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Sample records for acid stimulated-like gene

  1. Modulating gene function with peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Crooke, Stanley T.

    2008-01-01

    A review on peptide nucleic acid (PNA) oligomers as modulators of gene expression ranging from gene silencing at the mRNAor the dsDNA (antigene) level, and redirection of mRNA splicing to gene activation through transcription bubble mimicking. PNA chem., anti-infective agents, cellular delivery, ...

  2. Mechanisms of gene regulation by fatty acids

    NARCIS (Netherlands)

    Georgiadi, A.; Kersten, A.H.

    2012-01-01

    Consumption of specific dietary fatty acids has been shown to influence risk and progression of several chronic diseases, such as cardiovascular disease, obesity, cancer, and arthritis. In recent years, insights into the mechanisms underlying the biological effects of fatty acids have improved consi

  3. Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference"...

  4. Gene quantification by the NanoGene assay is resistant to inhibition by humic acids.

    Science.gov (United States)

    Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong

    2011-10-15

    NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (captured by the probe and signaling DNA.

  5. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    Science.gov (United States)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2016-07-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  6. Inducible gene expression and environmentally regulated genes in lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1996-01-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transc

  7. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

    Directory of Open Access Journals (Sweden)

    Zhimin Dai

    Full Text Available Biological nitrogen fixation is an essential function of acid mine drainage (AMD microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

  8. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

    Science.gov (United States)

    Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

    2014-01-01

    Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

  9. Quorum sensing-controlled gene expression in lactic acid bacteria

    NARCIS (Netherlands)

    Kuipers, Oscar P.; Ruyter, Pascalle G.G.A. de; Kleerebezem, Michiel; Vos, Willem M. de

    1998-01-01

    Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the struc

  10. Tomato ABSCISIC ACID STRESS RIPENING (ASR) gene family revisited.

    Science.gov (United States)

    Golan, Ido; Dominguez, Pia Guadalupe; Konrad, Zvia; Shkolnik-Inbar, Doron; Carrari, Fernando; Bar-Zvi, Dudy

    2014-01-01

    Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.

  11. Tomato ABSCISIC ACID STRESS RIPENING (ASR gene family revisited.

    Directory of Open Access Journals (Sweden)

    Ido Golan

    Full Text Available Tomato ABSCISIC ACID RIPENING 1 (ASR1 was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each, whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons. ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA. Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.

  12. Complex modulation of androgen responsive gene expression by methoxyacetic acid

    Directory of Open Access Journals (Sweden)

    Stanley Kerri A

    2011-03-01

    Full Text Available Abstract Background Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA, the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known. Methods A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale. Results MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes. Conclusions These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model.

  13. Repeats in transforming acidic coiled-coil (TACC) genes.

    Science.gov (United States)

    Trivedi, Seema

    2013-06-01

    Transforming acidic coiled-coil proteins (TACC1, 2, and 3) are essential proteins associated with the assembly of spindle microtubules and maintenance of bipolarity. Dysregulation of TACCs is associated with tumorigenesis, but studies of microsatellite instability in TACC genes have not been extensive. Microsatellite or simple sequence repeat instability is known to cause many types of cancer. The present in silico analysis of SSRs in human TACC gene sequences shows the presence of mono- to hexa-nucleotide repeats, with the highest densities found for mono- and di-nucleotide repeats. Density of repeats is higher in introns than in exons. Some of the repeats are present in regulatory regions and retained introns. Human TACC genes show conservation of many repeat classes. Microsatellites in TACC genes could be valuable markers for monitoring numerical chromosomal aberrations and or cancer.

  14. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  15. Structure of the human 4-hydroxyphenylpyruvic acid dioxygenase gene (HPD)

    Energy Technology Data Exchange (ETDEWEB)

    Awata, H.; Endo, F.; Matsuda, I. [Kumamoto Univ. (Japan)

    1994-10-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and developmentally regulated in mammals, and a genetic deficiency in this enzyme in humans and mice leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human gene libraries. The human HPD gene is over 30 kb long and is split into 14 exons. The extract sizes and boundaries of exon blocks were determined, and all of the splice donor and acceptor sites conformed to the GT/AG rule. Analysis of the 5{prime} flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5{prime} flanking region of the gene are similar to those of other genes that are specifically expressed in hepatocytes and that are developmentally regulated. 41 refs., 2 figs., 1 tab.

  16. The pea gene NA encodes ent-kaurenoic acid oxidase.

    Science.gov (United States)

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  17. Impact of methoxyacetic acid on mouse Leydig cell gene expression

    Directory of Open Access Journals (Sweden)

    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  18. Inducible gene expression and environmentally regulated genes in lactic acid bacteria.

    Science.gov (United States)

    Kok, J

    1996-10-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed.

  19. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

  20. Hepatic bile acids and bile acid-related gene expression in pregnant and lactating rats

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    Qiong N. Zhu

    2013-08-01

    Full Text Available Background. Significant physiological changes occur during pregnancy and lactation. Intrahepatic cholestasis of pregnancy (ICP is a liver disease closely related to disruption of bile acid homeostasis. The objective of this study was to examine the regulation of bile acid synthesis and transport in normal pregnant and lactating rats.Materials and Methods. Livers from timed pregnant SD rats were collected on gestational days (GD 10, 14 and 19, and postnatal days (PND 1, 7, 14 and 21. Total bile acids were determined by the enzymatic method, total RNA was isolated and subjected to real time RT-PCR analysis. Liver protein was extracted for western-blot analysis.Results. Under physiological conditions hepatic bile acids were not elevated during pregnancy but increased during lactation in rats. Bile acid synthesis rate-limiting enzyme Cyp7a1 was unchanged on gestational days, but increased on PND14 and 21 at mRNA and protein levels. Expression of Cyp8b1, Cyp27a1 and Cyp7b1 was also higher during lactation. The mRNA levels of small heterodimer partner (SHP and protein levels of farnesoid X receptor (FXR were increased during pregnancy and lactation. Bile acid transporters Ntcp, Bsep, Mrp3 and Mrp4 were lower at gestation, but increased during lactation. Hepatic Oatp transporters were decreased during pregnancy and lactation.Conclusion. Hepatic bile acid homeostasis is maintained during normal pregnancy in rats, probably through the FXR-SHP regulation. The expression of bile acid synthesis genes and liver bile acid accumulation were increased during lactation, together with increased expression of bile acid efflux transporter Bsep, Mrp3 and Mrp4.

  1. A gene network engineering platform for lactic acid bacteria.

    Science.gov (United States)

    Kong, Wentao; Kapuganti, Venkata S; Lu, Ting

    2016-02-29

    Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.

  2. Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

    Institute of Scientific and Technical Information of China (English)

    Peter J. van der Spek; Andreas Kremer; Lynn Murry; Michael G. Walker

    2003-01-01

    Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.

  3. Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

    Institute of Scientific and Technical Information of China (English)

    PeterJ.vanderSpek; AndreasKremer; LynnMurry; MichaelG.Walker

    2003-01-01

    Microarray analyses of gene expression are widely used,but reports of the same analyses by different groups give widely divergent results,and raise questions regarding reproducibility and reliability.We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes.These reports show substantial differences in their results.In this article,we review the methodology,results,and potential causes of differences in these applications of microarrays.Finally,we suggest practices to improve the reliability and reproducibility of microarray experiments.

  4. Fatty acid-gene interactions, adipokines and obesity.

    Science.gov (United States)

    Stryjecki, C; Mutch, D M

    2011-03-01

    It is now recognized that the low-grade inflammation observed with obesity is associated with the development of a wide range of downstream complications. As such, there is considerable interest in elucidating the regulatory mechanisms underlying the production of inflammatory molecules to improve the prevention and treatment of obesity and its co-morbidities. White adipose tissue is no longer considered a passive reservoir for storing lipids, but rather an important organ influencing energy metabolism, insulin sensitivity and inflammation by the secretion of proteins, commonly referred to as adipokines. Dysregulation of several adipokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and adiponectin, contributes to the low-grade inflammation that is a hallmark of obesity. Evidence now suggests that fatty acids represent a class of molecules that can modulate adipokine production, thereby influencing inflammatory status. Although the precise molecular mechanisms by which dietary fats regulate adipokine production remain unclear, recent findings indicate that diet-gene interactions may have an important role in the transcriptional and secretory regulation of adipokines. Single-nucleotide polymorphisms in the genes encoding TNF-α, IL-6 and adiponectin can modify circulating levels of these adipokines and, subsequently, obesity-related phenotypes. This genetic variation can also alter the influence of dietary fatty acids on adipokine production. Therefore, the current review will show that it is paramount to consider both genetic information and dietary fat intake to unravel the inter-individual variability in inflammatory response observed in intervention protocols targeting obesity.

  5. Escherichia coli and Salmonella typhimurium supX genes specify deoxyribonucleic acid topoisomerase I.

    OpenAIRE

    Trucksis, M; Golub, E I; Zabel, D J; Depew, R E

    1981-01-01

    Mutations of the Escherichia coli or Salmonella typhimurium supX genes eliminated deoxyribonucleic acid topoisomerase I. Suppression of a supX amber mutation partially restored the topoisomerase. Multicopy plasmids carrying supX+ caused overproduction of topoisomerase. Thus, these supX genes were identified as topA genes which specify deoxyribonucleic acid topoisomerase I.

  6. Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA.

    Science.gov (United States)

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

    2014-05-01

    The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.

  7. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    Energy Technology Data Exchange (ETDEWEB)

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  8. Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

    Directory of Open Access Journals (Sweden)

    Lei Anping

    2012-03-01

    Full Text Available Abstract Background Biofuel has been the focus of intensive global research over the past few years. The development of 4th generation biofuel production (algae-to-biofuels based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation. Results We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP, 3-ketoacyl-ACP-synthase (KAS, and acyl-ACP thioesterase (FATA gene expression had significant correlations with monounsaturated FA (MUFA synthesis and polyunsaturated FA (PUFA synthesis. Conclusions We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.

  9. Combining eicosapentaenoic acid, decosahexaenoic acid and arachidonic acid, using a fully crossed design, affect gene expression and eicosanoid secretion in salmon head kidney cells in vitro.

    Science.gov (United States)

    Holen, Elisabeth; He, Juyun; Espe, Marit; Chen, Liqiou; Araujo, Pedro

    2015-08-01

    Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses.

  10. Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

    Science.gov (United States)

    An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

    2015-03-01

    Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

  11. Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of Escherichia coli K-12

    OpenAIRE

    Andrianopoulos, Kanella; Wang, Lei; Reeves, Peter R.

    1998-01-01

    GDP–l-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP–d-mannose. l-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP–l-fucose biosynthetic pathway genes, gmd. We report here the identification ...

  12. The rice OsLpa1 gene encodse a novel protein involved in phytic acid metabolism

    Science.gov (United States)

    The rice low phytic acid 1 (OsLpa1) gene was originally identified using a forward genetics approach. Mutation of this gene resulted in a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, the rice lpa1 mutant does not appear to differ significa...

  13. Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

    Science.gov (United States)

    Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

    2000-01-01

    Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed. PMID:10869089

  14. Overexpression of Fatty-Acid-β-Oxidation-Related Genes Extends the Lifespan of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Shin-Hae Lee

    2012-01-01

    Full Text Available A better understanding of the aging process is necessary to ensure that the healthcare needs of an aging population are met. With the trend toward increased human life expectancies, identification of candidate genes affecting the regulation of lifespan and its relationship to environmental factors is essential. Through misexpression screening of EP mutant lines, we previously isolated several genes extending lifespan when ubiquitously overexpressed, including the two genes encoding the fatty-acid-binding protein and dodecenoyl-CoA delta-isomerase involved in fatty-acid β-oxidation, which is the main energy resource pathway in eukaryotic cells. In this study, we analyzed flies overexpressing the two main components of fatty-acid β-oxidation, and found that overexpression of fatty-acid-β-oxidation-related genes extended the Drosophila lifespan. Furthermore, we found that the ability of dietary restriction to extend lifespan was reduced by the overexpression of fatty-acid-β-oxidation-related genes. Moreover, the overexpression of fatty-acid-β-oxidation-related genes enhanced stress tolerance to oxidative and starvation stresses and activated the dFOXO signal, indicating translocation to the nucleus and transcriptional activation of the dFOXO target genes. Overall, the results of this study suggest that overexpression of fatty-acid-β-oxidation-related genes extends lifespan in a dietary-restriction-related manner, and that the mechanism of this process may be related to FOXO activation.

  15. Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Meilan Xue

    2012-12-01

    Full Text Available Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3, which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells. Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

  16. Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Meilan; Ge, Yinlin; Zhang, Jinyu [Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao Shandong (China); Wang, Qing [Affiliated Hospital of Qingdao University, Qingdao Shandong (China); Hou, Lin [Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao Shandong (China)

    2012-09-14

    Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

  17. Detailed transcriptomics analysis of the effect of dietary fatty acids on gene expression in the heart.

    Science.gov (United States)

    Georgiadi, Anastasia; Boekschoten, Mark V; Müller, Michael; Kersten, Sander

    2012-03-19

    Fatty acids comprise the primary energy source for the heart and are mainly taken up via hydrolysis of circulating triglyceride-rich lipoproteins. While most of the fatty acids entering the cardiomyocyte are oxidized, a small portion is involved in altering gene transcription to modulate cardiometabolic functions. So far, no in vivo model has been developed enabling study of the transcriptional effects of specific fatty acids in the intact heart. In the present study, mice were given a single oral dose of synthetic triglycerides composed of one single fatty acid. Hearts were collected 6 h thereafter and used for whole genome gene expression profiling. Experiments were conducted in wild-type and peroxisome proliferator-activated receptor (PPAR)α-/- mice to allow exploration of the specific contribution of PPARα. It was found that: 1) C18:3 had the most pronounced effect on cardiac gene expression. 2) The largest similarity in gene regulation was observed between C18:2 and C18:3. Large similarity was also observed between PPARα agonist Wy14643 and C22:6. 3) Many genes were regulated by one particular treatment only. Genes regulated by one particular treatment showed large functional divergence. 4) The majority of genes responding to fatty acid treatment were regulated in a PPARα-dependent manner, emphasizing the importance of PPARα in mediating transcriptional regulation by fatty acids in the heart. 5) Several genes were robustly regulated by all or many of the fatty acids studied, mostly representing well-described targets of PPARs (e.g., Acot1, Angptl4, Ucp3) but also including Zbtb16/PLZF, a transcription factor crucial for natural killer T cell function. 6) Deletion and activation of PPARα had a major effect on expression of numerous genes involved in metabolism and immunity. Our analysis demonstrates the marked impact of dietary fatty acids on gene regulation in the heart via PPARα.

  18. Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

    Science.gov (United States)

    O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

    2015-08-01

    The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms.

  19. Association of an ACSL1 gene variant with polyunsaturated fatty acids in bovine skeletal muscle

    Directory of Open Access Journals (Sweden)

    Widmann Philipp

    2011-11-01

    Full Text Available Abstract Background The intramuscular fat deposition and the fatty acid profiles of beef affect meat quality. High proportions of unsaturated fatty acids are related to beef flavor and are beneficial for the nutritional value of meat. Moreover, a variety of clinical and epidemiologic studies showed that particularly long-chain omega-3 fatty acids from animal sources have a positive impact on human health and disease. Results To screen for genetic factors affecting fatty acid profiles in beef, we initially performed a microsatellite-based genome scan in a F2 Charolais × German Holstein resource population and identified a quantitative trait locus (QTL for fatty acid composition in a region on bovine chromosome 27 where previously QTL affecting marbling score had been detected in beef cattle populations. The long-chain acyl-CoA synthetase 1 (ACSL1 gene was identified as the most plausible functional and positional candidate gene in the QTL interval due to its direct impact on fatty acid metabolism and its position in the QTL interval. ACSL1 is necessary for synthesis of long-chain acyl-CoA esters, fatty acid degradation and phospholipid remodeling. We validated the genomic annotation of the bovine ACSL1 gene by in silico comparative sequence analysis and experimental verification. Re-sequencing of the complete coding, exon-flanking intronic sequences, 3' untranslated region (3'UTR and partial promoter region of the ACSL1 gene revealed three synonymous mutations in exons 6, 7, and 20, six noncoding intronic gene variants, six polymorphisms in the promoter region, and four variants in the 3' UTR region. The association analysis identified the gene variant in intron 5 of the ACSL1 gene (c.481-233A>G to be significantly associated with the relative content of distinct fractions and ratios of fatty acids (e.g., n-3 fatty acids, polyunsaturated, n-3 long-chain polyunsaturated fatty acids, trans vaccenic acid in skeletal muscle. A tentative association

  20. Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells.

    Science.gov (United States)

    Lampen, A; Meyer, S; Nau, H

    2001-10-31

    Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.

  1. Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas

    Directory of Open Access Journals (Sweden)

    Gu Keyu

    2012-07-01

    Full Text Available Abstract Background Temporal and spatial expression of fatty acid and lipid biosynthetic genes are associated with the accumulation of storage lipids in the seeds of oil plants. In jatropha (Jatropha curcas L., a potential biofuel plant, the storage lipids are mainly synthesized and accumulated in the endosperm of seeds. Although the fatty acid and lipid biosynthetic genes in jatropha have been identified, the expression of these genes at different developing stages of endosperm has not been systemically investigated. Results Transmission electron microscopy study revealed that the oil body formation in developing endosperm of jatropha seeds initially appeared at 28 days after fertilization (DAF, was actively developed at 42 DAF and reached to the maximum number and size at 56 DAF. Sixty-eight genes that encode enzymes, proteins or their subunits involved in fatty acid and lipid biosynthesis were identified from a normalized cDNA library of jatropha developing endosperm. Gene expression with quantitative reverse-transcription polymerase chain reaction analysis demonstrated that the 68 genes could be collectively grouped into five categories based on the patterns of relative expression of the genes during endosperm development. Category I has 47 genes and they displayed a bell-shaped expression pattern with the peak expression at 28 or 42 DAF, but low expression at 14 and 56 DAF. Category II contains 8 genes and expression of the 8 genes was constantly increased from 14 to 56 DAF. Category III comprises of 2 genes and both genes were constitutively expressed throughout endosperm development. Category IV has 9 genes and they showed a high expression at 14 and 28 DAF, but a decreased expression from 42 to 56 DAF. Category V consists of 2 genes and both genes showed a medium expression at 14 DAF, the lowest expression at 28 or 42 DAF, and the highest expression at 56 DAF. In addition, genes encoding enzymes or proteins with similar function were

  2. Effects of Long Chain Fatty Acid Synthesis and Associated Gene Expression in Microalga Tetraselmis sp.

    Directory of Open Access Journals (Sweden)

    T. Catalina Adarme-Vega

    2014-06-01

    Full Text Available With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA to C20:4 eicosatetraenoic acid (ETA, correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding β-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4, but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt.

  3. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  4. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    Science.gov (United States)

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective.

  5. Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene.

    Science.gov (United States)

    Merritt, C M; Board, P G

    1988-06-15

    Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein. The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions. Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported. In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx. 3.3 kb downstream from AGP1. The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences. It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.

  6. Detailed transcriptomics analysis of the effect of dietary fatty acids on gene expression in the heart

    NARCIS (Netherlands)

    Georgiadi, A.; Boekschoten, M.V.; Muller, M.R.; Kersten, A.H.

    2012-01-01

    Fatty acids comprise the primary energy source for the heart and are mainly taken up via hydrolysis of circulating triglyceride-rich lipoproteins. While most of the fatty acids entering the cardiomyocyte are oxidized, a small portion is involved in altering gene transcription to modulate cardiometab

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  8. Regulation of the expression of key genes involved in HDL metabolism by unsaturated fatty acids

    Science.gov (United States)

    The aim of this study was to determine the effects, and possible mechanisms of action, of unsaturated fatty acids on the expression of genes involved in HDL metabolism in HepG2 cells. The mRNA concentration of target genes was assessed by real time PCR. Protein concentrations were determined by wes...

  9. Use of the alr gene as a food-grade selection marker in lactic acid bacteria

    NARCIS (Netherlands)

    Bron, P.A.; Benchimol, M.G.; Lambert, J.; Palumbo, E.; Deghorain, M.; Delcour, J.; Vos, de W.M.; Kleerebezem, M.; Hols, P.

    2002-01-01

    Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection marker

  10. Common variation in fatty acid metabolic genes and risk of incident sudden cardiac arrest

    NARCIS (Netherlands)

    R.N. Lemaitre (Rozenn ); C.O. Johnson (Catherine); S. Hesselson (Stephanie); N. Sotoodhenia (Nona); B. McKnight (Barbara); C.M. Sitlani (Colleen); D. Rea (Dan); I.B. King (Irena); P.-Y. Kwok (Pui-Yan); A. Mak (Angel); G. Li (Guo); J. Brody (Jennifer); E.B. Larson (Eric); D. Mozaffarian (Dariush); B.M. Psaty (Bruce); A. Huertas-Vazquez (Adriana); J.-C. Tardif (Jean-Claude); C.M. Albert (Christine); L.-P. Lyytikäinen (Leo-Pekka); D.E. Arking (Dan); S. Kääb (Stefan); H.V. Huikuri (Heikki); B.P. Krijthe (Bouwe); M. Eijgelsheim (Mark); Y.A. Wang (Ying); K. Reinier (Kyndaron); T. Lehtimäki (Terho); S.L. Pulit (Sara); R. Brugada (Ramon); M. Müller-Nurasyid (Martina); C. Newton-Cheh (Christopher); P.J. Karhunen (Pekka); B.H.Ch. Stricker (Bruno); P. Goyette (Philippe); J.I. Rotter (Jerome); S.S. Chugh (Sumeet); A. Chakravarti (Aravinda); X. Jouven (Xavier); D.S. Siscovick (David)

    2014-01-01

    textabstractBackground There is limited information on genetic factors associated with sudden cardiac arrest (SCA). Objective To assess the association of common variation in genes in fatty acid pathways with SCA risk. Methods We selected 85 candidate genes and 1155 single nucleotide polymorphisms (

  11. Regulation of hepatic gene expression by saturated fatty acids.

    Science.gov (United States)

    Vallim, T; Salter, A M

    2010-01-01

    Diets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently, they have also been suggested to promote the development of non-alcoholic fatty liver disease. While there is now considerable evidence to suggest that polyunsaturated fatty acids exert many of their effects through regulating the activity of transcription factors, including peroxisome proliferator activated receptors, sterol regulatory binding proteins (SREBPs) and liver X receptor, our understanding of how saturated fatty acids act is still limited. Here we review the potential mechanisms whereby saturated fatty acids modulate hepatic lipid metabolism thereby impacting on the synthesis, storage and secretion of lipids. Evidence is presented that their effects are, at least partly, mediated through modulation of the activity of the SREBP family of transcription factors.

