert J Erives

    Full Text Available The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i sixteen distinct biosynthetic functions; (ii the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.

  12. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

    Science.gov (United States)

    Anjum, Awais; Brathwaite, Kelly J; Aidley, Jack; Connerton, Phillippa L; Cummings, Nicola J; Parkhill, Julian; Connerton, Ian; Bayliss, Christopher D

    2016-06-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5'CCCGA and 5'CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. PMID:26786317

  13. Evolution of the 4-coumarate:coenzyme A ligase (4CL) gene family:Conserved evolutionary pattern and two new gene classes in gymnosperms

    Institute of Scientific and Technical Information of China (English)

    Hui GAO; Dong-Mei GUO; Wen-Juan LIU; Jin-Hua RAN; Xiao-Quan WANG

    2012-01-01

    The 4-coumarate:coenzyme A ligase (4CL) is the branch point enzyme that channels the general phenylpropanoid metabolism into specific lignin and flavonoid biosynthesis branches.Genetic engineering experiments on the 4CL gene have been carried out in many species,but the precise functions of different gene members are still unresolved.To investigate the evolutionary relationships and functional differentiation of the 4CL gene family,we made a comprehensive evolutionary analysis of this gene family from 27 species representing the major lineages of land plants.The phylogenetic analysis indicates that both vascular and seed plant 4CL genes form monophyletic groups,and that three and two 4CL classes can be recognized in gymnosperms and angiosperms,respectively.The evolutionary rate and frequency of duplication of the 4CL gene family are much more conserved than that of the CAD/SAD (cinnamyl/sinapyl alcohol dehydrogenase) gene family,which catalyzes the last step in monolignol biosynthesis.This may be due to different selective pressures on these genes whose products catalyze different steps in the biosynthesis pathway.In addition,we found two new major classes of 4CL genes in gymnosperms.

  14. Screening for and isolation and identification of malathion-degrading bacteria: cloning and sequencing a gene that potentially encodes the malathion-degrading enzyme, carboxylestrase in soil bacteria.

    Science.gov (United States)

    Goda, Sayed K; Elsayed, Iman E; Khodair, Taha A; El-Sayed, Walaa; Mohamed, Mervat E

    2010-11-01

    Five malathion-degrading bacterial strains were enriched and isolated from soil samples collected from different agricultural sites in Cairo, Egypt. Malathion was used as a sole source of carbon (50 mg/l) to enumerate malathion degraders, which were designated as IS1, IS2, IS3, IS4, and IS5. They were identified, based on their morphological and biochemical characteristics, as Pseudomonas sp., Pseudomonas putida, Micrococcus lylae, Pseudomonas aureofaciens, and Acetobacter liquefaciens, respectively. IS1 and IS2, which showed the highest degrading activity, were selected for further identification by partial sequence analysis of their 16S rRNA genes. The 16S rRNA gene of IS1 shared 99% similarity with that of Alphaprotoebacterium BAL284, while IS2 scored 100% similarity with that of Pseudomonas putida 32zhy. Malathion residues almost completely disappeared within 6 days of incubation in IS2 liquid cultures. LC/ESI-MS analysis confirmed the degradation of malathion to malathion monocarboxylic and dicarboxylic acids, which formed as a result of carboxylesterase activity. A carboxylesterase gene (CE) was amplified from the IS2 genome by using specifically designed PCR primers. The sequence analysis showed a significant similarity to a known CE gene in different Pseudomonas sp. We report here the isolation of a new malathion-degrading bacteria from soils in Egypt that may be very well adapted to the climatic and environmental conditions of the country. We also report the partial cloning of a new CE gene. Due to their high biodegradation activity, the bacteria isolated from this work merit further study as potential biological agents for the remediation of soil, water, or crops contaminated with the pesticide malathion. PMID:20401686

  15. Branching problems of unitary representations

    OpenAIRE

    Kobayashi, Toshiyuki

    2003-01-01

    The irreducible decomposition of a unitary representation often contains continuous spectrum when restricted to a non-compact subgroup. The author singles out a nice class of branching problems where each irreducible summand occurs discretely with finite multiplicity (admissible restrictions). Basic theory and new perspectives of admissible restrictions are presented from both analytic and algebraic view points. We also discuss some applications of admissible restrictions to modular varieties...

  16. Cash efficiency for bank branches

    OpenAIRE

    Cabello, Julia García

    2013-01-01

    Bank liquidity management has become a major issue during the financial crisis as liquidity shortages have intensified and have put pressure on banks to diversity and improve their liquidity sources. While a significant strand of the literature concentrates on wholesale liquidity generation and on the alternative to deposit funding, the management of an inventory of cash holdings within the banks’ branches is also a relevant issue as any significant improvement in cash management at the bank ...

  17. Solid State Photovoltaic Research Branch

    Energy Technology Data Exchange (ETDEWEB)

    1990-09-01

    This report summarizes the progress of the Solid State Photovoltaic Research Branch of the Solar Energy Research Institute (SERI) from October 1, 1988, through September 30,l 1989. Six technical sections of the report cover these main areas of SERIs in-house research: Semiconductor Crystal Growth, Amorphous Silicon Research, Polycrystalline Thin Films, III-V High-Efficiency Photovoltaic Cells, Solid-State Theory, and Laser Raman and Luminescence Spectroscopy. Sections have been indexed separately for inclusion on the data base.

  18. Creep of welded branched pipes

    OpenAIRE

    Rayner, Glen

    2004-01-01

    Creep failure of welds in high-temperature power plant steam piping systems is known to be a potential cause of plant failure. Creep behaviour of plain pipes with circumferential welds and cross-weld specimens have received fairly extensive attention. However, research into the creep behaviour of welded thick-walled branched steam pipes has received less attention. Consequently, this thesis addresses improving the understanding of the creep behaviour for this type of geometry. Numerical and a...

  19. Interactions between Axillary Branches of Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Veronica Ongaro; Katherine Bainbridge; Lisa Williamson; Ottoline Leyser

    2008-01-01

    Studies of apical dominance have benefited greatly from two-branch assays in pea and bean,in which the shoot system is trimmed back to leave only two active cotyledonary axillary branches.In these two-branch shoots,a large body of evidence shows that one actively growing branch is able to inhibit the growth of the other,prompting studies on the nature of the inhibitory signals,which are still poorly understood.Here,we describe the establishment of two-branch assays in Arabidopsis,using consecutive branches on the bolting stem.As with the classical studies in pea and bean,these consecutive branches are able to inhibit one another's growth.Not only can the upper branch inhibit the lower branch,but also the lower branch can inhibit the upper branch,illustrating the bi-directional action of the inhibitory signals.Using mutants,we show that the inhibition is partially dependent on the MAX pathway and that while the inhibition is clearly transmitted across the stem from the active to the inhibited branch,the vascular connectivity of the two branches is weak,and the MAX pathway is capable of acting unilaterally in the stem.

  20. Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Hearst, J.E. (Univ. of California, Berkeley (United States) Lawrence Berkeley Lab., CA (United States)); Alberti, M. (Lawrence Berkeley Lab., CA (United States))

    1990-12-01

    Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C{sub 40} carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. The authors report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to the carotenoid branch of general isoprenoid metabolism. All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding {beta}{alpha}{beta} fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionary diverse organisms.