  12. The Relationship between Dietary Fatty Acids and Inflammatory Genes on the Obese Phenotype and Serum Lipids

    OpenAIRE

    Yael T. Joffe; Malcolm Collins; Goedecke, Julia H.

    2013-01-01

    Obesity, a chronic low-grade inflammatory condition is associated with the development of many comorbidities including dyslipidemia. This review examines interactions between single nucleotide polymorphisms (SNP) in the inflammatory genes tumor necrosis alpha (TNFA) and interleukin-6 (IL-6) and dietary fatty acids, and their relationship with obesity and serum lipid levels. In summary, dietary fatty acids, in particular saturated fatty acids and the omega-3 and omega-6 polyunsaturated fatty a...

  13. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats.

    Science.gov (United States)

    Merritt, C M; Easteal, S; Board, P G

    1990-04-01

    There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.

  14. Polymorphisms in fatty acid metabolism-related genes are associated with colorectal cancer risk

    DEFF Research Database (Denmark)

    Hoeft, B.; Linseisen, J.; Beckmann, L.

    2010-01-01

    Colorectal cancer (CRC) is the third most common malignant tumor and the fourth leading cause of cancer death worldwide. The crucial role of fatty acids for a number of important biological processes suggests a more in-depth analysis of inter-individual differences in fatty acid metabolizing genes...... as contributing factor to colon carcinogenesis. We examined the association between genetic variability in 43 fatty acid metabolism-related genes and colorectal risk in 1225 CRC cases and 2032 controls participating in the European Prospective Investigation into Cancer and Nutrition study. Three hundred...

  15. The relationship between dietary fatty acids and inflammatory genes on the obese phenotype and serum lipids.

    Science.gov (United States)

    Joffe, Yael T; Collins, Malcolm; Goedecke, Julia H

    2013-05-21

    Obesity, a chronic low-grade inflammatory condition is associated with the development of many comorbidities including dyslipidemia. This review examines interactions between single nucleotide polymorphisms (SNP) in the inflammatory genes tumor necrosis alpha (TNFA) and interleukin-6 (IL-6) and dietary fatty acids, and their relationship with obesity and serum lipid levels. In summary, dietary fatty acids, in particular saturated fatty acids and the omega-3 and omega-6 polyunsaturated fatty acids, impact the expression of the cytokine genes TNFA and IL-6, and alter TNFα and IL-6 production. In addition, sequence variants in these genes have also been shown to alter their gene expression and plasma levels, and are associated with obesity, measures of adiposity and serum lipid concentrations. When interactions between dietary fatty acids and TNFA and IL-6 SNPs on obesity and serum lipid were analyzed, both the quantity and quality of dietary fatty acids modulated the relationship between TNFA and IL-6 SNPs on obesity and serum lipid profiles, thereby impacting the association between phenotype and genotype. Researching these diet-gene interactions more extensively, and understanding the role of ethnicity as a confounder in these relationships, may contribute to a better understanding of the inter-individual variability in the obese phenotype.

  16. The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism.

    Science.gov (United States)

    Brämer, C O; Steinbüchel, A

    2001-08-01

    From Ralstonia eutropha HF39 null-allele mutants were created by Tn5 mutagenesis and by homologous recombination which were impaired in growth on propionic acid and levulinic acid. From the molecular, physiological and enzymic analysis of these mutants it was concluded that in this bacterium propionic acid is metabolized via the methylcitric acid pathway. The genes encoding enzymes of this pathway are organized in a cluster in the order prpR, prpB, prpC, acnM, ORF5 and prpD, with prpR transcribed divergently from the other genes. (i) prpC encodes a 2-methylcitric acid synthase (42720 Da) as shown by the measurement of the respective enzyme activity, complementation of a prpC mutant of Salmonella enterica serovar Typhimurium and high sequence similarity. (ii) For the translational product of acnM the function of a 2-methyl-cis-aconitic acid hydratase (94726 Da) is proposed. This protein and also the ORF5 translational product are essential for growth on propionic acid, as revealed by the propionic-acid-negative phenotype of Tn5-insertion mutants, and are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid as shown by the accumulation of the latter, which could be purified as its calcium salt from the supernatants of these mutants. In contrast, inactivation of prpD did not block the ability of the cell to use propionic acid as carbon and energy source, as shown by the propionic acid phenotype of a null-allele mutant. It is therefore unlikely that prpD from R. eutropha encodes a 2-methyl-cis-aconitic acid dehydratase as proposed recently for the homologous prpD gene from S. enterica. (iii) The translational product of prpB encodes 2-methylisocitric acid lyase (32314 Da) as revealed by measurement of the respective enzyme activity and by demonstrating accumulation of methylisocitric acid in the supernatant of a prpB null-allele mutant. (iv) The expression of prpC and probably also of the other enzymes is regulated and is induced during

  17. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

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

    Directory of Open Access Journals (Sweden)

    Jia-Hong Zhu

    2015-02-01

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

  19. Human nutrigenomics of gene regulation by dietary fatty acids

    NARCIS (Netherlands)

    Afman, L.A.; Muller, M.R.

    2012-01-01

    Nutrigenomics employs high-throughput genomics technologies to unravel how nutrients modulate gene and protein expression and ultimately influence cellular and organism metabolism. The most often-applied genomics technique so far is transcriptomics, which allows quantifying genome-wide changes in ge

  20. Bile acid-induced virulence gene expression of Vibrio parahaemolyticus reveals a novel therapeutic potential for bile acid sequestrants.

    Directory of Open Access Journals (Sweden)

    Kazuyoshi Gotoh

    Full Text Available Vibrio parahaemolyticus, a bacterial pathogen, causes human gastroenteritis. A type III secretion system (T3SS2 encoded in pathogenicity island (Vp-PAI is the main contributor to enterotoxicity and expression of Vp-PAI encoded genes is regulated by two transcriptional regulators, VtrA and VtrB. However, a host-derived inducer for the Vp-PAI genes has not been identified. Here, we demonstrate that bile induces production of T3SS2-related proteins under osmotic conditions equivalent to those in the intestinal lumen. We also show that bile induces vtrA-mediated vtrB transcription. Transcriptome analysis of bile-responsive genes revealed that bile strongly induces expression of Vp-PAI genes in a vtrA-dependent manner. The inducing activity of bile was diminished by treatment with bile acid sequestrant cholestyramine. Finally, we demonstrate an in vivo protective effect of cholestyramine on enterotoxicity and show that similar protection is observed in infection with a different type of V. parahaemolyticus or with non-O1/non-O139 V. cholerae strains of vibrios carrying the same kind of T3SS. In summary, these results provide an insight into how bacteria, through the ingenious action of Vp-PAI genes, can take advantage of an otherwise hostile host environment. The results also reveal a new therapeutic potential for widely used bile acid sequestrants in enteric bacterial infections.

  1. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ §

    Science.gov (United States)

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.

    2008-01-01

    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  2. Analysis of the aspartic acid metabolic pathway using mutant genes.

    Science.gov (United States)

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  3. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.

    Science.gov (United States)

    Haun, William; Coffman, Andrew; Clasen, Benjamin M; Demorest, Zachary L; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre; Cedrone, Frederic; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2014-09-01

    Soybean oil is high in polyunsaturated fats and is often partially hydrogenated to increase its shelf life and improve oxidative stability. The trans-fatty acids produced through hydrogenation pose a health threat. Soybean lines that are low in polyunsaturated fats were generated by introducing mutations in two fatty acid desaturase 2 genes (FAD2-1A and FAD2-1B), which in the seed convert the monounsaturated fat, oleic acid, to the polyunsaturated fat, linoleic acid. Transcription activator-like effector nucleases (TALENs) were engineered to recognize and cleave conserved DNA sequences in both genes. In four of 19 transgenic soybean lines expressing the TALENs, mutations in FAD2-1A and FAD2-1B were observed in DNA extracted from leaf tissue; three of the four lines transmitted heritable FAD2-1 mutations to the next generation. The fatty acid profile of the seed was dramatically changed in plants homozygous for mutations in both FAD2-1A and FAD2-1B: oleic acid increased from 20% to 80% and linoleic acid decreased from 50% to under 4%. Further, mutant plants were identified that lacked the TALEN transgene and only carried the targeted mutations. The ability to create a valuable trait in a single generation through targeted modification of a gene family demonstrates the power of TALENs for genome engineering and crop improvement.

  4. Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas.

    Science.gov (United States)

    Kaur, Simran; Norkina, Oxana; Ziemer, Donna; Samuelson, Linda C; De Lisle, Robert C

    2004-08-01

    The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.

  5. Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

    Science.gov (United States)

    Cohen, S; Tzuri, G; Harel-Beja, R; Itkin, M; Portnoy, V; Sa'ar, U; Lev, S; Yeselson, L; Petrikov, M; Rogachev, I; Aharoni, A; Ophir, R; Tadmor, Y; Lewinsohn, E; Burger, Y; Katzir, N; Schaffer, A A

    2012-07-01

    Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of 6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

  6. Cloning and molecular characterization of △12-fatty acid desaturase gene from Mortierella isabellina

    Institute of Scientific and Technical Information of China (English)

    Ming-Chun Li; Hang Li; Dong-Sheng Wei; Lai-Jun Xing

    2006-01-01

    AIM: To clone △12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of △12-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli(E.coli) strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1.RESULTS: Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △12-fatty acid desaturase,which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION: Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

  7. Polymorphisms in the fatty acid desaturase genes and diet are important determinants of infant docosahexaenoic acid status

    DEFF Research Database (Denmark)

    Lauritzen, L.; Harsløf, L.; Larsen, L.H.;

    2013-01-01

    Tissue docosahexaenoic acid (DHA) accretion in early infancy is supported by DHA in breast-milk and may thus decrease once complementary feeding takes over. Endogenous synthesis of DHA from alphalinolenic acid is low and polymorphisms in the genes that encodes the fatty acid desaturases (FADS) has...... been shown to have little effect on DHA-status in adults. It is unclear to what extent endogenous DHA-synthesis contributes to infant DHAstatus. We aim to investigate the role of diet and FADS-polymorphisms on DHA-status at 9 months and 3 years. Methods: This cross-sectional study with Danish infants...... use data from two prospective studies (EFiON and the SKOTcohort). We measured erythrocyte (RBC) DHA-status at 9 months (n=409) and 3 years (n=176) and genotyped 4 FADS tagSNPs, rs3834458, rs1535, rs174575 and rs174448 (n=401)....

  8. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    Science.gov (United States)

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  9. Genome‐wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus

    Science.gov (United States)

    Medema, Marnix H.; Alam, Mohammad T.; Heijne, Wilbert H. M.; van den Berg, Marco A.; Müller, Ulrike; Trefzer, Axel; Bovenberg, Roel A. L.; Breitling, Rainer; Takano, Eriko

    2011-01-01

    Summary To increase production of the important pharmaceutical compound clavulanic acid, a β‐lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome‐wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild‐type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology. PMID:21342474

  10. Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus.

    Science.gov (United States)

    Medema, Marnix H; Alam, Mohammad T; Heijne, Wilbert H M; van den Berg, Marco A; Müller, Ulrike; Trefzer, Axel; Bovenberg, Roel A L; Breitling, Rainer; Takano, Eriko

    2011-03-01

    To increase production of the important pharmaceutical compound clavulanic acid, a β-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild-type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology.

  11. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

    Directory of Open Access Journals (Sweden)

    Hamid-Reza Samadlouie

    2014-06-01

    Full Text Available The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  12. Involvement of Sp1 in Butyric Acid-Induced HIV-1 Gene Expression

    Directory of Open Access Journals (Sweden)

    Kenichi Imai

    2015-09-01

    Full Text Available Background/Aims: The ability of human immunodeficiency virus-1(HIV-1 to establish latent infection and its re-activation is considered critical for progression of HIV-1 infection. We previously reported that a bacterial metabolite butyric acid, acting as a potent inhibitor of histone deacetylases (HDACs, could lead to induction of HIV-1 transcription; however, the molecular mechanism remains unclear. The aim of this study was to investigate the effect of butyric acid on HIV-1 gene expression. Methods: Butyric acid-mediated HIV-1 gene expression was determined by luciferase assay and Chromatin immunoprecipitation assay. Western blot analysis and ELISA were used for the detection of HIV-1. Results: We found that Sp1 binding sites within the HIV-1 promoter are primarily involved in butyric acid-mediated HIV-1 activation. In fact, Sp1 knockdown by small interfering RNA and the Sp1 inhibitor mithramycin A abolished the effect of butyric acid. We also observed that cAMP response element-binding-binding protein (CBP was required for butyric acid-induced HIV-1 activation. Conclusions: These results suggest that butyric acid stimulates HIV-1 promoter through inhibition of the Sp1-associated HDAC activity and recruitment of CBP to the HIV-1 LTR. Our findings suggest that Sp1 should be considered as one of therapeutic targets in anti-viral therapy against HIV-1 infection aggravated by butyric acid-producing bacteria.

  13. Cloning and phylogenetic analysis of a fatty acid elongase gene from Nannochloropsis oculata CS179

    Science.gov (United States)

    Pan, Kehou; Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Yang, Guanpin

    2009-12-01

    Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.

  14. Cloning and Phylogenetic Analysis of a Fatty Acid Elongase Gene from Nannochloropsis oculata CS179

    Institute of Scientific and Technical Information of China (English)

    PAN Kehou; MA Xiaolei; YU Jianzhong; ZHU Baohua; YANG Guanpin

    2009-01-01

    Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the N J-tree revealed that the N. oculata CS 179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.

  15. Nucleic acid modulation of gene expression: approaches for nucleic acid therapeutics against cancer.

    Science.gov (United States)

    Nakata, Yuji; Kim, Tae-Kon; Shetzline, Susan; Gewirtz, Alan M

    2005-01-01

    Most cancers are characterized by abnormal gene expression, which is thought to contribute to the pathogenesis and maintenance of the malignant phenotype; abnormal proliferation, maturation, and apoptosis. Silencing such genes would appear to be a rational approach to the therapy of cancer, and some preliminary clinical studies support this concept. Of the strategies available, the anti-mRNA gene silencing approach has attracted much attention and is the focus of this review. This strategy includes three types of agents: (1) single-stranded antisense oligonucleotides; (2) catalytically active oligonucleotides, such as ribozymes, and DNAzymes that possess inherent RNA cleaving activity; and (3) small interfering RNA (siRNA) molecules that induce RNA interference (RNAi). Among these agents, antisense oligonucleotides, especially phosphorothioate (PS) oligonucleotides, have been the most frequently used in clinical trials. In this article, we provide an overview of anti-mRNA gene silencing agents and their development for use as cancer therapeutics.

  16. Gene classification based on amino acid motifs and residues: the DLX (distal-less test case.

    Directory of Open Access Journals (Sweden)

    Nuno A Fonseca

    Full Text Available BACKGROUND: Comparative studies using hundreds of sequences can give a detailed picture of the evolution of a given gene family. Nevertheless, retrieving only the sequences of interest from public databases can be difficult, in particular, when working with highly divergent sequences. The difficulty increases substantially when one wants to include in the study sequences from many (or less well studied species whose genomes are non-annotated or incompletely annotated. METHODOLOGY/PRINCIPAL FINDINGS: In this work we evaluate the usefulness of different approaches of gene retrieval and classification, using the distal-less (DLX gene family as a test case. Furthermore, we evaluate whether the use of a large number of gene sequences from a wide range of animal species, the use of multiple alternative alignments, and the use of amino acids aligned with high confidence only, is enough to recover the accepted DLX evolutionary history. CONCLUSIONS/SIGNIFICANCE: The canonical DLX homeobox gene sequence here derived, together with the characteristic amino acid variants here identified in the DLX homeodomain region, can be used to retrieve and classify DLX genes in a simple and efficient way. A program is made available that allows the easy retrieval of synteny information that can be used to classify gene sequences. Maximum likelihood trees using hundreds of sequences can be used for gene identification. Nevertheless, for the DLX case, the proposed DLX evolutionary is not recovered even when multiple alignment algorithms are used.

  17. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

    Science.gov (United States)

    Campos, Laura; Granell, Pablo; Tárraga, Susana; López-Gresa, Pilar; Conejero, Vicente; Bellés, José María; Rodrigo, Ismael; Lisón, Purificación

    2014-04-01

    We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

  18. Genes involved in cell adhesion and signaling: a new repertoire of retinoic acid receptor target genes in mouse embryonic fibroblasts.

    Science.gov (United States)

    Al Tanoury, Ziad; Piskunov, Aleksandr; Andriamoratsiresy, Dina; Gaouar, Samia; Lutzing, Régis; Ye, Tao; Jost, Bernard; Keime, Céline; Rochette-Egly, Cécile

    2014-02-01

    Nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

  19. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    OpenAIRE

    Lidiia Astakhova; Mtakai Ngara; Olga Babich; Aleksandr Prosekov; Lyudmila Asyakina; Lyubov Dyshlyuk; Tore Midtvedt; Xiaoying Zhou; Ingemar Ernberg; Liudmila Matskova

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell l...

  20. Increase of Clavulanic acid production by using recombinant Streptomyces clavuligerus strain including claR gene

    Directory of Open Access Journals (Sweden)

    Maryam Kay

    2014-07-01

    Full Text Available   Introduction : Clavulanic acid is a major β-lactam antibiotic which is produced by Streptomyces clavuligerus. Clavulanic acid is used in combination of strong but sensitive to β-lactamase antibiotics. The claR gene has an important role in regulation of clavulanic acid production and is needed for the expression of the genes in final step of clavulanic acid biosynthesis.   Materials and methods: The recombinant construct pMTclaR which contains claR gene is obtained from Isfahan University and plasmid extraction was done from Streptomyces lividans for next steps. The Streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR. Finally, bioassay method was used to survey the effect of extra copy of claR on clavulanic acid production .   Results : The typical chalky white colony of Streptomyces clavuligerus was seen on GYME plates containing thiostrepton antibiotic. Plasmid extraction was initially carried out. Furthermore, PCR reaction was done by claR specific primers and the 1334 bp band which was belonging to claR was detected. Finally, the bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the claR gene in multicopy plasmids resulted in a 2.5 fold increase in clavulanic acid production .   Discussion and conclusion : In this study the 3.3 fold increase in clavulanic acid production was obtained by using an expression vector containing claR. According to the clinical use of clavulanic acid, production of bacterial strains which are able to produce high level of antibiotic can help significantly in customization of antibiotic production.

  1. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

    Science.gov (United States)

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  2. Characterization of the Shewanella oneidensis Fur gene: roles in iron and acid tolerance response

    OpenAIRE

    Wu Liyou; Luo Feng; Harris Daniel P; Yang Yunfeng; Parsons Andrea B; Palumbo Anthony V; Zhou Jizhong

    2008-01-01

    Abstract Background Iron homeostasis is a key metabolism for most organisms. In many bacterial species, coordinate regulation of iron homeostasis depends on the protein product of a Fur gene. Fur also plays roles in virulence, acid tolerance, redox-stress responses, flagella chemotaxis and metabolic pathways. Results We conducted physiological and transcriptomic studies to characterize Fur in Shewanella oneidensis, with regard to its roles in iron and acid tolerance response. A S. oneidensisf...

  3. Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

    Directory of Open Access Journals (Sweden)

    Mahdi Ebrahimi

    2014-09-01

    Full Text Available Alteration of the lipid content and fatty acid (FA composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA and α-linolenic acid (LNA for 100 days. Inclusion of flaxseed oil increased (p < 0.05 the α-linolenic acid (C18:3n-3 concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05 decreased the arachidonic acid (C20:4n-6 and conjugated linolenic acid (CLA c-9 t-11 content in the ST muscle. There was a significant (p < 0.05 upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression.

  4. Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption

    Institute of Scientific and Technical Information of China (English)

    Marco Montagnani; Anna Abrahamsson; Cecilia G(a)lman; G(o)sta Eggertsen; Hanns-Ulrich Marschall; Elisa Ravaioli; Curt Einarsson; Paul A Dawson

    2006-01-01

    The etiology of most cases of idiopathic bile acid malabsorption (TBAM) is unknown. Tn this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR)and peroxisome proliferator activated receptor alpha (PPARα). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using 75Se-homocholic acid taurine(SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4). The ASBT,FXR, and PPARα genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC,and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARα genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.

  5. Nucleic acids--genes, drugs, molecular lego and more.

    Science.gov (United States)

    Häner, Robert

    2010-01-01

    Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results.

  6. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Science.gov (United States)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  7. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  8. Molecular cloning and functional characterization of a Δ6-fatty acid desaturase gene from Rhizopus oryzae.

    Science.gov (United States)

    Zhu, Yu; Zhang, Bi-Bo

    2013-09-01

    The objective was to screen for and isolate a novel enzyme with the specific activity of a Δ6-fatty acid desaturase from Rhizopus oryzae. In this study, R. oryzae was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated here as RoD6D, with high homology to fungal Δ6-fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited Δ6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The corresponding genomic sequence of RoD6D was 1565 bp in length, with five introns. This is the first report on the characterization and gene cloning of a Δ6-fatty acid desaturase of R. oryzae from Douchi.

  9. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

  10. Monitoring Gene Expression In Vivo with Nucleic Acid Molecular Switches

    Energy Technology Data Exchange (ETDEWEB)

    David C. Ward; Patricia Bray-Ward

    2005-01-26

    The overall objectives of this project were (1) to develop allosteric ribozymes capable of acting as molecular switches for monitoring the levels of both wild-type and mutant mRNA species in living cells and whole animals and (2) to develop highly efficient reagents to deliver nucleic acid molecular switches into living cells, tissues and animals with the ultimate goal of expression profiling specific mRNAs of diagnostic or prognostic value within tumors in animals. During the past year, we have moved our laboratory to Nevada and in the moving process we have lost electronic and paper copies of prior progress reports concerning the construction and biological properties of the molecular switches. Since there was minimal progress during the last year on molecular switches, we are relying on past project reports to provide a summary of our data on this facet of the grant. Here we are summarizing the work done on the delivery reagents and their application to inducing mutations in living cells, which will include work done during the no cost extension.

  11. [Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

    Science.gov (United States)

    Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

    2014-09-01

    Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

  12. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    Science.gov (United States)

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

  13. Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus

    NARCIS (Netherlands)

    Medema, M.H.; Alam, M.T.; Heijne, W.H.M.; Berg, M.A. van den; Müller, U.; Trefzer, A.; Bovenberg, R.A.L.; Breitling, R.; Takano, E.

    2011-01-01

    To increase production of the important pharmaceutical compound clavulanic acid, a beta-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expressi

  14. Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus.

    NARCIS (Netherlands)

    Medema, M.H.; Alam, M.T.; Heijne, W.H.; Berg, M.A.M.C. van den; Muller, U.; Trefzer, A.; Bovenberg, R.A.; Breitling, R.; Takano, E.

    2011-01-01

    To increase production of the important pharmaceutical compound clavulanic acid, a beta-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expressi

  15. Modulation of antimicrobial host defense peptide gene expression by free fatty acids.

    Directory of Open Access Journals (Sweden)

    Lakshmi T Sunkara

    Full Text Available Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP genes in the chicken (PLoS One 2011, 6: e27225. In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.

  16. Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria.

    Science.gov (United States)

    Tomoeda, K; Awata, H; Matsuura, T; Matsuda, I; Ploechl, E; Milovac, T; Boneh, A; Scott, C R; Danks, D M; Endo, F

    2000-11-01

    The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of 'hawkinsin,' may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

  17. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    Science.gov (United States)

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  18. Relative gene expression in acid-adapted Escherichia coli O157:H7 during lactoperoxidase and lactic acid challenge in Tryptone Soy Broth.

    Science.gov (United States)

    Parry-Hanson, Angela A; Jooste, Piet J; Buys, Elna M

    2010-09-20

    Cross-protection of acid-adapted Escherichia coli O157:H7 against inimical stresses is mediated by the glucose-repressed sigma factor RpoS. However, many food systems in which E. coli O157:H7 occurs are complex and contain glucose. This study was aimed at investigating the contribution of acid and lactoperoxidase (LP)-inducible genes to cross-protection of E. coli O157:H7 against LP system and lactic acid (LA) in Tryptone Soy Broth (TSB). Acid-adapted and non-adapted E. coli O157:H7 were challenged to activated LP and LA at pH 4.0 and 5.0 in TSB for 6h at 25°C followed by expression of acid and LP-inducible genes. Acid-adapted E. coli showed cross-protection against activated LP and LA. All the acid-inducible genes tested were repressed at pH 4.0 with or without activated LP system. At pH 7.4, gadA, ompC and ompF were induced in acid-adapted cells. Induction of corA occurred in non-adapted cells but was repressed in acid-adapted cells. Although acid-inducible genes were repressed at pH 4.0, high resistance of acid-adapted cells indicates that expression of acid-inducible genes occurred during acid adaptation and not the actual challenge. Repression of rpoS indicates that RpoS-independent systems contribute to cross-protection in acid-adapted E. coli O157:H7.

  19. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  20. The influence of gene transfer on the lactic acid bacteria evolution

    Directory of Open Access Journals (Sweden)

    Višnja Bačun-Družina

    2009-09-01

    Full Text Available In the case of preparing various dairy products, the exploitation of lactic acid bacteria has been essential in the course of past millennia in all known nations. Numerous comparative analyses of gene and genome sequences reveal that the exchange of genetic material within and between bacterial species is far more general and frequent than has previously been thought. Consequently, the horizontal gene transfer between distant species or within the same species is an important factor in the Lactobacillales evolution. Knowledge about the exchange of lactobacillus genetic information through horizontal gene transfer, mobile genetic elements, and its evolution is very important due to characterizations and stability maintenance of autochthonous as well as industrial lactic acid bacteria strains in dairy products that benefit human health.

  1. Docosahexaenoic acid regulates gene expression in HUVEC cells treated with polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Gdula-Argasińska, Joanna; Czepiel, Jacek; Totoń-Żurańska, Justyna; Jurczyszyn, Artur; Perucki, William; Wołkow, Paweł

    2015-07-16

    The molecular mechanism of inflammation and carcinogenesis induced by exposure of polycyclic aromatic hydrocarbons (PAHs) is not clearly understood. Our study was undertaken due to the strong pro-carcinogenic potential and reactivity of PAH-metabolites, as well as the susceptibility of polyunsaturated fatty acids to oxidation. The aim of this study was to evaluate the pro- or anti-inflammatory impact of n-3 docosahexaenoic acid on human primary umbilical vein endothelial cells (HUVEC) exposed to polycyclic aromatic hydrocarbons. We analysed the influence of docosahexaenoic acid (DHA) and/or PAHs supplementation on the fatty acid profile of cell membranes, on cyclooxygenase-2 (COX-2), aryl hydrocarbon receptor (AHR), and glutathione S transferase Mu1 (GSTM1) protein expression as well as on the prostaglandin synthase 2 (PTGS2), AHR, GSTM1, PLA2G4A, and cytochrome P450 CYP1A1 gene expression. We observed that COX-2 and AHR protein expression was increased while GSTM1 expression was decreased in cells exposed to DHA and PAHs. Docosahexaenoic acid down-regulated CYP1A1 and up-regulated the AHR and PTGS2 genes. Our findings suggested that DHA contributes significantly to alleviate the harmful effects caused by PAHs in endothelial cells. Moreover, these results suggest that a diet rich in n-3 fatty acids is helpful to reduce the harmful effects of PAHs exposure on human living in heavily polluted areas.

  2. Characterization of genomic rearrangements of the alpha1-acid glycoprotein/orosomucoid gene in Ghanaians.

    Science.gov (United States)

    Yuasa, I; Nakamura, H; Henke, L; Henke, J; Nakagawa, M; Irizawa, Y; Umetsu, K

    2001-01-01

    In this study, the structure of the alpha1-acid glycoprotein (AGP), or orosomucoid (ORM), gene was investigated in a Ghanaian mother and her child, who shared an unusual variant, ORM1 S2(C), found by isoelectric focusing. Three remarkable changes of nucleotide sequence were observed: (1) The two ORM1 alleles, ORMI*S and ORMI*S2(C), had the AGP2 gene-specific sequence at one and three regions, respectively, in exon 5 to intron 5. The variant allele originating from ORMi*S was characterized by a G-to-A transition, resulting in an amino acid change from valine to methionine, which is also detected in ORM1 F2, a form that is common in Europeans. (2) The AGP2 gene of the child, inherited from the father, was duplicated, as revealed by long-range polymerase chain reaction. (3) Three new mutations were observed in two exons of the AGP2 genes of the mother and child. All of these novel genomic rearrangements, which were not observed in Japanese subjects, may have arisen through point mutation, gene conversion, and unequal crossover events. It is likely that the rearrangement of the AGP gene has often occurred in Africans.

  3. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    Science.gov (United States)

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

  4. Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

    Science.gov (United States)

    Benoit, Isabelle; Asther, Michèle; Bourne, Yves; Navarro, David; Canaan, Stéphane; Lesage-Meessen, Laurence; Herweijer, Marga; Coutinho, Pedro M; Asther, Marcel; Record, Eric

    2007-09-01

    The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

  5. Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids1[OPEN

    Science.gov (United States)

    Deason, Nicholas; DellaPenna, Dean

    2017-01-01

    Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed’s nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants. PMID:27872244

  6. [Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast].

    Science.gov (United States)

    He, Dongqin; Xiao, Dongguang; Lv, Ye

    2008-02-01

    The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

  7. Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus).

    Science.gov (United States)

    Meyer, Eli; Manahan, Donal T

    2009-08-01

    Transport of amino acids from low concentrations in seawater by marine invertebrates has been extensively studied, but few of the genes involved in this physiological process have been identified. We have characterized three amino acid transporter genes cloned from embryos of the sea urchin Strongylocentrotus purpuratus. These genes show phylogenetic proximity to classical amino acid transport systems, including Gly and B0+, and the inebriated gene (INE). Heterologous expression of these genes in frog oocytes induced a 40-fold increase in alanine transport above endogenous levels, demonstrating that these genes mediate alanine transport. Antibodies specific to one of these genes (Sp-AT1) inhibited alanine transport, confirming the physiological activity of this gene in larvae. Whole-mount antibody staining of larvae revealed expression of Sp-AT1 in the ectodermal tissues associated with amino acid transport, as independently demonstrated by autoradiographic localization of radioactive alanine. Maximum rates of alanine transport increased 6-fold during early development, from embryonic to larval stages. Analysis of gene expression during this developmental period revealed that Sp-AT1 transcript abundance remained nearly constant, while that of another transporter gene (Sp-AT2) increased 11-fold. The functional characterization of these genes establishes a molecular biological basis for amino acid transport by developmental stages of marine invertebrates.

  8. Rapid Cloning and Expression of Glutaryl-7-Aminocephalosporanic Acid Acylase Genes from Soil Samples

    Institute of Scientific and Technical Information of China (English)

    LUO Hui; YU Huimin; LI Qiang; SHEN Zhongyao

    2005-01-01

    A polymerase chain reaction (PCR)-based strategy was developed to rapidly obtain the gene encoding for an industrially important enzyme, glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase. Different soil samples were cultured with a Pseudomonas selective medium to enrich specific microorganisms, and then the genomic DNA was extracted to serve as PCR templates. PCR primers for GL-7-ACA acylase gene amplification were designed on the basis of bioinformatics searches and analyses. The method was used to successfully amplify three GL-7-ACA acylase genes from different soil samples. The GL-7-ACA acylase genes were then cloned and overexpressed in Escherichia coli with a relatively high level of 266 unit·L-1.

  9. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    DEFF Research Database (Denmark)

    Birkedal, Henrik; Nielsen, Peter E

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine...... interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression...... suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair...

  10. Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

    Science.gov (United States)

    Allam, Uday Sankar; Krishna, M Gopala; Sen, Minakshi; Thomas, Rony; Lahiri, Amit; Gnanadhas, Divya Prakash; Chakravortty, Dipshikha

    2012-01-01

    During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the ΔSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The ΔSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the ΔSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the ΔSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella.

  11. Stress-Survival Gene Identification From an Acid Mine Drainage Algal Mat Community

    Science.gov (United States)

    Urbina-Navarrete, J.; Fujishima, K.; Paulino-Lima, I. G.; Rothschild-Mancinelli, B.; Rothschild, L. J.

    2014-12-01

    Microbial communities from acid mine drainage environments are exposed to multiple stressors to include low pH, high dissolved metal loads, seasonal freezing, and desiccation. The microbial and algal communities that inhabit these niche environments have evolved strategies that allow for their ecological success. Metagenomic analyses are useful in identifying species diversity, however they do not elucidate the mechanisms that allow for the resilience of a community under these extreme conditions. Many known or predicted genes encode for protein products that are unknown, or similarly, many proteins cannot be traced to their gene of origin. This investigation seeks to identify genes that are active in an algal consortium during stress from living in an acid mine drainage environment. Our approach involves using the entire community transcriptome for a functional screen in an Escherichia coli host. This approach directly targets the genes involved in survival, without need for characterizing the members of the consortium.The consortium was harvested and stressed with conditions similar to the native environment it was collected from. Exposure to low pH (DNA (cDNA) libraries in E. coli. The transformed E. coli were exposed to the same stressors as the original algal consortium to select for surviving cells. Successful cells incorporated the transcripts that encode survival mechanisms, thus allowing for selection and identification of the gene(s) involved. Initial selection screens for freeze and desiccation tolerance have yielded E. coli that are 1 order of magnitude more resistant to freezing (0.01% survival of control with no transcript, 0.2% survival of E. coli with transcript) and 3 orders of magnitude more resistant to desiccation (0.005% survival of control cells with no transcripts, 5% survival of cells with transcript).This work is transformative because genetic functions can be selected without having prior knowledge of the genes or of the organisms involved

  12. Response of fatty acid synthesis genes to the binding of human salivary amylase by Streptococcus gordonii.

    Science.gov (United States)

    Nikitkova, Anna E; Haase, Elaine M; Vickerman, M Margaret; Gill, Steven R; Scannapieco, Frank A

    2012-03-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.

  13. A Systems genetics approach identifies gene regulatory networks associated with fatty acid composition in brassica rapa seed

    NARCIS (Netherlands)

    Basnet, Ram Kumar; Pino Del Carpio, Dunia; Xiao, Dong; Bucher, Johan; Jin, Mina; Boyle, Kerry; Fobert, Pierre; Visser, R.G.F.; Maliepaard, Chris; Bonnema, Guusje

    2016-01-01

    Fatty acids in seeds affect seed germination and seedling vigor, and fatty acid composition determines the quality of seed oil. In this study, quantitative trait locus (QTL) mapping of fatty acid and transcript abundance was integrated with gene network analysis to unravel the genetic regulation

  14. Comparison of the △12 fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes

    Institute of Scientific and Technical Information of China (English)

    ShanlinYu; Lijuan Pan; Qingli Yang; Ping Min; Zengkai Ren; Hongsheng Zhang

    2008-01-01

    △12fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds. By RT-PCR method, the full-length cDNAs of △12 fatty acid desaturase gene were isolated from peanut (Arachis hypogaea L.) genotypes with normal and high ratio of oleic to linoleic acid, which were designated AhFAD2B and AhFAD2B', respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position +442 bp of AhFAD2B' sequence of high oleic acid genotypes, which resulted in the shift of open reading frame and a truncated protein AhFAD2B', with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of △12 fatty acid desaturase gene in high oleic acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with AhFAD2B or AhFAD2B' proved that only AhFAD2B gene product showed significant A12 fatty acid desaturase activity,but AhFAD2B' gene product did not. These results suggested that the change of AhFAD2B' gene sequence resulted in lower activity or deactivation of △12 fatty acid desaturase in high oleic acid genotype.

  15. Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

    Science.gov (United States)

    Mamoon, Abulkhair; Subauste, Angela; Subauste, Maria C; Subauste, Jose

    2014-10-25

    Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver.

  16. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    Science.gov (United States)

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  17. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds

    Directory of Open Access Journals (Sweden)

    Yusuke Tagashira

    2015-04-01

    Full Text Available The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP6 biosynthesis-related genes, as InsP6 is a major storage form of P in seeds. The rice (Oryza sativa L. low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP6 biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP6 and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP6 biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP6 biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  18. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    Science.gov (United States)

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  19. Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

    Directory of Open Access Journals (Sweden)

    Daniel Estanislao Goszczynski

    2014-12-01

    Full Text Available LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01, and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01, may be helpful to improve the quality of meat and improve health.

  20. Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

    Science.gov (United States)

    Goszczynski, Daniel Estanislao; Mazzucco, Juliana Papaleo; Ripoli, María Verónica; Villarreal, Edgardo Leopoldo; Rogberg-Muñoz, Andrés; Mezzadra, Carlos Alberto; Melucci, Lilia Magdalena; Giovambattista, Guillermo

    2014-01-01

    LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health. PMID:25606458

  1. Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat.

    Science.gov (United States)

    Goszczynski, Daniel Estanislao; Mazzucco, Juliana Papaleo; Ripoli, María Verónica; Villarreal, Edgardo Leopoldo; Rogberg-Muñoz, Andrés; Mezzadra, Carlos Alberto; Melucci, Lilia Magdalena; Giovambattista, Guillermo

    2014-12-01

    LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health.

  2. Identification and heterologous expression of a Δ4-fatty acid desaturase gene from Isochrysis sphaerica.

    Science.gov (United States)

    Guo, Bing; Jiang, Mulan; Wan, Xia; Gong, Yangmin; Liang, Zhuo; Hu, Chuanjiong

    2013-10-28

    The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

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

    Science.gov (United States)

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

    2003-07-01

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

  4. Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

    Directory of Open Access Journals (Sweden)

    Igor R. Costa

    2014-12-01

    Full Text Available Essential amino acids (EAA consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS and betaine-homocysteine S-methyltransferase (BHMT diverged from the expected Tree of Life (ToL relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  5. Gene expression in retinoic acid-induced neural tube defects A cDNA mieroarray analysis

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Long; Zhong Yang; Yi Zeng; Hongli Li; Yangyun Han; Chao You

    2009-01-01

    BACKGROUND: Neural tube defects can be induced by abnormal factors in vivo or in vitro during development. However, the molecular mechanisms of neural tube defect induction, and the related gene expression and regulation are still unknown.OBJECTIVE: To compare the differences in gene expression between normal embryos and those with neural tube defects.DESIGN, TIME AND SETTING: A neural development study was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA between January 2006 and October 2007.MATERIALS: Among 120 adult Kunming mice, 60 pregnant mice were randomly and evenly divided into a retinoic acid group (n = 30) and a normal control group (n =30). The retinoic acid was produced by Sigma, USA, the gene microarray by the Amersham Pharmacia Company, Hong Kong, and the gene sequence was provided by the Incyte database, USA.METHODS: Retinoic acid was administered to prepare models of neural tube defects, and corn oil was similady administered to the normal control group. Total RNA was extracted from embryonic tissue of the two groups using a Trizol kit, and a cDNA microarray containing 1 100 known genes was used to compare differences in gene expression between the normal control group and the retinoic acid group on embryonic (E) clay 10.5 and 11.5. Several differentially expressed genes were randomly selected from the two groups for Northern blotting, to verify the results of the cDNA microarray.MAIN OUTCOME MEASURES: Morphological changes and differential gene expression between the normal control group and the retinoic acid group.RESULTS: Anatomical microscopy demonstrated that an intact closure of the brain was formed in the normal mouse embryos by days E10.5 and E11.5. The cerebral appearance was full and smooth, and the surface of the spine was intact. However, in the retinoic acid group on days E10.5 and E11.5, there were more dead embryos. Morphological malformations typically included non-closure at the top of

  6. Effect of chronic valproic Acid treatment on hepatic gene expression profile in wfs1 knockout mouse.

    Science.gov (United States)

    Punapart, Marite; Eltermaa, Mall; Oflijan, Julia; Sütt, Silva; Must, Anne; Kõks, Sulev; Schalkwyk, Leonard C; Fernandes, Catherine; Vasar, Eero; Soomets, Ursel; Terasmaa, Anton

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

  7. Structure of 4-hydrophenylpyruvic acid dioxygenase (HPD) gene and its mutation in tyrosinemic mouse strain III

    Energy Technology Data Exchange (ETDEWEB)

    Awata, H.; Endo, F.; Matsuda, I. [Kumamoto Univ. Medical School (Japan)] [and others

    1994-09-01

    4-Hydroxphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and is developmentally regulated in mammals. A genetic deficiency of the enzyme in man and mouse leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human and mouse gene libraries. The human HPD gene is over 30 kilo-bases long and is split into 14 exons. Analysis of the 5{prime} flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5{prime} flanking region of the gene are similar to those of other genes which are specifically expressed in hepatocytes and which are developmentally regulated. The gene for mouse HPD has a similar structure and we obtained evidence for a nucleotide substitution which generates a termination codon in exon 7 of the HPD gene in III mice. This mutation associates a partial exon skipping and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Thus, mouse strain III can serve as a genetic model for human tyrosinemia type 3. Ongoing studies are expected to elucidate the disease process involved in hereditary tyrosinemia type 1 and to shed light on mechanisms that mediate developmental regulation of HPD gene expression. In addition, mouse strain III together with recently established models for tyrosinemia type 1 will facilitate studies on hereditary tyrosinemias.

  8. ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

  9. Gene-nutrient interactions: importance of folic acid and vitamin B12 during early embryogenesis.

    Science.gov (United States)

    Finnell, Richard H; Shaw, Gary M; Lammer, Edward J; Rosenquist, Thomas H

    2008-06-01

    The role that nutritional factors play in mammalian development has received renewed attention over the past two decades as the scientific literature has exploded with reports that folic acid supplementation in the periconceptional period can protect embryos from a number of highly significant malformations. As is often the case, the relationship between B vitamin supplementation and improved pregnancy outcomes is more complicated than initially perceived, as the interaction between nutritional factors and selected genes must be considered. In this review, we attempt to summarize the complex clinical and experimental literature on nutritional factors, their biological transport mechanisms, and interactions with genetic polymorphisms that impact early embryogenesis. While not exhaustive, our goal was to provide an overview of important gene-nutrient interactions, focusing on folic acid and vitamin B12, to serve as a framework for understanding the multiple roles they play in early embryogenesis.

  10. Effects of candidate gene polymorphisms on the detailed fatty acids profile determined by gas chromatography in bovine milk.

    Science.gov (United States)

    Pegolo, S; Cecchinato, A; Mele, M; Conte, G; Schiavon, S; Bittante, G

    2016-06-01

    Association analyses between candidate genes and bovine milk fatty acids can improve our understanding of genetic variation in milk fatty acid profiles and reveal potential opportunities to tailor milk fat composition through selection strategies. In this work, we investigated the association of 51 single nucleotide polymorphisms (SNP) selected from 37 candidate genes using a functional and positional approach, with 47 fatty acids, 9 fatty acid groups, and 5 Δ(9)-desaturation indices in milk samples from Brown Swiss cows. Individual milk samples were collected from 1,158 Italian Brown Swiss cows, and gas chromatography was used to obtain detailed milk fatty acid compositions. A GoldenGate assay system (Illumina, San Diego, CA) was used to perform genotype 96 selected SNP located in 54 genes across 22 chromosomes. In total, 51 polymorphic SNP in 37 candidate genes were retained for the association analysis. A Bayesian linear animal model was used to estimate the contribution of each SNP. A total of 129 tests indicated relevant additive effects between a given SNP and a single fatty acid trait; 38 SNP belonging to 30 genes were relevant for a total of 57 fatty acid traits. Most of the studied fatty acid traits (~81%) were relevantly associated with multiple SNP. Relevantly associated SNP were mainly found in genes related to fat metabolism, linked to or contained in previously identified quantitative trait loci for fat yield or content, or associated with genes previously identified in association analyses with milk fatty acid profiles in other cow breeds. The most representative candidate genes were LEP, PRL, STAT5A, CCL3, ACACA, GHR, ADRB2, LPIN1, STAT1, FABP4, and CSN2. In particular, relevant associations with SNP located on bovine chromosome 19 (BTA19) were found. Two candidate genes on BTA19 (CCL3 and ACACA) were relevantly associated with de novo short- and medium-chain fatty acids, likely explaining the high heritability values found for these fatty acids

  11. Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

    Science.gov (United States)

    Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

    2016-01-01

    Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues.

  12. Interaction between γ-aminobutyric acid A receptor genes: new evidence in migraine susceptibility.

    Directory of Open Access Journals (Sweden)

    Marlene Quintas

    Full Text Available Migraine is a common neurological episodic disorder with a female-to-male prevalence 3- to 4-fold higher, suggesting a possible X-linked genetic component. Our aims were to assess the role of common variants of gamma-aminobutyric acid A receptor (GABAAR genes, located in the X-chromosome, in migraine susceptibility and the possible interaction between them. An association study with 188 unrelated cases and 286 migraine-free controls age- and ethnic matched was performed. Twenty-three tagging SNPs were selected in three genes (GABRE, GABRA3 and GABRQ. Allelic, genotypic and haplotypic frequencies were compared between cases and controls. We also focused on gene-gene interactions. The AT genotype of rs3810651 of GABRQ gene was associated with an increased risk for migraine (OR: 4.07; 95% CI: 1.71-9.73, p=0.002, while the CT genotype of rs3902802 (OR: 0.41; 95% CI: 0.21-0.78, p=0.006 and GA genotype of rs2131190 of GABRA3 gene (OR: 0.53; 95% CI: 0.32-0.88, p=0.013 seem to be protective factors. All associations were found in the female group and maintained significance after Bonferroni correction. We also found three nominal associations in the allelic analyses although there were no significant results in the haplotypic analyses. Strikingly, we found strong interactions between six SNPs encoding for different subunits of GABAAR, all significant after permutation correction. To our knowledge, we show for the first time, the putative involvement of polymorphisms in GABAAR genes in migraine susceptibility and more importantly we unraveled a role for novel gene-gene interactions opening new perspectives for the development of more effective treatments.

  13. Profiling of hepatic gene expression in rats treated with fibric acid analogs

    Energy Technology Data Exchange (ETDEWEB)

    Cornwell, Paul D.; Souza, Angus T. de; Ulrich, Roger G

    2004-05-18

    Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors whose ligands include fatty acids, eicosanoids and the fibrate class of drugs. In humans, fibrates are used to treat dyslipidemias. In rodents, fibrates cause peroxisome proliferation, a change that might explain the observed hepatomegaly. In this study, rats were treated with multiple dose levels of six fibric acid analogs (including fenofibrate) for up to two weeks. Pathological analysis identified hepatocellular hypertrophy as the only sign of hepatotoxicity, and only one compound at the highest dose caused any significant increase in serum ALT or AST activity. RNA profiling revealed that the expression of 1288 genes was related to dose or length of treatment and correlated with hepatocellular hypertrophy. This gene list included expression changes that were consistent with increased mitochondrial and peroxisomal {beta}-oxidation, increased fatty acid transport, increased hepatic uptake of LDL-cholesterol, decreased hepatic uptake of glucose, decreased gluconeogenesis and decreased glycolysis. These changes are likely linked to many of the clinical benefits of fibrate drugs, including decreased serum triglycerides, decreased serum LDL-cholesterol and increased serum HDL-cholesterol. In light of the fact that all six compounds stimulated similar or identical changes in the expression of this set of 1288 genes, these results indicate that hepatomegaly is due to PPAR{alpha} activation, although signaling through other receptors (e.g. PPAR{gamma}, RXR) or through non-receptor pathways cannot be excluded.

  14. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  15. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakočiūnė, Dzuiga; Herrero-Fresno, Ana; Jelsbak, Lotte;

    2016-01-01

    RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis...

  16. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    Science.gov (United States)

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-29

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

  17. Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

    Science.gov (United States)

    Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

    2017-12-01

    This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l(-1), monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

  18. Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis

    Institute of Scientific and Technical Information of China (English)

    Haixiong Liu; Shifeng Li; Yunbin Zhang; Yuanchang Yan; Yiping Li

    2009-01-01

    Glutamate decarboxylase 65 (GAD65) produces γ-amino-butyric acid,the main inhibitory neurotransmitter in adult mammalian brain.Previous experiments,per-formed in brain,showed that GAD65 gene possesses two TATA-less promoters,although the significance is unknown.Here,by rapid amplification of cDNA ends method,two distinct GAD65 mRNA isoforms transcribed from two independent clusters of transcription start sites were identified in post-natal rat testis.RT-PCR results revealed that the two mRNA isoforms had distinct expression patterns during post-natal testis maturation,suggesting that GAD65 gene expression was regulated by alternative promoters at the transcription level.By using GAD65-speciflc antibodies,western blotting analysis showed that the 58-kDa GAD65,N-terminal 69 amino acids truncated form of full-length GAD65 protein,was developmentally expressed during post-natal testis matu-ration,suggesting that GAD65 gene expression in testis may also be regulated by post-translational processing.Confocal immunofluorescence microscopy revealed that GAD65 protein was presented in Leydig cells of Day 1 testis,primary spermatocytes and spermatids of post-natal of Day 90 testis.The above results suggested that GAD65 gene expression is dynamically regulated at mul-tiple levels during post-natal testis maturation.

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

    Indian Academy of Sciences (India)

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

    2009-06-01

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

  20. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  1. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

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

    Science.gov (United States)

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

    2009-06-01

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

  3. Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

    Science.gov (United States)

    Kurioka, T; Mizutari, K; Niwa, K; Fukumori, T; Inoue, M; Hasegawa, M; Shiotani, A

    2016-02-01

    Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.

  4. Role of a liver fatty acid-binding protein gene in lipid metabolism in chicken hepatocytes.

    Science.gov (United States)

    Gao, G L; Na, W; Wang, Y X; Zhang, H F; Li, H; Wang, Q G

    2015-01-01

    This study investigated the role of the chicken liver fatty acid-binding protein (L-FABP) gene in lipid metabolism in hepatocytes, and the regulatory relationships between L-FABP and genes related to lipid metabolism. The short hairpin RNA (shRNA) interference vector with L-FABP and an eukaryotic expression vector were used. Chicken hepatocytes were subjected to shRNA-mediated knockdown or L-FABP cDNA overexpression. Expression levels of lipid metabolism-related genes and biochemical parameters were detected 24, 36, 48, 60, and 72 h after transfection with the interference or overexpression plasmids for L-FABP, PPARα and L-BABP expression levels, and the total amount of cholesterol, were significantly affected by L-FABP expression. L-FABP may affect lipid metabolism by regulating PPARα and L-BABP in chicken hepatocytes.

  5. THE EXPRESSION OF CONNEXIN GENES IN NASOPHARYNGEAL CARCINOMA CELLS AND THE EFFECT OF RETINOIC ACID ON THE REGULATION OF THOSE GENES

    Institute of Scientific and Technical Information of China (English)

    JIANG Ning; BIN Liang-hua; TANG Xiang-na; ZHOU Ming; ZENG Zhao-yang; Li Gui-yuan

    1999-01-01

    Objective: To detect which members in the connexin gene family are expressed in nasopharyngeal carcinoma (NPC) cell line HNE1, and the mechanism by which those genes are specifically switched on and off during retinoic acid (RA) induction. Methods: Establishing the cell growth curves of NPC cells. Observing the effect of RA on connexin genes by Northern hybridization. Results: Two genes Cx46 and Cx37, belonging to the connexin gene family, were expressed in HNE, The down-regulation of Cx46 and Cx37, up-regulation of RARa and growth inhibition was observed in HNE1, after exposure to RA. The gene expression and cell growth in HNE1 cells was restored after removal of RA. Conclusion: Two members of the connexin gene family: Cx37 and Cx46 were expressed in HNE1 cells, RA can inhibit the expression of those two genes mediated by RARa, and the effects of RA on HNE1 are reversible.

  6. 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.

  7. QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

    Directory of Open Access Journals (Sweden)

    Charcosset Alain

    2010-01-01

    Full Text Available Abstract Background Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes. Results The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP and five novel 9-cis-epoxycarotenoid dioxygenase (NCED related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in

  8. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    Directory of Open Access Journals (Sweden)

    Shewmaker Christine K

    2010-10-01

    Full Text Available Abstract Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD 2 and fatty acid elongase (FAE 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general

  9. Expression of a cyanobacterial {del}{sup 6}-desaturase gene results in {gamma}-linolenic acid production in transgenic plants

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A.S.; Thomas, T.L. [Texas A & M Univ., College Station, TX (United States)

    1996-05-01

    Gamma-linolenic acid (GLA), a nutritionally important fatty acid in human and animal diets, is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a fatty acid that could be converted to GLA by the enzyme {del}{sup 6}-desaturase if it were present. As a first step to producing GLA in oil seed crops, we have cloned a cyanobacterial {del}{sup 6}-desaturase gene. Expression of this gene in transgenic tobacco resulted in GLA accumulation. Octadecatetraenoic acid, a highly unsaturated, industrially important fatty acid, was also found in transgenic tobacco plants expressing the cyanobacterial {del}{sup 6}-desaturase. This is the first example of engineering the production of `novel` polyunsaturated fatty acids in transgenic plants. 28 refs., 4 figs., 1 tab.

  10. OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH

    Directory of Open Access Journals (Sweden)

    Alimuddin Alimuddin

    2008-12-01

    Full Text Available Eicosapentaenoic acid (EPA, 20:5n-3 and docosahexaenoic acid (DHA, 22:6n-3 have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3 rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD, Δ5-desaturase-like (OmΔ5FAD and elongase-like (MELO encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou were individually transferred into zebrafish (Danio rerio as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05 than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05 than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05 than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over

  11. Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05.

    Science.gov (United States)

    Myszka, Kamila; Schmidt, Marcin T; Olejnik-Schmidt, Agnieszka K; Leja, Katarzyna; Czaczyk, Katarzyna

    2014-12-01

    The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 μg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene.

  12. Polymorphism in the fatty acid desaturase genes and diet are important determinants of infant n-3 fatty acid status

    DEFF Research Database (Denmark)

    Harsløf, L.B.S.; Larsen, L.H.; Ritz, C.;

    and polymorphism in the genes that encodes the fatty acid desaturases (FADS) has little effect on DHA-status in adults. It is however unclear to what extent endogenous DHA-synthesis contributes to infant DHA-status. Aim: To investigate the role of diet and FADS polymorphism on DHA-status at 9 months and 3 years....... Methods: This cross-sectional study with Danish infants use data from two prospective studies (EFiON and the SKOT cohort). We measured erythrocyte (RBC) DHA-status at 9 months (n=409) and 3 years (n=176) and genotyped 4 FADS tagSNPs, rs3834458, rs1535, rs174575 and rs174448 (n=401). Information about...... breastfeeding was obtained by questionnaires and fish intake was assessed by 7-day pre-coded food diaries. Results: FADS-genotype, breastfeeding, and fish intake were found to explain 25% of the variation in infant RBC DHA-status (mean±SD: 6.6±1.9% of the fatty acids (FA%)). Breastfeeding was the most important...

  13. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    Science.gov (United States)

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  14. Polymorphisms in lipogenic genes and milk fatty acid composition in Holstein dairy cattle.

    Science.gov (United States)

    Nafikov, Rafael A; Schoonmaker, Jon P; Korn, Kathleen T; Noack, Kristin; Garrick, Dorian J; Koehler, Kenneth J; Minick-Bormann, Jennifer; Reecy, James M; Spurlock, Diane E; Beitz, Donald C

    2014-12-01

    Changing bovine milk fatty acid (FA) composition through selection can decrease saturated FA (SFA) consumption, improve human health and provide a means for manipulating processing properties of milk. Our study determined associations between milk FA composition and genes from triacylglycerol (TAG) biosynthesis pathway. The GC dinucleotide allele of diacylglycerol O-acyltransferase 1:g.10433-10434AA >GC was associated with lower palmitic acid (16:0) concentration but higher oleic (18:1 cis-9), linoleic (18:2 cis-9, cis-12) acid concentrations, and elongation index. Accordingly, the GC dinucleotide allele was associated with lower milk fat percentage and SFA concentrations but higher monounsaturated FA and polyunsaturated FA (PUFA) concentrations. The glycerol-3-phosphate acyltransferase, mitochondrial haplotypes were associated with higher myristoleic acid (14:1 cis-9) concentration and C14 desaturation index. The 1-acylglycerol-3-phosphate acyltransferase 1 haplotypes were associated with higher PUFA and linoleic acid concentrations. The results of this study provide information for developing genetic tools to modify milk FA composition in dairy cattle.

  15. Identification of Genes in Saccharomyces cerevisiae that Are Haploinsufficient for Overcoming Amino Acid Starvation

    Directory of Open Access Journals (Sweden)

    Nancy S. Bae

    2017-04-01

    Full Text Available The yeast Saccharomyces cerevisiae responds to amino acid deprivation by activating a pathway conserved in eukaryotes to overcome the starvation stress. We have screened the entire yeast heterozygous deletion collection to identify strains haploinsufficient for growth in the presence of sulfometuron methyl, which causes starvation for isoleucine and valine. We have discovered that cells devoid of MET15 are sensitive to sulfometuron methyl, and loss of heterozygosity at the MET15 locus can complicate screening the heterozygous deletion collection. We identified 138 cases of loss of heterozygosity in this screen. After eliminating the issues of the MET15 loss of heterozygosity, strains isolated from the collection were retested on sulfometuron methyl. To determine the general effect of the mutations for a starvation response, SMM-sensitive strains were tested for the ability to grow in the presence of canavanine, which induces arginine starvation, and strains that were MET15 were also tested for growth in the presence of ethionine, which causes methionine starvation. Many of the genes identified in our study were not previously identified as starvation-responsive genes, including a number of essential genes that are not easily screened in a systematic way. The genes identified span a broad range of biological functions, including many involved in some level of gene expression. Several unnamed proteins have also been identified, giving a clue as to possible functions of the encoded proteins.

  16. Molecular and biochemical characterization of the jasmonic acid methyltransferase gene from black cottonwood (Populus trichocarpa)

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Yao, Jianzhuang [University of Tennessee, Knoxville (UTK); Chaiprasongsuk, Minta [University of Tennessee, Knoxville (UTK); Li, Guanglin [University of Tennessee, Knoxville (UTK); Guan, Ju [University of Tennessee, Knoxville (UTK); Tschaplinski, Timothy J [ORNL; Guo, Hong [University of Tennessee, Knoxville (UTK); Chen, Feng [University of Tennessee, Knoxville (UTK)

    2013-01-01

    Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175 lM and 341 lM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

  17. Myristic Acid (MA) Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

    Institute of Scientific and Technical Information of China (English)

    LU Nai-sheng; ZHANG Yong-liang; JIANG Qing-yan; SHU Gang; XIE Qiu-ping; ZHU Xiao-tong; GAO Ping; ZHOU Gui-xuan; WANG Song-bo; WANG Li-na; XI Qian-yun

    2014-01-01

    Intramuscular fat (IMF) content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid (MA) on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle (LDM) and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ(PPARγ) and adipose-related genes, such as glucose transporter 1 (GLUT1), lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4/aP2), fatty acid translocase (FAT), acetyl-CoA carboxylaseα(ACCα), adipose triglyceride lipase (ATGL) and fatty acid synthase (FASN). However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α(C/EBPα) or hormone sensitive lipase (HSL). The expression of pyruvate dehydrogenase kinase 4 (PDK4) was increased by MA during the early stages of differentiation (day 1-3). In addition, MA also increased the absolute content of C14 (P<0.001) and saturated fatty acids (SFA) (P<0.05) to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.

  18. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    Science.gov (United States)

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  19. Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism

    OpenAIRE

    Motoko Maekawa; Yoshimi Iwayama; Tetsuo Ohnishi; Manabu Toyoshima; Chie Shimamoto; Yasuko Hisano; Tomoko Toyota; Shabeesh Balan; Hideo Matsuzaki; Yasuhide Iwata; Shu Takagai; Kohei Yamada; Motonori Ota; Satoshi Fukuchi; Yohei Okada

    2015-01-01

    The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SL...

  20. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

    Science.gov (United States)

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-05-12

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  1. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

    Directory of Open Access Journals (Sweden)

    Kiyohito Yoshida

    2016-05-01

    Full Text Available The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase, the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  2. Dietary conjugated linoleic acid modify gene expression in liver, muscles, and fat tissues of finishing pigs.

    Science.gov (United States)

    Tous, N; Theil, P K; Lauridsen, C; Lizardo, R; Vilà, B; Esteve-Garcia, E

    2012-12-01

    The aim of this study was to investigate underlying mechanisms of dietary conjugated linoleic acid (CLA) on lipid metabolism in various tissues of pigs. Sixteen gilts (73 ± 3 kg) were fed a control (containing sunflower oil) or an experimental diet in which 4% of sunflower oil was replaced by CLA, and slaughtered at an average BW of 117 ± 4.9 kg. Transcription of peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), sterol regulatory element binding protein (SREBP1), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), delta-6-desaturase (D6D), and stearoyl CoA desaturase (SCD) were determined by real-time PCR in longissimus thoracis (LT) and semimembranosus (SM) muscles, LT subcutaneous and SM intermuscular fat, and in the liver. Fatty acid (FA) composition was analyzed using gas chromatography in these tissues, except for SM intermuscular fat. Dietary CLA increased PPARγ in LT muscle (P tissues studied (P fat. Transcription of genes related to FA synthesis was reduced by CLA in SM muscle and liver (SREBP1, both P fat. Saturated FA were increased in all studied tissues (P tissue-specific way by CLA. It was concluded that dietary CLA affected transcription of genes and fat metabolism in a tissue-specific manner.

  3. Colored petri nets to model gene mutation and amino acids classification.

    Science.gov (United States)

    Yang, Jinliang; Gao, Rui; Meng, Max Q-H; Tarn, Tzyh-Jong

    2012-05-07

    The genetic code is the triplet code based on the three-letter codons, which determines the specific amino acid sequences in proteins synthesis. Choosing an appropriate model for processing these codons is a useful method to study genetic processes in Molecular Biology. As an effective modeling tool of discrete event dynamic systems (DEDS), colored petri net (CPN) has been used for modeling several biological systems, such as metabolic pathways and genetic regulatory networks. According to the genetic code table, CPN is employed to model the process of genetic information transmission. In this paper, we propose a CPN model of amino acids classification, and further present the improved CPN model. Based on the model mentioned above, we give another CPN model to classify the type of gene mutations via contrasting the bases of DNA strands and the codons of amino acids along the polypeptide chain. This model is helpful in determining whether a certain gene mutation will cause the changes of the structures and functions of protein molecules. The effectiveness and accuracy of the presented model are illustrated by the examples in this paper.

  4. Sesamin modulates gene expression without corresponding effects on fatty acids in Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Schiller Vestergren, A; Wagner, L; Pickova, J; Rosenlund, G; Kamal-Eldin, A; Trattner, S

    2012-09-01

    This study examined the effects of sesamin inclusion in vegetable oil-based diets fed to Atlantic salmon (Salmo salar L.). The diets used differed in n-6/n-3 fatty acid (FA) ratio (0.5 and 1) and sesamin content (high 5.8 g/kg, low 1.16 g/kg and no sesamin). The oils used in the feeds were a mixture of rapeseed, linseed and palm oil. Fish were fed for 4 months. Fatty acids and expression of hepatic genes involved in transcription, lipid uptake, desaturation, elongation and β-oxidation were measured. No major effects on the percentage of DHA in white muscle, liver triacylglycerol and phospholipid fraction were detected. Genes involved in β-oxidation, elongation and desaturation were affected by sesamin addition. Limited effects were seen on any of the transcription factors tested and no effect was seen on the expression of peroxisome proliferator-activated receptors (PPAR). Expression of both SREBP-1 and SREBP-2 increased with sesamin addition. It was concluded that supplementation of fish feed with a high level of sesamin had a negative effect on the growth rate and live weight and did not alter the proportions of DHA in tissues even though gene expression was affected. Thus, more studies are needed to formulate a diet that would increase the percentage of DHA in fish without negative effects on fish growth.

  5. Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure

    Directory of Open Access Journals (Sweden)

    Sahbaie Peyman

    2007-06-01

    Full Text Available Abstract Background Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. Methods Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. Results The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. Conclusion Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.

  6. Gene expression signature of DMBA-induced hamster buccal pouch carcinomas: modulation by chlorophyllin and ellagic acid.

    Directory of Open Access Journals (Sweden)

    Ramamurthi Vidya Priyadarsini

    Full Text Available Chlorophyllin (CHL, a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA, a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA-induced hamster buccal pouch (HBP carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy.

  7. Gene expression signature of DMBA-induced hamster buccal pouch carcinomas: modulation by chlorophyllin and ellagic acid.

    Science.gov (United States)

    Vidya Priyadarsini, Ramamurthi; Kumar, Neeraj; Khan, Imran; Thiyagarajan, Paranthaman; Kondaiah, Paturu; Nagini, Siddavaram

    2012-01-01

    Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy.

  8. Gene-gene interactions of fatty acid synthase (FASN) using multifactor-dimensionality reduction method in Korean cattle.

    Science.gov (United States)

    Lee, Jeayoung; Jin, Mehyun; Lee, Yoonseok; Ha, Jaejung; Yeo, Jungsou; Oh, Dongyep

    2014-01-01

    We examined the gene-gene interactions of five exonic single nucleotide polymorphisms (SNPs) in the gene encoding fatty acid synthase using 513 Korean cattle and using the model free and the non-parametrical multifactor dimensionality reduction method for the analysis. The five SNPs of g.12870 T>C, g.13126 T>C, g.15532 C>A, g.16907 T>C and g.17924 G>A associated with a variety of fatty acid compositions and marbling score were used in this study. The two-factor interaction between g.13126 T>C and g.15532 C>A had the highest training-balanced among the five-factor models and a testing-balanced accuracy at 70.18 % on C18:1 with a cross-validation consistency of 10 out of 10. Also, the two-factor interaction between g.13126 T>C and g.15532 C>A had the highest testing-balanced accuracy at 68.59 % with a 10 out of 10 cross-validation consistency, than any other models on MUFA. In MS, a single SNP g.15532 C>A had the best accuracy at 58.85 % and the two-factor interaction model g.12870 T>C and g.15532 C>A had the highest testing-balanced accuracy at 64.00 %. The three-factor interaction model g.12870 T>C, g.13126 T>C and g.15532 C>A was recorded as having a high testing-balanced accuracy of 63.24 %, but it was lower than the two-factor interaction model. We used likelihood ratio tests for interaction, and Chi square tests to validate our results, with all tests showing statistical significance. We also compared this with mean scores between the high-risk trait group and low-risk trait group. The genotypes of TTCA, TTAA and TCAA at g.15532 and g.13126 on C18:1, genotypes TTCC, TTCA, TTAA, TCAA CCAA at g.15532 and g.13126 on MUFA and genotypes CCCC, TCCA, CCCA, TTAA, TCAA and CCAA at g.15532 and g.12870 on MS were recommended for the genetic improvement of beef quality.

  9. Arachidonic acid has a dominant effect to regulate lipogenic genes in 3T3-L1 adipocytes compared to omega-3 fatty acids

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    Hitesh Vaidya

    2015-03-01

    Full Text Available Background: The effects of long-chain n-3 and n-6 polyunsaturated fatty acids (PUFA on the regulation of adipocytes metabolism are well known. These fatty acids are generally consumed together in our diets; however, the metabolic regulation of adipocytes in the presence of these fatty acids when given together is not known. Objective: To investigate the effects of n-3 PUFA and arachidonic acid (AA, an n-6 PUFA, on the regulation of adipogenic and lipogenic genes in mature 3T3-L1 adipocytes. Methods: 3T3-L1 adipocytes were incubated in the presence or absence of 100 µM of eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA; docosapentaenoic acid, DPA and AA, either alone or AA+n-3 PUFA; control cells received bovine serum albumin alone. The mRNA expression of adipogenic and lipogenic genes was measured. The fatty acid composition of adipocytes was analyzed using gas chromatography. Results: Individual n-3 PUFA or AA had no effect on the mRNA expression of peroxisome-proliferator-activated receptor-γ; however, AA+EPA and AA+DPA significantly increased (P<0.05 the expression compared to control cells (38 and 42%, respectively. AA and AA+EPA increased the mRNA expression of acetyl-CoA carboxylase 1 (P<0.05. AA treatment decreased the mRNA expression of stearoyl-CoA desaturase (SCD1 (P<0.01, while n-3 PUFA, except EPA, had no effect compared to control cells. AA+DHA and AA+DPA inhibited SCD1 gene expression (P<0.05 suggesting a dominant effect of AA. Fatty acids analysis of adipocytes revealed a higher accretion of AA compared to n-3 PUFA. Conclusions: Our findings reveal that AA has a dominant effect on the regulation of lipogenic genes in adipocytes.

  10. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

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    Yuan, Yuan; Wang, Zhouyong; Jiang, Chao; Wang, Xumin; Huang, Luqi

    2014-01-25

    Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species.

  11. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  12. Epigenetic regulation of the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs

    NARCIS (Netherlands)

    Corominas, J.; Marchesi, J.A.; Puig-Oliveras, A.; Revilla, M.; Estelle, J.; Alves, E.; Folch, J.M.; Ballester, M.

    2015-01-01

    BACKGROUND: In previous studies on an Iberian x Landrace cross, we have provided evidence that supported the porcine ELOVL6 gene as the major causative gene of the QTL on pig chromosome 8 for palmitic and palmitoleic acid contents in muscle and backfat. The single nucleotide polymorphism (SNP) ELOVL

  13. Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

    NARCIS (Netherlands)

    Mach Casellas, N.; Jacobs, A.A.A.; Kruijt, L.; Baal, van J.; Smits, M.C.J.

    2014-01-01

    The aim of this study was to determine the effects of unprotected dietary unsaturated fatty acids (UFA) from different plant oils on gene expression in the mammary gland of grazing dairy cows. Milk composition and gene expression in the mammary gland tissue were evaluated in grazing dairy cows suppl

  14. Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

    Science.gov (United States)

    Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

    2015-09-30

    Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

  15. Obstructive heart defects associated with candidate genes, maternal obesity, and folic acid supplementation.

    Science.gov (United States)

    Tang, Xinyu; Cleves, Mario A; Nick, Todd G; Li, Ming; MacLeod, Stewart L; Erickson, Stephen W; Li, Jingyun; Shaw, Gary M; Mosley, Bridget S; Hobbs, Charlotte A

    2015-06-01

    Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1,644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity.

  16. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

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    Morral Núria

    2011-01-01

    Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  17. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

    Science.gov (United States)

    Creelman, R A; Tierney, M L; Mullet, J E

    1992-06-01

    Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  19. An acute dose of gamma-hydroxybutyric acid alters gene expression in multiple mouse brain regions.

    Science.gov (United States)

    Schnackenberg, B J; Saini, U T; Robinson, B L; Ali, S F; Patterson, T A

    2010-10-13

    Gamma-hydroxybutyric acid (GHB) is normally found in the brain in low concentrations and may function as a neurotransmitter, although the mechanism of action has not been completely elucidated. GHB has been used as a general anesthetic and is currently used to treat narcolepsy and alcoholism. Recreational use of GHB is primarily as a "club drug" and a "date rape drug," due to its amnesic effects. For this study, the hypothesis was that behavioral and neurochemical alterations may parallel gene expression changes in the brain after GHB administration. Adult male C57/B6N mice (n=5/group) were administered a single dose of 500 mg/kg GHB (i.p.) and were sacrificed 1, 2 and 4 h after treatment. Control mice were administered saline. Brains were removed and regionally dissected on ice. Total RNA from the hippocampus, cortex and striatum was extracted, amplified and labeled. Gene expression was evaluated using Agilent whole mouse genome 4x44K oligonucleotide microarrays. Microarray data were analyzed by ArrayTrack and differentially expressed genes (DEGs) were identified using P or = 1.7 as the criteria for significance. Principal component analysis (PCA) and Hierarchical Cluster Analysis (HCA) showed that samples from each time point clustered into distinct treatment groups with respect to sacrifice time. Ingenuity pathways analysis (IPA) was used to identify involved pathways. The results show that GHB induces gene expression alterations in hundreds of genes in the hippocampus, cortex and striatum, and the number of affected genes increases throughout a 4-h time course. Many of these DEGs are involved in neurological disease, apoptosis, and oxidative stress.

  20. Induction of YdeO, a regulator for acid resistance genes, by ultraviolet irradiation in Escherichia coli.

    Science.gov (United States)

    Yamanaka, Yuki; Ishihama, Akira; Yamamoto, Kaneyoshi

    2012-01-01

    YdeO, an AraC-type transcription factor, is an important regulator in the induction of acid-resistance genes in Escherichia coli. In this study, we found that ydeO expression was induced 20 min after exposure to UV irradiation. This required the evgA and gadE genes in vivo. YdeO, induced by UV, controls the expression of a total of 21 genes. This accompanies SOS response in E. coli.

  1. Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

    Science.gov (United States)

    Jnawali, Hum Nath; Yoo, Jin Cheol; Sohng, Jae Kyung

    2011-06-01

    To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

  2. Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione.

    Science.gov (United States)

    Han, Yi; Mhamdi, Amna; Chaouch, Sejir; Noctor, Graham

    2013-06-01

    Glutathione is a determinant of cellular redox state with roles in defence and detoxification. Emerging concepts suggest that this compound also has functions in cellular signalling. Here, we report evidence that glutathione plays potentially important roles in setting signalling strength through the jasmonic acid (JA) pathway. Firstly, we show that basal expression of JA-related genes is correlated with leaf glutathione content when the latter is manipulated either genetically or pharmacologically. Secondly, analyses of an oxidative stress signalling mutant, cat2, reveal that up-regulation of the JA pathway triggered by intracellular oxidation requires accompanying glutathione accumulation. Genetically blocking this accumulation in a cat2 cad2 line largely annuls H2 O2 -induced expression of JA-linked genes, and this effect can be rescued by exogenously supplying glutathione. While most attention on glutathione functions in biotic stress responses has been focused on the thiol-regulated protein NPR1, a comparison of JA-linked gene expression in cat2 cad2 and cat2 npr1 double mutants provides evidence that glutathione acts through other components to regulate the response of this pathway to oxidative stress. Our study provides new information implicating glutathione as a factor determining basal JA gene expression and suggests novel glutathione-dependent control points that regulate JA signalling in response to intracellular oxidation.

  3. Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

    Science.gov (United States)

    Rosic, Nedeljka N

    2012-04-01

    Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

  4. Protein and Amino Acid Supplementation Does Not Alter Proteolytic Gene Expression following Immobilization

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    Jennifer A. Bunn

    2011-01-01

    Full Text Available Objective. To determine if supplementation of protein and amino acids (PAA decreases skeletal muscle expression of atrophy-related genes, muscle mass, and strength during immobilization in humans. Methods. Twenty males wore a lower-limb immobilization boot for 28 days and consumed either a PAA supplement (28 g protein or carbohydrate placebo (28 g maltodextrose, while consuming their normal daily diet. Testing sessions included dietary analysis, lower-leg girth and body composition measurements, strength testing, and gastrocnemius muscle biopsies. Muscle was analyzed for mRNA expression of markers in the ubiquitin and calpain systems, myostatin, TNF-α, and NF-κB. Results. All genes of interest increased over time (P<.05, but there was no difference between groups. Lower-leg girth decreased over time (P=0.02; however, there were no significant changes in body composition or strength. Conclusion. Short-term lower-limb disuse, despite the absence of significant muscle atrophy, is associated with increases in skeletal muscle gene expression of several proteolysis-related genes. These changes do not appear to be altered by oral PAA supplementation.

  5. Polyethylenimine-polyacrylic acid nanocomposites: Type of bonding does influence the gene transfer efficacy and cytotoxicity.

    Science.gov (United States)

    Tripathi, Sushil K; Ahmadi, Zeba; Gupta, Kailash C; Kumar, Pradeep

    2016-04-01

    The main aim of the current study is to compare the physicochemical properties, cytotoxicity and gene-transfer ability of electrostatically and covalently linked nanocomposites of polyethylenimine (PEI) and polyacrylic acid (PAA) on mammalian cells. Two series of nanocomposites, ionic PEI-PAA (iPP) and covalent PEI-PAA (cPP), were synthesized by varying the amounts of polyacrylic acid (PAA). Physicochemical characterization revealed that iPP nanopcomposites were of bigger sized than cPP nanocomposites with zeta potential almost comparable. Nucleic acid binding assay displayed that iPP and cPP nanocomposites, having sufficient cationic charge, efficiently interacted with plasmid DNA and completely retarded its electrophoretic mobility on agarose gel. In vitro MTT assay showed slightly higher cell viability of cPP/pDNA complexes over their ionic counterparts. Both the series of nanocomposite/pDNA complexes exhibited considerably higher transfection efficacy compared to pDNA complexes of native bPEI and the standard transfection reagent, Lipofectamine, with cPP/pDNA complexes performed much better than iPP/pDNA complexes. Flow cytometry further confirmed these findings where cPP-4/pDNA complex showed transfection in ∼ 85% HEK293 cells, while iPP-2/pDNA complex transfected ∼ 67% HEK293 cells. Lipofectamine/pDNA and bPEI/pDNA complexes could transfect just ∼ 35% and ∼ 26% HEK293 cells. All these results demonstrate the superiority of covalently linked nanocomposites (cPP) which could be used as efficient carriers for nucleic acids in future gene delivery applications.

  6. High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2

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    Hellberg Michael E

    2010-05-01

    Full Text Available Abstract Background Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. Results In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus, a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella possess proteins structurally similar to tachylectin-2. Conclusions Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1 part of Oculina's innate immunity repertoire, and 2 evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.

  7. Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene

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    Hiroshi eMasuda

    2013-05-01

    Full Text Available Iron deficiency is a serious problem around the world, especially in developing countries. The production of iron-biofortified rice will help ameliorate this problem. Previously, expression of the iron storage protein, ferritin, in rice using an endosperm-specific promoter resulted in a two-fold increase in iron concentration in the resultant transgenic seeds. However, further overexpression of ferritin did not produce an additional increase in the seed iron concentration, and symptoms of iron deficiency were noted in the leaves of the transgenic plants. In the present study, we aimed to further increase the iron concentration in rice seeds without increasing the sensitivity to iron deficiency by enhancing the uptake and transport of iron via a ferric iron chelator, mugineic acid. To this end, we introduced the soybean ferritin gene (SoyferH2 driven by two endosperm-specific promoters, along with the barley nicotianamine synthase gene (HvNAS1, two nicotianamine aminotransferase genes (HvNAAT-A and -B, and a mugineic acid synthase gene (IDS3 to enhance mugineic acid production in rice plants. A marker-free vector was utilized as a means of increasing public acceptance. Representative lines were selected from 102 transformants based on the iron concentration in polished seeds and ferritin accumulation in the seeds. These lines were grown in both commercially supplied soil (iron-sufficient conditions and calcareous soil (iron-deficient conditions. Lines expressing both ferritin and mugineic acid biosynthetic genes showed signs of iron-deficiency tolerance in calcareous soil. The iron concentration in polished T3 seeds was increased by 4 and 2.5 times, as compared to that in non-transgenic lines grown in normal and calcareous soil, respectively. These results indicate that the concomitant introduction of the ferritin gene and mugineic acid biosynthetic genes effectively increased the seed iron level without causing iron sensitivity under iron

  8. Frequent mutations of lysophosphatidic acid receptor-1 gene in rat liver tumors

    Energy Technology Data Exchange (ETDEWEB)

    Obo, Yumi; Yamada, Takanori; Furukawa, Mami; Hotta, Mayuko [Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Honoki, Kanya [Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Fukushima, Nobuyuki [Laboratory of Molecular Neurobiology, Department of Life Science, Faculty of Science and Technology, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi [Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)], E-mail: ttujiuch@life.kindai.ac.jp

    2009-01-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA1 gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N-nitrosodiethylamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCs induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) - polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%) induced by DEN. Five out of 12 HCCs (41.7%) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved in rat liver carcinogenesis.

  9. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Srivathsa C Venugopal

    2009-07-01

    Full Text Available Resistance (R protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1, non-race-specific disease resistance 1 (NDR1, phytoalexin deficient 4 (PAD4, senescence associated gene 101 (SAG101, and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  10. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    Science.gov (United States)

    Venugopal, Srivathsa C; Jeong, Rae-Dong; Mandal, Mihir K; Zhu, Shifeng; Chandra-Shekara, A C; Xia, Ye; Hersh, Matthew; Stromberg, Arnold J; Navarre, DuRoy; Kachroo, Aardra; Kachroo, Pradeep

    2009-07-01

    Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  11. Tbx1 and Brn4 regulate retinoic acid metabolic genes during cochlear morphogenesis

    Directory of Open Access Journals (Sweden)

    Braunstein Evan M

    2009-05-01

    Full Text Available Abstract Background In vertebrates, the inner ear is comprised of the cochlea and vestibular system, which develop from the otic vesicle. This process is regulated via inductive interactions from surrounding tissues. Tbx1, the gene responsible for velo-cardio-facial syndrome/DiGeorge syndrome in humans, is required for ear development in mice. Tbx1 is expressed in the otic epithelium and adjacent periotic mesenchyme (POM, and both of these domains are required for inner ear formation. To study the function of Tbx1 in the POM, we have conditionally inactivated Tbx1 in the mesoderm while keeping expression in the otic vesicle intact. Results Conditional mutants (TCre-KO displayed malformed inner ears, including a hypoplastic otic vesicle and a severely shortened cochlear duct, indicating that Tbx1 expression in the POM is necessary for proper inner ear formation. Expression of the mesenchyme marker Brn4 was also lost in the TCre-KO. Brn4-;Tbx1+/-embryos displayed defects in growth of the distal cochlea. To identify a potential signal from the POM to the otic epithelium, expression of retinoic acid (RA catabolizing genes was examined in both mutants. Cyp26a1 expression was altered in the TCre-KO, while Cyp26c1 showed reduced expression in both TCre-KO and Brn4-;Tbx1+/- embryos. Conclusion These results indicate that Tbx1 expression in the POM regulates cochlear outgrowth potentially via control of local retinoic acid activity.

  12. Transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides.

    Science.gov (United States)

    Pakpour, Nazzy; Cheung, Kong Wai; Souvannaseng, Lattha; Concordet, Jean-Paul; Luckhart, Shirley

    2010-12-26

    Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development (1). From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in (2)). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice (3,4). We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

  13. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    Directory of Open Access Journals (Sweden)

    Wen-Bin Xue

    2016-07-01

    Full Text Available The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA. To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively.

  14. Folic Acid supplementary reduce the incidence of adenocarcinoma in a mouse model of colorectal cancer: microarray gene expression profile

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    Lin Yan-Wei

    2011-12-01

    Full Text Available Abstract Background Whether Folic acid is a potential drug that may prevent the progression of colorectal carcinoma and when to use are important healthy issues we focus on. Our study is to examine the effect of folic acid on the development of the CRC and the optimal time folic acid should be provided in a mouse-ICR model induced by 1, 2-Dimethylhydrazine. Also, we investigated the gene expression profile of this model related to folic acid. Method Female ICR mouse (n = 130 were divided into 7 groups either with the treatment of 1, 2-Dimethylhydrazine (20 mg/kg bodyweight weekly or folic acid (8 mg/kg bodyweight twice a week for 12 or 24 weeks. Using a 4 × 44 K Agilent whole genome oligo microarray assay, different gene expression among groups (NS, DMH, FA2, FA3 were identified and selected genes were validated by real-time polymerase chain reaction. Results Animals with a supplementary of folic acid showed a significant decrease in the incidence, the maximum diameter and multiplicity of adenocarcinomas (P Conclusion Our study demonstrated that folic acid supplementary was significantly associated with the decrease risk of CRC. And the subgroup of providing folic acid without precancerous lesions was more effective than that with precancerous lesions.

  15. Identification of target genes of transcription factor CEBPB in acute promyelocytic leukemia cells induced by all-trans retinoic acid

    Institute of Scientific and Technical Information of China (English)

    Lei Yu; Yang-De Zhang; Jun Zhou; De-Ming Yao; Xiang Li

    2013-01-01

    Objective: To indentify target genes of transcription factor CCAAT enhancer-binding proteinβ (CEBPB) in acute promyelocytic leukemia cells induced by all-trans retinoic acid. Methods:A new strategy for high-throughput identification of direct target genes was established by combining chromatin immunoprecipitation (ChIP) with in vitro selection. Then, 106 potential CEBPB binding fragments from the genome of the all-trans retinoic acid (ATRA)-treated NB4 cells were identified. Results: Of them, 82 were mapped in proximity to known or previously predicted genes; 7 were randomly picked up for further confirmation by ChIP-PCR and 3 genes (GALM, ITPR2 and ORM2) were found to be specifically up-regulated in the ATRA-treated NB4 cells, indicating that they might be the down-stream target genes of ATRA. Conclusions: Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.

  16. Uric acid stimulates endothelin-1 gene expression associated with NADPH oxidase in human aortic smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Hung-hsing CHAO; Ju-chi LIU; Jia-wei LIN; Cheng-hsien CHEN; Chieh-hsi WU; Tzu-hurng CHENG

    2008-01-01

    Aim: Recent experimental and human studies have shown that hyperuricemia is associated with hypertension and cardiovascular diseases. Elevated levels of endotheliu-1 (ET-1) has been regarded as one of the most powerful indepen-dent predictors of cardiovascular diseases. For investigating whether uric acidinduced vascular diseases are related to ET-1, the uric acid-induced ET-1 expression in human aortic smooth muscle cells (HASMC) was examined. Methods: Cultured HASMC treated with uric acid, cell proliferation and ET-1 expression were examined. Antioxidant pretreatments on uric acid-induced extracellular signal-regulated kinases (ERK) phosphorylation were carried out to elucidate the redox-sensitive pathway in proliferation and ET-1 gene expression. Results: Uric acid was found to increase HASMC proliferation, ET-1 expression and reactive oxygen species production. The ability of both N-acetylcysteine and apocynin (1-[4-hydroxy-3-methoxyphenyl]ethanone, a NADPH oxidase inhibitor) to inhibit uric acid-induced ET-1 secretion and cell proliferation suggested the involvement of intracellular redox pathways. Furthermore, apocynin, and p47phox small interfering RNA knockdown inhibited ET-1 secretion and cell proliferation induced by uric acid. Inhibition of ERK by U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) significantly suppressed uric acid-induced ET-I expression, implicating this pathway in the response to uric acid. In addition, uric acid increased the transcription factor activator protein-1 (AP-1) medi-ated reporter activity, as well as the ERK phosphorylation. Mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression. Conclusion: This is the first observation of ET-1 regulation by uric acid in HASMC, which implicates the important role of uric acid in the vascular changes associated with hypertension and vascular diseases.

  17. Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

    Science.gov (United States)

    Xie, Yun; Zhao, Jun-Long; Wang, Chuan-Wei; Yu, Ai-Xin; Liu, Niu; Chen, Li; Lin, Fei; Xu, Han-Hong

    2016-05-18

    Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional characterization of candidate AAT genes in future studies.

  18. Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.

  19. Fatty acid esters of phloridzin induce apoptosis of human liver cancer cells through altered gene expression.

    Directory of Open Access Journals (Sweden)

    Sandhya V G Nair

    Full Text Available Phloridzin (phlorizin or phloretin 2'-O-glucoside is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2, growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK, cell cycle machinery (CDKs, TERT, TOP2A, TOP2B as well as epigenetics regulators (HDACs. These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects

  20. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.

    Science.gov (United States)

    Liu, Shu-Wen; Li, Kai; Yang, Shi-Ling; Tian, Shu-Fen; He, Ling

    2015-03-01

    A sequence characterised amplified region marker was developed to determine an acid resistance-related gene in Lactobacillus plantarum. A random amplified polymorphic DNA marker named S116-680 was reported to be closely related to the acid resistance of the strains. The DNA band corresponding to this marker was cloned and sequenced with the induction of specific designed PCR primers. The results of PCR test helped to amplify a clear specific band of 680 bp in the tested acid-resistant strains. S116-680 marker would be useful to explore the acid-resistant mechanism of L. plantarum and to screen desirable malolactic fermentation strains.

  1. Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum.

    Science.gov (United States)

    Inagaki, K; Nakahira, K; Mukai, K; Tamura, T; Tanaka, H

    1998-06-01

    The gene xynA encoding an acid endo-beta-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Eschrichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65 degrees C, and is stable pH between 3.0 and 8.0. The K(m) and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30 degrees C are 3.5 mg/ml and 403 mumol/min/mg.

  2. Increased expression of fatty-acid and calcium metabolism genes in failing human heart.

    Directory of Open Access Journals (Sweden)

    Vanessa García-Rúa

    Full Text Available BACKGROUND: Heart failure (HF involves alterations in metabolism, but little is known about cardiomyopathy-(CM-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA uptake and oxidation or in calcium-(Ca(2+-handling in the human heart. METHODS: RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36 without diabetes mellitus of ischaemic (ICM, n = 16 or dilated (DCM, n = 20 cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6. RESULTS: Significant increases in mRNA of genes regulating FA uptake (CD36 and intracellular transport (Heart-FA-Binding Protein (HFABP were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA, PPAR-gamma coactivator-1-alpha (PGC1A and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca(2+-handling genes (Two-Pore-Channel 1 (TPCN1, Two-Pore-Channel 2 (TPCN2, and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1 increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca(2+-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL: three were common to and three distinct from ICM. CONCLUSION: DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca(2+-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca(2

  3. Comparative analysis of RNA regulatory elements of amino acid metabolism genes in Actinobacteria

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2005-10-01

    Full Text Available Abstract Background Formation of alternative structures in mRNA in response to external stimuli, either direct or mediated by proteins or other RNAs, is a major mechanism of regulation of gene expression in bacteria. This mechanism has been studied in detail using experimental and computational approaches in proteobacteria and Firmicutes, but not in other groups of bacteria. Results Comparative analysis of amino acid biosynthesis operons in Actinobacteria resulted in identification of conserved regions upstream of several operons. Classical attenuators were predicted upstream of trp operons in Corynebacterium spp. and Streptomyces spp., and trpS and leuS genes in some Streptomyces spp. Candidate leader peptides with terminators were observed upstream of ilvB genes in Corynebacterium spp., Mycobacterium spp. and Streptomyces spp. Candidate leader peptides without obvious terminators were found upstream of cys operons in Mycobacterium spp. and several other species. A conserved pseudoknot (named LEU element was identified upstream of leuA operons in most Actinobacteria. Finally, T-boxes likely involved in the regulation of translation initiation were observed upstream of ileS genes from several Actinobacteria. Conclusion The metabolism of tryptophan, cysteine and leucine in Actinobacteria seems to be regulated on the RNA level. In some cases the mechanism is classical attenuation, but in many cases some components of attenuators are missing. The most interesting case seems to be the leuA operon preceded by the LEU element that may fold into a conserved pseudoknot or an alternative structure. A LEU element has been observed in a transposase gene from Bifidobacterium longum, but it is not conserved in genes encoding closely related transposases despite a very high level of protein similarity. One possibility is that the regulatory region of the leuA has been co-opted from some element involved in transposition. Analysis of phylogenetic patterns

  4. Screening for Glucosyltransferase gene (gtf from exopolysaccahride producing lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Donna M. Ariestanti

    2008-04-01

    Full Text Available Glucosyltransferase (GTF is an enzyme involved in exopolysaccharide (EPS polymer synthesis in microbes. One example of EPS that has been used in pharmaceutical and medical application is dextran. Dextran has been used in conjugated-drug delivery system as matrix. As a group of microbes producing EPS, lactic acid bacteria (LAB have been well reported carrying sucrase genes glucosyltransferase (gtf, as well as fructosyltransferases (ftf. In an attempt to search for novel gtf genes as the aim of this study, LAB collection isolated from local sources yielded from previous study were screened performing PCR using degenerate primers DegFor and DegRev. An approximately 660 base pairs (bp amplicons were obtained by using genomic DNAs of those LAB isolates as templates with conserved region of gtf genes catalytic domain as target. Two out of 20 LAB strains were yielded no amplicon as observed on agarose gel, while one strain exhibited non-specific amplicon DNA bands with sizes other than 660 bp. The two negative ones were isolated from soil obtained from dairy product waste field and from waste of soy sauce from previous study, while the latter was isolated from waste of soy sauce.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Genes associated with long-chain omega-3 fatty acids in bovine skeletal muscle.

    Science.gov (United States)

    Perez, R; Cañón, J; Dunner, S

    2010-01-01

    Long-chain omega-3 fatty acids (n-3 FAs) influence meat tenderness, juiciness, and flavor, and are beneficial to human health. The percentage of long-chain n-3 FAs in total FAs is termed the omega-3 index (O3I). It is thus of great interest to favor rising this index in bovine skeletal muscle, to obtain healthier, tastier, and more nutritive meat. This study was aimed to detect transcriptomic variations related to O3I in muscles in 15-month-old males of 4 Spanish cattle breeds raised under the same conditions. Through the analysis of extreme O3I phenotypes, 3 genes of interest (AANAT, UCP2 and AHA1) were identified. AANAT and UCP2 were strongly up-regulated, while AHA1 was repressed in animals with a high O3I. Moreover, gene expression differed between GDF8-null animal muscles (tested for nt821del11 and Q204X mutations) and the wild-type muscles for genes GDH1, IGF2R, FADS1, ASPH, and AIM1, all showing down-regulation in Asturiana de los Valles calves with muscle hypertrophy (GDF8-null). This shows that in GDF8-null animals other pathways are used for FA synthesis.

  7. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    Science.gov (United States)

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae.

  8. Tryptophan hydroxylase gene 1 (TPH1) variants associated with cerebrospinal fluid 5-hydroxyindole acetic acid and homovanillic acid concentrations in healthy volunteers

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas;

    2010-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin synthesis. We investigated possible relationships between five TPH1 gene polymorphisms and cerebrospinal fluid (CSF) concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), the major dopamine metab...

  9. Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes.

    Directory of Open Access Journals (Sweden)

    Youjun Feng

    Full Text Available Escherichia coli FadR plays two regulatory roles in fatty acid metabolism. FadR represses the fatty acid degradation (fad system and activates the unsaturated fatty acid synthetic pathway. Cross-talk between E. coli FadR and the ArcA-ArcB oxygen-responsive two-component system was observed that resulted in diverse regulation of certain fad regulon β-oxidation genes. We have extended such analyses to the fadL and fadD genes, the protein products of which are required for long chain fatty acid transport and have also studied the role of a third global regulator, the CRP-cAMP complex. The promoters of both the fadL and fadD genes contain two experimentally validated FadR-binding sites plus binding sites for ArcA and CRP-cAMP. Despite the presence of dual binding sites FadR only modestly regulates expression of these genes, indicating that the number of binding sites does not determine regulatory strength. We report complementary in vitro and in vivo studies indicating that the CRP-cAMP complex directly activates expression of fadL and fadD as well as the β-oxidation gene, fadH. The physiological relevance of the fadL and fadD transcription data was validated by direct assays of long chain fatty acid transport.

  10. Analysis of Global Expression Profiles of Arabidopsis Genes Under Abscisic Acid and H2O2 Applications

    Institute of Scientific and Technical Information of China (English)

    Peng-Cheng Wang; Yan-Yan Du; Guo-Yong An; Yun Zhou; Chen Miao; Chun-Peng Song

    2006-01-01

    To gain insight into the coordination of gene expression profiles under abscisic acid (ABA) and H2O2 applications,global changes in gene expression in response to ABA and H2O2 in Arabidopsis seedlings were investigated using GeneChip (Santa Clara, CA, USA) arrays. Among over 24 000 genes present in the arrays, 459 transcripts were found to be significantly increased, whereas another 221 decreased following H2O2 treatment compared with control. Similar to treatment with H2O2, ABA treatment elevated the transcription of 391 genes and repressed that of 322 genes. One hundred and forty-three upregulated genes and 75 downregulated genes were shared between the two treatments and these genes were mainly involved in metabolism, signal transduction, transcription, defense, and resistance. Only two genes, which encode an APETALA2/dehydration-responsive element binding protein (AP2/DREBP) family transcriptional factor and a late embryogenesisabundant protein, were downregulated by H2O2, but upregulated by ABA. These results suggest that, similar to ABA, H2O2 plays a global role in gene transcription of Arabidopsisseedlings. The transcriptional responses induced by the application of exogenous ABA and H2O2 overlapped substantially. These two treatments regulated most of the downstream genes in a coordinated manner.

  11. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    Science.gov (United States)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  12. Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

    Directory of Open Access Journals (Sweden)

    Royah Vaezi

    2013-12-01

    Full Text Available In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4 from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15. These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA in marine microalgae.

  13. Analysis of the porcine APOA2 gene expression in liver, polymorphism identification and association with fatty acid composition traits.

    Science.gov (United States)

    Ballester, M; Revilla, M; Puig-Oliveras, A; Marchesi, J A P; Castelló, A; Corominas, J; Fernández, A I; Folch, J M

    2016-10-01

    APOA2 is a protein implicated in triglyceride, fatty acid and glucose metabolism. In pigs, the APOA2 gene is located on pig chromosome 4 (SSC4) in a QTL region affecting fatty acid composition, fatness and growth traits. In this study, we evaluated APOA2 as a candidate gene for meat quality traits in an Iberian × Landrace backcross population. The APOA2:c.131T>A polymorphism, located in exon 3 of APOA2 and determining a missense mutation, was associated with the percentage of hexadecenoic acid [C16:1(n-9)], linoleic acid [C18:2(n-6)], α-linolenic acid [C18:3(n-3)], dihomo-gamma-linolenic acid [C20:3(n-6)] and polyunsaturated fatty acids (PUFAs) in backfat. Furthermore, this SNP was associated with the global mRNA expression levels of APOA2 in liver and was used as a marker to determine allelic expression imbalance by pyrosequencing. We determined an overexpression of the T allele in heterozygous samples with a mean ratio of 2.8 (T/A), observing a high variability in the allelic expression among individuals. This result suggests that complex regulatory mechanisms, beyond a single polymorphism (e.g. epigenetic effects or multiple cis-acting polymorphisms), may be regulating APOA2 gene expression.

  14. Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase.

    Science.gov (United States)

    Jusoh, Malinna; Loh, Saw Hong; Chuah, Tse Seng; Aziz, Ahmad; Cha, Thye San

    2015-03-01

    Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.

  15. Polymorphism in the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs.

    Directory of Open Access Journals (Sweden)

    Jordi Corominas

    Full Text Available BACKGROUND: The ELOVL fatty acid elongase 6 (ELOVL6, the only elongase related to de novo lipogenesis, catalyzes the rate-limiting step in the elongation cycle by controlling the fatty acid balance in mammals. It is located on pig chromosome 8 (SSC8 in a region where a QTL affecting palmitic, and palmitoleic acid composition was previously detected, using an Iberian x Landrace intercross. The main goal of this work was to fine-map the QTL and to evaluate the ELOVL6 gene as a positional candidate gene affecting the percentages of palmitic and palmitoleic fatty acids in pigs. METHODOLOGY AND PRINCIPAL FINDINGS: The combination of a haplotype-based approach and single-marker analysis allowed us to identify the main, associated interval for the QTL, in which the ELOVL6 gene was identified and selected as a positional candidate gene. A polymorphism in the promoter region of ELOVL6, ELOVL6:c.-533C>T, was highly associated with the percentage of palmitic and palmitoleic acids in muscle and backfat. Significant differences in ELOVL6 gene expression were observed in backfat when animals were classified by the ELOVL6:c.-533C>T genotype. Accordingly, animals carrying the allele associated with a decrease in ELOVL6 gene expression presented an increase in C16:0 and C16:1(n-7 fatty acid content and a decrease of elongation activity ratios in muscle and backfat. Furthermore, a SNP genome-wide association study with ELOVL6 relative expression levels in backfat showed the strongest effect on the SSC8 region in which the ELOVL6 gene is located. Finally, different potential genomic regions associated with ELOVL6 gene expression were also identified by GWAS in liver and muscle, suggesting a differential tissue regulation of the ELOVL6 gene. CONCLUSIONS AND SIGNIFICANCE: Our results suggest ELOVL6 as a potential causal gene for the QTL analyzed and, subsequently, for controlling the overall balance of fatty acid composition in pigs.

  16. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes.

    Science.gov (United States)

    Thaller, M C; Lombardi, G; Berlutti, F; Schippa, S; Rossolini, G M

    1995-01-01

    The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

  17. Mapping of a gene for epidermolytic palmoplantar keratoderma to the region of the acidic keratin gene cluster at 17q12-q21.

    Science.gov (United States)

    Reis, A; Küster, W; Eckardt, R; Sperling, K

    1992-01-01

    Epidermolytic palmoplantar keratoderma (EPPK) (Vörner-Unna-Thost) is an autosomal dominantly inherited skin disease of unknown etiology characterized by diffuse severe hyperkeratosis of the palms and soles and, histologically, by cellular degeneration. We have mapped a gene for EPPK to chromosome 17q11-q23, with linkage analysis using microsatellite DNA-polymorphisms, in a single large family of 7 generations. A maximum lod score of z = 6.66 was obtained with the probe D17S579 at a recombination fraction of theta = 0.00. This locus maps to the same region as the type I (acidic) keratin gene cluster. Keratins, members of the intermediate filament family, the major proteins of the cytoskeleton in epidermis, are differentially expressed in a tissue-specific manner. One acidic keratin, keratin 9 (KRT9), is expressed only in the terminally differentiated epidermis of palms and soles. The KRT9 gene has not yet been cloned; however, since the genes for most acidic keratins are clustered, it is highly probable that it too will map to this region. We therefore propose KRT9 as the candidate gene for EPPK.

  18. 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

    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...

  19. Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids.

    Science.gov (United States)

    Olaetxea, Maite; Mora, Verónica; Bacaicoa, Eva; Garnica, María; Fuentes, Marta; Casanova, Esther; Zamarreño, Angel M; Iriarte, Juan C; Etayo, David; Ederra, Iñigo; Gonzalo, Ramón; Baigorri, Roberto; García-Mina, Jose M

    2015-12-01

    The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.

  20. Varying the nucleic acid composition of siRNA molecules dramatically varies the duration and degree of gene silencing.

    Science.gov (United States)

    Lamberton, Janelle S; Christian, Allen T

    2003-06-01

    The utility of short interfering RNA (siRNA) as a means of gene silencing depends on several factors. These include the degree to which a gene can be silenced, the length of time for which the gene remains silenced, the degree of recovery of gene function, and the effects of the silencing process on general cell functions. We hypothesized that changing the nucleic acid composition of the siRNA constructs used for silencing would affect these parameters. With siRNA gene silencing of the glucose-6-phosphate dehydrogenase gene as a baseline, we found that siDNA molecules have an effect that is similar in duration but lesser in degree, whereas hybrid DNA:RNA molecules have an effect that is enormously greater in both duration and degree.

  1. Alpha-synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

    DEFF Research Database (Denmark)

    Golovko, Mikhail Y; Rosenberger, Thad A; Feddersen, Søren;

    2007-01-01

    Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unknown....... In the present study, we examined the effect of Snca gene ablation on brain 22:6n-3 metabolism. We determined 22:6n-3 uptake and incorporation into brain phospholipids by infusing awake, wild-type and Snca-/- mice with [1-14C]22:6n-3 using steady-state kinetic modeling. In addition, because Snca modulates 20:4n...

  2. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    Science.gov (United States)

    Yaacob, Norhayati; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  3. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    Directory of Open Access Journals (Sweden)

    Norhayati Yaacob

    2016-03-01

    Full Text Available Background. Not all yeast alcohol dehydrogenase 2 (ADH2 are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2 and S. cerevisiae (SceADH2 in response to 2% (w/v glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell

  4. Usage of U7 small nuclear ribonucleic acid in gene therapy of hemoglobin D Punjab disorder: Rationale?

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2013-01-01

    Full Text Available Background: Hemoglobin (Hb D Punjab disorder is a congenital hemoglobinopathy described in India. It is a disorder due to defect in beta-globin gene. Materials and Methods: Here, the author assesses the possibility of U7.623 gene therapy for Hb D Punjab disorder. A standard bioinformatic analysis to study the effect of co-expression between nucleic acid sequence for human Hb D Punjab beta-globin chain and U7.623 was performed. Result: It can be seen that fully recovery of Hb function and biological process can be derived via gene ontology study. Conclusion: Here, there is a rationale to use U7 small nuclear ribonucleic acid as a possible tool for gene therapy in Hb D Punjab disorder.

  5. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Directory of Open Access Journals (Sweden)

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  6. Association with amino acids does not enhance efficacy of polymerized liposomes as a system for lung gene delivery

    OpenAIRE

    Elga eBernardo Bandeira De Melo; Miquéias eLopes-Pacheco; Nadia eChiaramoni; Débora eFerreira; Maria Julieta eFernandez-Ruocco; Maria Jimena ePrieto; Tatiana eMaron-Gutierrez; Perrotta, Ramiro M.; Hugo C Castro-Faria-Neto; Patricia Rieken Macedo Rocco; Silvia del Valle Alonso; Marcelo Marcos Morales

    2016-01-01

    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study,...

  7. Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.

    Science.gov (United States)

    Jiang, Wen Zhi; Henry, Isabelle M; Lynagh, Peter G; Comai, Luca; Cahoon, Edgar B; Weeks, Donald P

    2016-11-11

    The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T2 and T3 generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T3 and T4 generation Camelina seeds was associated with a combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.

  8. Promoter strength of folic acid synthesis genes affects sulfa drug resistance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Iliades, Peter; Berglez, Janette; Meshnick, Steven; Macreadie, Ian

    2003-01-01

    The enzyme dihydropteroate synthase (DHPS) is an important target for sulfa drugs in both prokaryotic and eukaryotic microbes. However, the understanding of DHPS function and the action of antifolates in eukaryotes has been limited due to technical difficulties and the complexity of DHPS being a part of a bifunctional or trifunctional protein that comprises the upstream enzymes involved in folic acid synthesis (FAS). Here, yeast strains have been constructed to study the effects of FOL1 expression on growth and sulfa drug resistance. A DHPS knockout yeast strain was complemented by yeast vectors expressing the FOL1 gene under the control of promoters of different strengths. An inverse relationship was observed between the growth rate of the strains and FOL1 expression levels. The use of stronger promoters to drive FOL1 expression led to increased sulfamethoxazole resistance when para-aminobenzoic acid (pABA) levels were elevated. However, high FOL1 expression levels resulted in increased susceptibility to sulfamethoxazole in pABA free media. These data suggest that up-regulation of FOL1 expression can lead to sulfa drug resistance in Saccharomyces cerevisiae.

  9. Circadian and Dopaminergic Regulation of Fatty Acid Oxidation Pathway Genes in Retina and Photoreceptor Cells

    Science.gov (United States)

    Vancura, Patrick; Wolloscheck, Tanja; Baba, Kenkichi; Tosini, Gianluca; Iuvone, P. Michael; Spessert, Rainer

    2016-01-01

    The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy—one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid β-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice—a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina. PMID:27727308

  10. Hydroxycinnamic acid functional ingredients and their biosynthetic genes in tubers of Solanum tuberosum Group Phureja

    Directory of Open Access Journals (Sweden)

    Liyao Ji

    2016-12-01

    Full Text Available Potato is an ideal candidate for the delivery of functional ingredients due to its high worldwide consumption. The metabolites in cooked tubers of eight diploid potato genotypes from Colombia were explored. Potato tubers were harvested, cooked,lyophilized, and then stored at −80°C. Metabolites were extracted from flesh samples and analyzed using liquid chromatography and high-resolution mass spectrometry. A total of 294 metabolites were putatively identified, of which 87 metabolites were associated with health-benefiting roles for humans, such as anticancer and anti-inflammatory properties. Two metabolites, chlorogenic acid and N-Feruloyltyramine were detected in high abundance and were mapped on to the potato metabolic pathways to predict the related biosynthetic enzymes: hydroxycinnamoyl-CoA quinate transferase (HQT and tyramine hydroxycinnamoyl transferase (THT, respectively. The coding genes of these enzymes identified nonsynonymous single-nucleotide polymorphisms (nsSNPs in AC09, AC64, and Russet Burbank, with the highest enzyme stability found in AC09. This is consistent with the highest presence of hydroxycinnamic acids in the AC09 genotype. The metabolites detected at high fold change, their functional ingredient properties, and their enhancement through breeding to improve health of the indigenous communities’ of Colombia are discussed.

  11. Genome-wide screening for genes associated with valproic acid sensitivity in fission yeast.

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    Lili Zhang

    Full Text Available We have been studying the action mechanisms of valproic acid (VPA in fission yeast Schizosaccharomyces pombe by developing a genetic screen for mutants that show hypersensitivity to VPA. In the present study, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 148 deletion strains to be VPA sensitive. Of the 148 strains, 93 strains also showed sensitivity to another aliphatic acids HDAC inhibitor, sodium butyrate (SB, and 55 strains showed sensitivity to VPA but not to SB. Interestingly, we found that both VPA and SB treatment induced a marked increase in the transcription activity of Atf1 in wild-type cells. However, in clr6-1, a mutant allele the clr6(+ gene encoding class I HDAC, neither VPA- nor SB induced the activation of Atf1 transcription activity. We also found that VPA, but not SB, caused an increase in cytoplasmic Ca(2+ level. We further found that the cytoplasmic Ca(2+ increase was caused by Ca(2+ influx from extracellular medium via Cch1-Yam8 channel complex. Altogether, our present study indicates that VPA and SB play similar but distinct roles in multiple physiological processes in fission yeast.

  12. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    Qian JIN; Li-Xia YANG; Hao-Mang JIAO; Bin LU; Yu-Qun WU; Yuan-Cong ZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E. Coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the nativeenzyme.

  13. Purification, Gene Cloning and Expression of an Acidic Phospholipase A2 from Agkistrodon shedaoensis Zhao

    Institute of Scientific and Technical Information of China (English)

    QianJIN; Li-XiaYANG; Hao-MangJIAO; BinLU; Yu-QunWU; Yuan-CongZHOU

    2004-01-01

    A protein with the activity of phospholipase A2 named asAPLA2 was pmified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column,Source S and Mono Q FPLC. Its molecular weight was estimated as 19kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1μmol/L ADP, and the IC50 was determined to be 6μmol/L. Degenerate primer was designed and synthesized according to the Nterminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares.The gene was then cloned into the expression plasmid pET-40b(+) and expressed in E.coli BL21(DE3).Western blot analysis indicated that the expressed protein cross-reacted with the antibc dy against the native enzyme.

  14. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    Science.gov (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  15. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery.

    Science.gov (United States)

    Liu, Ya; Kong, Ming; Cheng, Xiao Jie; Wang, Qian Qian; Jiang, Li Ming; Chen, Xi Guang

    2013-04-15

    The present work described nanoparticles (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) using coacervation process as novel potential carriers for gene delivery. An N/P ratio of 5 and OCMCS/HA weight ratio of 4 were the optimal conditions leading to the smallest (164.94 nm), positive charged (+14.2 mV) and monodispersed NPs. OCMCS-HA/DNA (OHD) NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The MTT survival assay indicated no significant cytotoxicity. The transfection efficiency of OHD NPs was 5-fold higher than OCMCS/DNA (OD) NPs; however, it decreased significantly in the presence of excess free HA. The results indicated that OHD NPs internalized in Caco-2 cells were mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OHD NPs for the targeting and further transfer of genes to the epithelial cells.

  16. Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge in vitro

    Science.gov (United States)

    Sulabh, Sourabh; Bhushan, Bharat; Panigrahi, Manjit; Verma, Ankita; Baba, Naseer Ahmad; Kumar, Pushpendra

    2016-01-01

    Aim: To study the effect of Staphylococcus aureus cell wall antigens, peptidoglycan (PGN) and lipoteichoic acid (LTA) challenge on immune cells present in bovine peripheral blood mononuclear cells (PBMCs). Materials and Methods: In this study, efforts have been made to investigate the effects of three combinations (10+10, 20+20 and 30+30 μg/ml) of PGN and LTA obtained from S. aureus. These antigens were used to challenge the bovine PBMCs. After 6 h of incubation quantitative, real time-polymerase chain reaction was used to study toll-like receptor 2 (TLR-2) and major cytokine mRNA expression in bovine PBMC challenged with three different antigen blends. Results: The results indicated that mRNA level of interferon gamma is influenced by the expression of TLR-2 gene. Tumor necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and IL-8 genes showed a maximum response at a dose of 10 μg of PGN and 10 μg of LTA challenge per ml of culture medium. The outcome also suggests that both IL-10 and IL-8 followed the expression pattern of TNF-α. Conclusion: A dose of 10 μg of PGN and 10 μg of LTA per ml of culture medium was found to be most suitable for challenging PBMC. PMID:27733800

  17. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  18. Transcriptional regulation of genes involved in retinoic acid metabolism in Senegalese sole larvae.

    Science.gov (United States)

    Boglino, Anaïs; Ponce, Marian; Cousin, Xavier; Gisbert, Enric; Manchado, Manuel

    2017-01-01

    The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.

  19. Deciphering ascorbic acid regulatory pathways in ripening tomato fruit using a weighted gene correlation network analysis approach.

    Science.gov (United States)

    Gao, Chao; Ju, Zheng; Li, Shan; Zuo, Jinhua; Fu, Daqi; Tian, Huiqin; Luo, Yunbo; Zhu, Benzhong

    2013-11-01

    Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.

  20. Deciphering Ascorbic Acid Regulatory Pathways in Ripening Tomato Fruit Using a Weighted Gene Correlation Network Analysis Approach

    Institute of Scientific and Technical Information of China (English)

    Chao Gao; Zheng Ju; Shan Li; Jinhua Zuo; Daqi Fu; Huiqin Tian; Yunbo Luo; Benzhong Zhu

    2013-01-01

    Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.

  1. Association of novel SNPs in the candidate genes affecting caprine milk fatty acids related to human health.

    Science.gov (United States)

    Dixit, S P; Sivalingam, Jayakumar; Tyagi, A K; Saroha, V; Sharma, A; Nagda, R K

    2015-06-01

    In the present investigation, 618 milk samples of Sirohi breed of goat were collected, and analyzed for conjugated linoleic acid (CLA, C18:2) and other fatty acids. The CLA in studied goat milk samples was 4.87 mg/g of milk fat and C18:2 cis-9, trans-11 contributes 2.9 mg/g of milk fat and trans10 cis12 contributes 0.82 mg/g of milk fat. The saturated fatty acids in the milk accounted for 69.55% and unsaturated fatty acid accounted for 28.50%. The unsaturated fatty acid was constituted by monounsaturated fatty acid (24.57%) and polyunsaturated fatty acids (3.96%.). The major contribution (45.56%) in total fatty acid was of C12:0, C14:0 and C16:0. C18:0 and short chain ones (C4:0, C6:0, C8:0, and C10:0) have a neutral or cholesterol-decreasing effect. The DNA sequence analysis of the genes (DGAT1, SCAP, PPARG, OLR, FABP3 and PRL) in a random panel of 8 Sirohi goats revealed 38 SNPs across the targeted regions. Out of the studied SNPs (38) across these genes, 22 SNPs had significant effect on one or a group of fatty acids including CLA. The genotypes at these loci showed significant differences in the least square means of a particular fatty acid or a group of fatty acids including CLA and its isomers.

  2. Adipocyte Accumulation of Long-Chain Fatty Acids in Obesity is Multifactorial, Resulting from Increased Fatty Acid Uptake and Decreased Activity of Genes Involved in Fat Utilization

    Science.gov (United States)

    Walewski, José L.; Ge, Fengxia; Gagner, Michel; Inabnet, William B.; Pomp, Alfons; Branch, Andrea D.

    2010-01-01

    Background The obesity epidemic causes significant morbidity and mortality. Knowledge of cellular function and gene expression in obese adipose tissue will yield insights into obesity pathogenesis and suggest therapeutic targets. The aim of this work is to study the processes determining fat accumulation in adipose tissue from obese patients. Methods Omental fat was collected from two cohorts of obese bariatric surgery patients and sex-matched normal-weight donors. Isolated adipocytes were compared for cell size, volume, and long-chain fatty acid (LCFA) uptake. Omental fat RNAs were screened by 10K microarray (cohort 1: three obese, three normal) or Whole Genome microarray (cohort 2: seven obese, four normal). Statistical differences in gene and pathway expression were identified in cohort 1 using the GeneSifter Software (Geospiza) with key results confirmed in cohort 2 samples by microarray, quantitative real-time polymerase chain reaction, and pathway analysis. Results Obese omental adipocytes had increased surface area, volume, and Vmax for saturable LCFA uptake. Dodecenoyl-coenzyme A delta isomerase, central to LCFA metabolism, was approximately 1.6-fold underexpressed in obese fat in cohorts 1 and 2. Additionally, the Kyoto Encyclopedia of Genes and Genomics pathway analysis identified oxidative phosphorylation and fatty acid metabolism pathways as having coordinate, nonrandom down-regulation of gene expression in both cohorts. Conclusions In obese omental fat, saturable adipocyte LCFA uptake was greater than in controls, and expression of key genes involved in lipolysis, β-oxidation, and metabolism of fatty acids was reduced. Thus, both increased uptake and reduced metabolism of LCFAs contribute to the accumulation of LCFAs in obese adipocytes. PMID:19866242

  3. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

    Science.gov (United States)

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  4. Structural Basis for Ether-a-go-go-Related Gene K+ Channel Subtype-Dependent Activation by Niflumic Acid[S

    OpenAIRE

    Fernandez, David; Sargent, John; Frank B Sachse; Sanguinetti, Michael C.

    2008-01-01

    Niflumic acid [2-((3-(trifluoromethyl)phenyl)amino)-3-pyridin-ecarboxylic acid, NFA] is a nonsteroidal anti-inflammatory drug that also blocks or modulates the gating of a wide spectrum of ion channels. Here we investigated the mechanism of channel activation by NFA on ether-a-go-go-related gene (ERG) K+ channel subtypes expressed in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. NFA acted from the extracellular side of the membrane to differentially enhance ERG channel ...

  5. Tryptophan hydroxylase gene 1 (TPH1) variants associated with cerebrospinal fluid 5-hydroxyindole acetic acid and homovanillic acid concentrations in healthy volunteers

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas;

    2010-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin synthesis. We investigated possible relationships between five TPH1 gene polymorphisms and cerebrospinal fluid (CSF) concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), the major dopamine...... metabolite homovanillic acid (HVA), and the major norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy volunteers (n=132). The G-allele of the TPH1 rs4537731 (A-6526G) polymorphism was associated with 5-HIAA and HVA, but not MHPG concentrations. None of the other four TPH1...

  6. Effect of eicosapentaenoic acid on the expression of ABCG1 gene in the human monocyte THP-1 cells.

    Directory of Open Access Journals (Sweden)

    Mostafa Moradi Sarabi

    2014-03-01

    Full Text Available Cardiovascular disease (CVD is the leading cause of death and disability in developed countries. Atherosclerosis is the major cause of CVD, accounting for about half of the attributed deaths. Cholesterol homeostasis is one of the most important factors in atherosclerosis. ATP-Binding cassette transporters cholesterol. Omega (ω 3 fatty acids are important ligands for regulation of ABC transporters such as ABCG1. Concern has been raised that the low absolute intakes of EPA and high ratios of ω-6 polyunsaturated fatty acids (ω-6 PUFA to EPA may predispose some individuals to CVD. Eicosapentaenoic acid (EPA is the most abundant ω3 fatty acid in the diet. The objective of this study was to evaluate the effect of different concentrations of EPA on the expression of ABCG1 gene in the human monocyte THP-1 cells. In this study, THP-1 cells were cultured in RPMI 1640 medium, THP-1 monocytes were then differentiated to macrophages with PMA (phorbol myristic acid and stimulated with 50, 75 and 100 μM of EPA for 24 h at 37°C. We examined the effects of EPA treatment on the expression of ABCG1 gene using Quantitative Real time RT-PCR (qRT-PCR. Our results, indicate that ABCG1 mRNA expression was significantly reduced by 50, 75 and 100 μM EPA fatty acid treatments as compared to the control cells (р = 0.009, р < 0.001 and р = 0.002, respectively. These results suggest that polyunsaturated fatty acids (PUFAs such as EPA have an effect on the cholesterol homeostasis in macrophages, and they can change the expression of ABCG1 gene. It seems that EPA has different effects on gene expression and lipid metabolism.

  7. Effect of eicosapentaenoic acid on the expression of ABCG1 gene in the human monocyte THP-1 cells.

    Science.gov (United States)

    Moradi Sarabi, Mostafa; Doosti, Mahmood; Einollahi, Nahid; Hesami, Soroush Shahryar; Dashti, Nasrin

    2014-01-01

    Cardiovascular disease (CVD) is the leading cause of death and disability in developed countries. Atherosclerosis is the major cause of CVD, accounting for about half of the attributed deaths. Cholesterol homeostasis is one of the most important factors in atherosclerosis. ATP-Binding cassette transporters cholesterol. Omega (ω) 3 fatty acids are important ligands for regulation of ABC transporters such as ABCG1. Concern has been raised that the low absolute intakes of EPA and high ratios of ω-6 polyunsaturated fatty acids (ω-6 PUFA) to EPA may predispose some individuals to CVD. Eicosapentaenoic acid (EPA) is the most abundant ω3 fatty acid in the diet. The objective of this study was to evaluate the effect of different concentrations of EPA on the expression of ABCG1 gene in the human monocyte THP-1 cells. In this study, THP-1 cells were cultured in RPMI 1640 medium, THP-1 monocytes were then differentiated to macrophages with PMA (phorbol myristic acid) and stimulated with 50, 75 and 100 μM of EPA for 24 h at 37°C. We examined the effects of EPA treatment on the expression of ABCG1 gene using Quantitative Real time RT-PCR (qRT-PCR). Our results, indicate that ABCG1 mRNA expression was significantly reduced by 50, 75 and 100 μM EPA fatty acid treatments as compared to the control cells (р = 0.009, р < 0.001 and р = 0.002, respectively). These results suggest that polyunsaturated fatty acids (PUFAs) such as EPA have an effect on the cholesterol homeostasis in macrophages, and they can change the expression of ABCG1 gene. It seems that EPA has different effects on gene expression and lipid metabolism.

  8. Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Zhichao eXu

    2016-02-01

    Full Text Available Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and 4 alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that 6 candidate cytochrome P450s and 5 candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

  9. Expression profiles of the genes associated with metabolism and transport of amino acids and their derivatives in rat liver regeneration.

    Science.gov (United States)

    Xu, C S; Chang, C F

    2008-01-01

    Amino acids (AA) are components of protein and precursors of many important biological molecules. To address effects of the genes associated with metabolism and transport of AA and their derivatives during rat liver regeneration (LR), we firstly obtained the above genes by collecting databases data and retrieving related thesis, and then analyzed their expression profiles during LR using Rat Genome 230 2.0 array. The LR-associated genes were identified by comparing the gene expression difference between partial hepatectomy (PH) and sham-operation (SO) rat livers. It was approved that 134 genes associated with metabolism of AA and their derivatives and 26 genes involved in transport of them were LR-associated. The initially and totally expressing number of these genes occurring in initial phase of LR (0.5-4 h after PH), G0/G1 (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure-function reconstruction of liver tissue (72-168 h after PH) were respectively 76, 17, 79, 5 and 162, 89, 564, 195, illustrating that these LR-associated genes were initially expressed mainly in initial stage, and functioned in different phases. Frequencies of up-regulation and down-regulation of them being separately 564 and 357 demonstrated that genes up-regulated outnumbered those down-regulated. Categorization of their expression patterns into 22 types implied the diversity of cell physiological and biochemical activities. According to expression changes and patterns of the above-mentioned genes in LR, it was presumed that histidine biosynthesis in the metaphase and anaphase, valine metabolism in the anaphase, and metabolism of glutamate, glutamine, asparate, asparagine, methionine, alanine, leucine and aromatic amino acid almost were enhanced in the whole LR; as for amino acid derivatives, transport of neutral amino acids, urea, gamma-aminobutyric acid, betaine and taurine, metabolism of dopamine, heme, S-adenosylmethionine, thyroxine, and

  10. Integrated systems biology analysis of transcriptomes reveals candidate genes for acidity control in developing fruits of sweet orange (Citrus sinensis L. Osbeck

    Directory of Open Access Journals (Sweden)

    Dingquan eHuang

    2016-04-01

    Full Text Available Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck with varying fruit acidity, Succari (acidless, Bingtang (low acid, and Newhall and Xinhui (normal acid. Fruits of these varieties at 45 days post anthesis (DPA, which corresponds to Stage I (cell division, had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion. Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis. Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AH10, degradation (such as APD2 and transcription (such as AIL6 and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control.

  11. Polymorphisms in genes encoding acetylsalicylic acid metabolizing enzymes are unrelated to upper gastrointestinal health in cardiovascular patients on acetylsalicylic acid.

    NARCIS (Netherlands)

    Oijen, M.G.H. van; Huybers, S.; Peters, W.H.M.; Drenth, J.P.H.; Laheij, R.J.F.; Verheugt, F.W.A.; Jansen, J.B.M.J.

    2005-01-01

    BACKGROUND: As acetylsalicylic acid is metabolized by UDP-glucuronosyltransferase 1A6 (UGT1A6) and cytochrome P450 2C9 (CYP2C9), interindividual differences in activity of these enzymes may modulate the effects and side-effects of acetylsalicylic acid. The objective of this study was to assess wheth

  12. The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells

    Science.gov (United States)

    KIKU, Yoshio; NAGASAWA, Yuya; TANABE, Fuyuko; SUGAWARA, Kazue; WATANABE, Atsushi; HATA, Eiji; OZAWA, Tomomi; NAKAJIMA, Kei-ichi; ARAI, Toshiro; HAYASHI, Tomohito

    2016-01-01

    Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes. PMID:27211287

  13. All-trans retinoic acid enhances bystander effect of suicide gene therapy in the treatment of breast cancer.

    Science.gov (United States)

    Kong, Heng; Liu, Xia; Yang, Liucheng; Qi, Ke; Zhang, Haoyun; Zhang, Jingwen; Huang, Zonghai; Wang, Hongxian

    2016-03-01

    All-trans retinoic acid (ATRA) has been shown to enhance the expression of connexin 43 (Cx43) and the bystander effect (BSE) in suicide gene therapy. These in turn improve effects of suicide gene therapies for several tumor types. However, whether ATRA can improve BSE remains unclear in suicide gene therapy for breast cancer. In the present study, MCF-7, human breast cancer cells were treated with ATRA in combination with a VEGFP-TK/CD gene suicide system developed by our group. We found that this combination enhances the efficiency of cell killing and apoptosis of breast cancer by strengthening the BSE in vitro. ATRA also promotes gap junction intercellular communication (GJIC) in MCF-7 cells by upregulation of the connexin 43 mRNA and protein in MCF-7 cells. These results indicate that enhancement of GJIC by ATRA in suicide gene system might serve as an attractive and cost-effective strategy of therapy for breast cancer cells.

  14. Identification of differences in human and great ape phytanic acid metabolism that could influence gene expression profiles and physiological functions

    Directory of Open Access Journals (Sweden)

    Siegmund Kimberly D

    2010-10-01

    Full Text Available Abstract Background It has been proposed that anatomical differences in human and great ape guts arose in response to species-specific diets and energy demands. To investigate functional genomic consequences of these differences, we compared their physiological levels of phytanic acid, a branched chain fatty acid that can be derived from the microbial degradation of chlorophyll in ruminant guts. Humans who accumulate large stores of phytanic acid commonly develop cerebellar ataxia, peripheral polyneuropathy, and retinitis pigmentosa in addition to other medical conditions. Furthermore, phytanic acid is an activator of the PPAR-alpha transcription factor that influences the expression of genes relevant to lipid metabolism. Results Despite their trace dietary phytanic acid intake, all great ape species had elevated red blood cell (RBC phytanic acid levels relative to humans on diverse diets. Unlike humans, chimpanzees showed sexual dimorphism in RBC phytanic acid levels, which were higher in males relative to females. Cultured skin fibroblasts from all species had a robust capacity to degrade phytanic acid. We provide indirect evidence that great apes, in contrast to humans, derive significant amounts of phytanic acid from the hindgut fermentation of plant materials. This would represent a novel reduction of metabolic activity in humans relative to the great apes. Conclusion We identified differences in the physiological levels of phytanic acid in humans and great apes and propose this is causally related to their gut anatomies and microbiomes. Phytanic acid levels could contribute to cross-species and sex-specific differences in human and great ape transcriptomes, especially those related to lipid metabolism. Based on the medical conditions caused by phytanic acid accumulation, we suggest that differences in phytanic acid metabolism could influence the functions of human and great ape nervous, cardiovascular, and skeletal systems.

  15. Non-viral gene delivery strategies for gene therapy: a 'menage a trois' among nucleic acids, materials, and the biological environment

    Energy Technology Data Exchange (ETDEWEB)

    Pezzoli, Daniele; Candiani, Gabriele, E-mail: gabriele.candiani@polimi.it [INSTM (National Interuniversity Consortium of Materials Science and Technology), Research Unit Milano Politecnico (Italy)

    2013-03-15

    Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription-translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

  16. The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2.

    Science.gov (United States)

    Nakamura, H; Yuasa, I; Umetsu, K; Nakagawa, M; Nanba, E; Kimura, K

    2000-09-24

    The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.

  17. Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes.

    Science.gov (United States)

    McCammon, Mark T; Epstein, Charles B; Przybyla-Zawislak, Beata; McAlister-Henn, Lee; Butow, Ronald A

    2003-03-01

    To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

  18. Maternal folic acid supplementation modulates DNA methylation and gene expression in the rat offspring in a gestation period-dependent and organ-specific manner.

    Science.gov (United States)

    Ly, Anna; Ishiguro, Lisa; Kim, Denise; Im, David; Kim, Sung-Eun; Sohn, Kyoung-Jin; Croxford, Ruth; Kim, Young-In

    2016-07-01

    Maternal folic acid supplementation can alter DNA methylation and gene expression in the developing fetus, which may confer disease susceptibility later in life. We determined which gestation period and organ were most sensitive to the modifying effect of folic acid supplementation during pregnancy on DNA methylation and gene expression in the offspring. Pregnant rats were randomized to a control diet throughout pregnancy; folic acid supplementation at 2.5× the control during the 1st, 2nd or 3rd week of gestation only; or folic acid supplementation throughout pregnancy. The brain, liver, kidney and colon from newborn pups were analyzed for folate concentrations, global DNA methylation and gene expression of the Igf2, Er-α, Gr, Ppar-α and Ppar-γ genes. Folic acid supplementation during the 2nd or 3rd week gestation or throughout pregnancy significantly increased brain folate concentrations (Pfolic acid supplementation throughout pregnancy significantly increased liver folate concentrations (P=.005), in newborn pups. Brain global DNA methylation incrementally decreased from early to late gestational folic acid supplementation and was the lowest with folic acid supplementation throughout pregnancy (P=.026). Folic acid supplementation in late gestation or throughout pregnancy significantly decreased Er-α, Gr and Ppar-α gene expression in the liver (Pfolic acid supplementation. Maternal folic acid supplementation affects tissue folate concentrations, DNA methylation and gene expression in the offspring in a gestation-period-dependent and organ-specific manner.

  19. Improvement of acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae by disruption of the FPS1 aquaglyceroporin gene.

    Science.gov (United States)

    Zhang, Jun-Guo; Liu, Xiu-Ying; He, Xiu-Ping; Guo, Xue-Na; Lu, Ying; Zhang, Bo-Run

    2011-02-01

    The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.

  20. Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya

    NARCIS (Netherlands)

    Malik, Amarila; Radji, Maksum; Kralj, Slavko; Dijkhuizen, Lubbert

    2009-01-01

    Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and f

  1. Increase of sulphur-containing amino acids in transgenic potato with 10 ku zein gene from maize

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The 10 k zein gene of maize was under control of patatin class ⅠPromoter of potato and transferred into potato genome by the leaf-disc method.The expression of 10 ku zein was determined in the tuber of transgenic plants by RT-PCR.Furthermore,the sulphur-containing amino acids in transgenic tuber increase remarkably.

  2. A Nonsense Mutation in the Acid α-Glucosidase Gene Causes Pompe Disease in Finnish and Swedish Lapphunds

    NARCIS (Netherlands)

    E.H. Seppälä (Eija); A.J.J. Reuser (Arnold); H. Lohi (Hannes)

    2013-01-01

    textabstractPompe disease is a recessively inherited and often fatal disorder caused by the deficiency of acid α-glucosidase, an enzyme encoded by the GAA gene and needed to break down glycogen in lysosomes. This glycogen storage disease type II has been reported also in Swedish Lapphund dogs. Here

  3. Polymorphisms in the genes involved in the arachidonic acid-pathway, fish consumption and the risk of colorectal cancer.

    NARCIS (Netherlands)

    Siezen, Christine L E; Bueno-de-Mesquita, H Bas; Peeters, Petra H M; Kram, Nicolien R; Doeselaar, Marina van; Kranen, Henk J van

    2006-01-01

    The objective of this study on colorectal cancer was to investigate the associations between SNPs in the genes involved in the arachidonic acid (AA)-pathway, their haplotypes and colorectal cancer. Moreover, interactions between SNPs and fish consumption were considered. In this study, a total of 50

  4. Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Wees, A.C.M. van; Hoffland, E.; Pelt, J.A. van; Loon, L.C. van

    1996-01-01

    Systemic acquired resistance is a pathogen-inducible defense mechanism in plants. The resistant state is dependent on endogenous accumulation of salicylic acid (SA) and is characterized by the activation of genes encoding pathogenesis-related (PR) proteins. Recently, selected nonpathogenic, root-col

  5. 9-CIS-RETINOIC ACID REPRESSES ESTROGEN-INDUCED EXPRESSION OF THE VERY-LOW-DENSITY APOLIPOPROTEIN-II GENE

    NARCIS (Netherlands)

    SCHIPPERS, IJ; KLOPPENBURG, M; SNIPPE, L; AB, G

    1994-01-01

    The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concent

  6. Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

    NARCIS (Netherlands)

    Mach Casellas, N.; Jacobs, A.A.A.; Kruijt, L.; Baal, van J.; Smits, M.A.

    2011-01-01

    The aim of this study was to determine the effects of supplementing unprotected dietary unsaturated fatty acids (UFAs) from different plant oils on gene expression in the mammary gland of grazing dairy cows. A total of 28 Holstein–Friesian dairy cows in mid-lactation were blocked according to parity

  7. Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator Gene nifA2 in Mesorhizobium loti MAFF303099

    OpenAIRE

    Nukui, Noriyuki; MINAMISAWA, KIWAMU; Ayabe, Shin-Ichi; Aoki, Toshio

    2006-01-01

    Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the β-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fix...

  8. Supplementation of essential fatty acids to Holstein calves during late uterine life and first month of life alters hepatic fatty acid profile and gene expression.

    Science.gov (United States)

    Garcia, M; Greco, L F; Lock, A L; Block, E; Santos, J E P; Thatcher, W W; Staples, C R

    2016-09-01

    Linoleic acid is an essential dietary fatty acid (FA). However, how the supplementation of linoleic acid during uterine and early life may modify the FA profile and transcriptome regulation of the liver, and performance of preweaned dairy calves is unknown. Our objective was to evaluate the effect of supplementation of essential FA to Holstein calves during late uterine and early life on their hepatic FA profile and global gene expression at 30 d of age. During the last 8 wk of pregnancy, Holstein cattle (n=96) were fed either no fat supplement (control), a saturated FA supplement enriched with C18:0, or an unsaturated FA supplement enriched with linoleic acid. Male calves (n=40) born from these dams were fed a milk replacer (MR) with either low (LLA) or high linoleic acid (HLA) concentration as the sole feedstuff during the first 30 d. Liver biopsy was performed at 30 d of age, and microarray analysis was performed on 18 liver samples. Total concentration of FA in liver were greater in calves fed LLA compared with those fed HLA MR (8.2 vs. 7.1%), but plasma concentrations of total FA did not differ due to MR diets. The FA profiles of plasma and liver of calves were affected differently by the prepartum diets. Specifically, the FA profile in liver was affected moderately by the feeding of fat prepartum, but the profiles did not differ due to the type of FA fed prepartum. The type of MR fed during the first 30 d of life had major effects on both plasma and liver FA profiles, resembling the type of fat fed. Plasma and liver of calves fed LLA MR had greater percentage of medium-chain FA (C12:0 and C14:0), whereas plasma and liver from calves fed HLA MR had greater percentages of linoleic and α-linolenic acids. Dams fed fat or a specific type of FA modified the expression of some genes in liver of calves, particularly those genes involved in biological functions and pathways related to upregulation of lipid metabolism and downregulation of inflammatory responses

  9. Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

    Directory of Open Access Journals (Sweden)

    Rivero Montserrat

    2011-04-01

    Full Text Available Abstract Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA, a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A or by oral gavage (Group B, supplemented just during suckling (Group C and control animals (Group D was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix. Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf, tissue inhibitor of metalloproteinase 1 (Timp1, galanin (Gal, synaptotagmin 1 (Syt1, growth factor receptor bound protein 2 (Grb2, actin gamma 2 (Actg2 and smooth muscle alpha actin (Acta2, as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

  10. Molecular Cloning and Characterization of a Novel Gene Involved in Fatty Acid Synthesis in Brassica napus L.

    Institute of Scientific and Technical Information of China (English)

    XIAO Gang; ZHANG Zhen-qian; LIU Rui-yang; YIN Chang-fa; WU Xian-meng; TAN Tai-long; GUAN Chun-yun

    2013-01-01

    Based on the sequence of a novel expressed sequence tag (EST), the full-length cDNA of 1 017 nucleotides was cloned from Brassica napus cv. Xiangyou 15 through rapid amplification of cDNA ends (RACE). The gene was designated as Bnhol34 (HQ585980), encoding a protein of 338 amino acids. BLAST analysis showed no high degree of sequence identity to any known gene. The calculated molecular weight of the Bnhol34 protein was 36.23 kDa, and the theoretical isoelectric point was 8.74. The Bnhol34 was also cloned from a high oleic acid mutant 854-1 through homologous cloning. There was no difference between the two Bnhol34 genes. Bnhol34 was localized in a tissue-specific manner in B. napus, and its expression level was about eight-fold greater in Xiangyou 15 seeds than in 854-1. The promoter region sequences of Bnhol34 were then isolated from Xiangyou 15 and 854-1, and a 93-bp deletion was found to occur in the Bnhol34 promoter region of 854-1. Three abscisic acid-responsive cis-elements (ABRE) were identified in the promoter region of Xiangyou 15. Real-time PCR analyses revealed that exogenous abscisic acid increased Bnhol34 expression by about four-fold in Xiangyou 15 seeds, yet did not change Bnhol34 expression in 854-1. It appeared that Bnhol34 might be abscisic acid insensitive in 854-1.

  11. Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

    Science.gov (United States)

    Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

    2010-07-01

    A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

  12. Characterization of the putative amino acid transporter genes AtCAT2, 3 &4: the tonoplast localized AtCAT2 regulates soluble leaf amino acids.

    Science.gov (United States)

    Yang, Huaiyu; Krebs, Melanie; Stierhof, York-Dieter; Ludewig, Uwe

    2014-05-01

    The plant vacuole constitutes a large transient storage compartment for nutrients, proteins and metabolites, and is a major cellular sink for toxic waste compounds. Amino acids can cross the vacuolar membrane via specific transport proteins, which are molecularly not well characterized. Two members of a small subfamily of the cationic amino acid transporters, AtCAT2 and AtCAT4, were primarily localized at the tonoplast when tagged with GFP. The closely related AtCAT3, by contrast, was detected in the endoplasmic reticulum membrane. The exchange of a di-acidic motif at the carboxy-tail affected their sub-cellular localization, with larger effects visible in transiently transformed protoplasts compared to stably expressing plant lines. The genes have broad, partially overlapping tissue expression, with CAT2 dominating in most tissues. Loss-of-function mutants of individual CATs showed no visible phenotype under various conditions, but the overall tissue concentration of amino acids was increased in soil-grown cat2 mutants. The data suggest that CAT2 is a critical target of leaf amino acid concentrations and manipulation of this tonoplast transporter can significantly alter total tissue amino acid concentrations.

  13. Identification of a novel C22-∆4-producing docosahexaenoic acid (DHA) specific polyunsaturated fatty acid desaturase gene from Isochrysis galbana and its expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shi, Tonglei; Yu, Aiqun; Li, Ming; Ou, Xiuyuan; Xing, Laijun; Li, Mingchun

    2012-12-01

    Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-∆4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ∆4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomyces cerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ∆4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-∆4-PUFAs-specific desaturase activity.

  14. PPARα signal pathway gene expression is associated with fatty acid content in yak and cattle longissimus dorsi muscle.

    Science.gov (United States)

    Qin, W; Liang, C N; Guo, X; Chu, M; Pei, J; Bao, P J; Wu, X Y; Li, T K; Yan, P

    2015-11-19

    Intramuscular fatty acid (FA) is related to meat qualities such as juiciness, tenderness, palatability, and shear force. PPARα plays an important role in lipid metabolism in the liver and skeletal muscle. This study investigated FA composition in yaks and cattle, in order to ascertain whether a correlation between PPARα signal pathway genes as candidate genes and meat FA composition in yaks and cattle exists. Statistical analyses revealed that levels of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in yaks were significantly higher than those in cattle (P cattle (P cattle. However, LPL expression in yaks was significantly higher than that in cattle (P cattle, the mRNA level of PLTP was positively correlated with SFA (P meat quality.

  15. Synthesis and evaluation of a glutamic acid-modified hPAMAM complex as a promising versatile gene carrier.

    Science.gov (United States)

    Hemmati, Mohammad; Kazemi, Bahram; Najafi, Farhood; Zarebkohan, Amir; Shirkoohi, Reza

    2016-01-01

    Hyperbranched poly(amidoamine) (HPAMAM), structurally analogous to polyamidoamine dendrimer (PAMAM) dendrimers, has been suggested to be an effective carrier for gene delivery. In the present study, glutamic acid-modified hPAMAM was developed as a novel non-viral gene carrier for the first time. The hPAMAM was synthesized by using a modified one-pot method. DNA was found to be bound to hPAMAM at different weight ratios (WhPAMAM/WDNA). The resulting HPAMAM-Glu20 was able to efficiently protect the encapsulated-DNA against degradation for over 2 h. In addition to low cytotoxicity, the transfection efficiency of hPAMAM-Glu20 represented much higher (p glutamic amino acid (Glu)-based gene delivery is an economical, effective and biocompatible method.

  16. Serum uric acid levels are associated with polymorphism in the SAA1 gene in Chinese subjects.

    Directory of Open Access Journals (Sweden)

    Xiang Xie

    Full Text Available OBJECTIVE: Serum uric acid (SUA is a cardiovascular risk marker associated with inflammation. The serum amyloid A protein (SAA is an inflammatory factor and is associated with cardiovascular disease (CVD. However, the relationship between genetic polymorphisms of SAA and SUA levels has not been studied. The objective of this study was to investigate the association between SUA levels and SAA genetic polymorphisms. METHODS: All participants were selected from subjects participating in the Cardiovascular Risk Survey (CRS study. The single nucleotide polymorphism (SNP rs12218 of the SAA1 gene was genotyped by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. The association of SUA levels with genotypes was assessed by using the general liner mode. RESULTS: The SNP rs12218 was associated with SUA levels by analyses of a dominate model (P = 0.002 and additive model (P = 0.005, and the difference remained significant after adjustment of sex, age, obesity, ethnicity, HDL-C, alcohol intake, smoking, and creatinine (P = 0.006 and P = 0.023, respectively. The TT genotype was associated with an increased SUA concentration of 39.34 mmol/L (95% confidence interval [CI], 3.61-75.06, P = 0.031 compared with the CC genotype, and the TT genotype was associated with an increased SUA concentration of 2.48 mmol/L (95% CI, 6.86-38.10; P = 0.005 compared with the CT genotype. CONCLUSIONS: The rs12218 SNP in the SAA1 gene was associated with SUA levels in Chinese subjects, indicating that carriers of the T allele of rs12218 have a high risk of hyperuricemia.

  17. Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

    Science.gov (United States)

    Yang, Xiaohong; Liu, Shaojie; Li, Siming; Wang, Pengzhen; Zhu, Weicong; Liang, Peihong; Tan, Jianrong; Cui, Shuliang

    2017-02-28

    Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule β-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

  18. Omega-3 Fatty Acid Enriched Chevon (Goat Meat Lowers Plasma Cholesterol Levels and Alters Gene Expressions in Rats

    Directory of Open Access Journals (Sweden)

    Mahdi Ebrahimi

    2014-01-01

    Full Text Available In this study, control chevon (goat meat and omega-3 fatty acid enriched chevon were obtained from goats fed a 50% oil palm frond diet and commercial goat concentrate for 100 days, respectively. Goats fed the 50% oil palm frond diet contained high amounts of α-linolenic acid (ALA in their meat compared to goats fed the control diet. The chevon was then used to prepare two types of pellets (control or enriched chevon that were then fed to twenty-male-four-month-old Sprague-Dawley rats (n=10 in each group for 12 weeks to evaluate their effects on plasma cholesterol levels, tissue fatty acids, and gene expression. There was a significant increase in ALA and docosahexaenoic acid (DHA in the muscle tissues and liver of the rats fed the enriched chevon compared with the control group. Plasma cholesterol also decreased (P<0.05 in rats fed the enriched chevon compared to the control group. The rat pellets containing enriched chevon significantly upregulated the key transcription factor PPAR-γ and downregulated SREBP-1c expression relative to the control group. The results showed that the omega-3 fatty acid enriched chevon increased the omega-3 fatty acids in the rat tissues and altered PPAR-γ and SREBP-1c genes expression.

  19. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin.

    Science.gov (United States)

    Liu, Xiao-Yan; Chi, Zhe; Liu, Guang-Lei; Madzak, Catherine; Chi, Zhen-Ming

    2013-02-01

    In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

  20. Transcriptomic profiling of linolenic acid-responsive genes in ROS signalling from RNA-seq data in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Capilla eMata-Pérez

    2015-03-01

    Full Text Available Linolenic acid (Ln released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA. The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signalling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2 fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs and Allene oxide cyclases (AOCs. In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding, such as WRKY, JAZ, MYC and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signalling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1, methionine sulfoxide reductase (MSR and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signalling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity and especially in stresses mediated by ROS.

  1. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    Science.gov (United States)

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  2. 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.

  3. A new hypotensive polyunsaturated fatty acid dietary combination regulates oleic acid accumulation by suppression of stearoyl CoA desaturase 1 gene expression in the SHR model of genetic hypertension

    NARCIS (Netherlands)

    Bellenger, J.; Bellenger, S.; Clement, L.; Mandard, S.J.; Diot, C.; Poisson, J.P.; Narce, M.

    2004-01-01

    Polyunsaturated fatty acids (PUFA) are known to repress SCD-1 gene expression, key enzyme of monounsaturated fatty acid biosynthesis. Alterations of the monounsaturated/saturated fatty acids ratio have been implicated in various diseases related to the metabolic syndrome, including hypertension. We

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

    Science.gov (United States)

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

    2013-01-01

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

  5. Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

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    Qin Lan

    2011-12-01

    Full Text Available Abstract Background The nonstructural protein 1 (NSP1 of rotavirus has been reported to block interferon (IFN signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs and (or the β-transducin repeat containing protein (β-TrCP. However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. Results The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I, but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS, indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. Conclusions Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

  6. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

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    Yunwon Moon

    Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

  7. Effects of dietary methionine on feed utilization, plasma amino acid profiles and gene expression in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Rolland, Marine

    performances. The reason for this is often suggested to be related to difference in amino acid uptake kinetics during digestion, resulting in a temporal mismatch in amino acid availability, resulting in poorer at protein synthesis site. In addition to their role as building blocks in protein synthesis, amino...... acids also serve as substrates for synthesis of metabolic intermediates, and increasing evidence shows that amino acids also function as signaling factors in the regulation of intermediary metabolism and growth related pathways. The present thesis comprises four supporting papers, based on two...... (free or bound) on hepatic intermediary metabolism using the same diets as in Paper III. The diets were fed to rainbow trout for 6 weeks, followed by sampling for i) hepatic gene expressions, ii) hepatosomatic index (HSI), iii) postprandial ammonia excretion, and iv) plasma methionine levels...

  8. Polymorphisms in Fatty Acid Desaturase (FADS Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA Supplementation

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    Patrick Couture

    2013-09-01

    Full Text Available Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM. Polymorphisms (SNPs in the fatty acid desaturase (FADS gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS and serum fatty acid composition. Objective: To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG and fasting insulin (FI responses following a 6-week n-3 polyunsaturated fatty acids (PUFA supplementation. Methods: 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g–2.2 g of EPA + 1.1 g of DHA. Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03 was performed. Results: Carriers of the minor allele for rs482548 (FADS2 had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008. For FI levels, a genotype effect was observed with one SNP (rs174456. For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively. Conclusion: Results suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation.

  9. Association with amino acids does not enhance efficacy of polymerized liposomes as a system for lung gene delivery

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    Elga eBernardo Bandeira De Melo

    2016-04-01

    Full Text Available Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids (1,2-bis-(tricosa-10,12-diynoyl-sn-glycero-3-phosphocholine associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with L-arginine, L-tryptophan, or L-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. L-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases.

  10. Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

    Science.gov (United States)

    Schlotz, Nina; Roulin, Anne; Ebert, Dieter; Martin-Creuzburg, Dominik

    2016-11-01

    Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

  11. Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato.

    Science.gov (United States)

    Plant, A L; Cohen, A; Moses, M S; Bray, E A

    1991-11-01

    The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.

  12. Gene structure and amino acid sequence of Latimeria chalumnae (coelacanth) myelin DM20: phylogenetic relation of the fish.

    Science.gov (United States)

    Tohyama, Y; Kasama-Yoshida, H; Sakuma, M; Kobayashi, Y; Cao, Y; Hasegawa, M; Kojima, H; Tamai, Y; Tanokura, M; Kurihara, T

    1999-07-01

    The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2-7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941-1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

  13. Antisense 2'-Deoxy, 2'-Fluoroarabino Nucleic Acid (2'F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids.

    Science.gov (United States)

    Souleimanian, Naira; Deleavey, Glen F; Soifer, Harris; Wang, Sijian; Tiemann, Katrin; Damha, Masad J; Stein, Cy A

    2012-01-01

    Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2'-deoxy-2'fluoroarabino nucleic acid (2'F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2'F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2'F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded oligos in vitro.

  14. Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression.

    Science.gov (United States)

    Yang, Yishan; Khoo, Wei Jie; Zheng, Qianwang; Chung, Hyun-Jung; Yuk, Hyun-Gyun

    2014-02-17

    The influence of growth temperature (10, 25, 37, and 42 °C) on the survival of Salmonella Enteritidis in simulated gastric fluid (SGF; pH=2.0) and during heat treatment (54, 56, 58, and 60 °C), on the membrane fatty acid composition, as well as on stress-/virulence-related gene expression was studied. Cells incubated at temperatures lower or higher than 37 °C did not increase their acid resistance, with the maximum D-value of 3.07 min in cells grown at 37 °C; while those incubated at higher temperature increased their heat resistance, with the maximum D60 °C-values of 1.4 min in cells grown at 42 °C. A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased. Compared to the control cells grown at 37 °C, the expression of rpoS was 16.5- and 14.4-fold higher in cells cultivated at 10 and 25 °C, respectively; while the expression of rpoH was 2.9-fold higher in those cultivated at 42 °C. The increased expression of stress response gene rpoH and the decreased ratio of unsaturated to saturated fatty acids correlated with the greater heat resistance of bacteria grown at 42 °C; while the decreased expression of stress response gene rpoS at 42 °C might contribute to the decrease in acid resistance. Virulence related genes-spvR, hilA, avrA-were induced in cells cultivated at 42 °C, except sefA which was induced in the control cells. This study indicates that environmental temperature may affect the virulence potential of S. Enteritidis, thus temperature should be well controlled during food storage.

  15. Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15

    NARCIS (Netherlands)

    A.H.M. Geurts van Kessel (Ad); H. de Leeuw (H.); E.J. Dekker (E.); J.M. Rijks (Jolianne); N. Spurr (N.); A.M. Ledbetter (Andrew M.); E. Kootwijk (E.); M.J. Vaessen (Marie-Josée)

    1991-01-01

    textabstractA human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17) (q22;q11

  16. An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

    Energy Technology Data Exchange (ETDEWEB)

    Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi; Horiuchi, Hiroyuki [Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657 (Japan); Ohta, Akinori, E-mail: aaohta@mail.ecc.u-tokyo.ac.jp [Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2010-11-26

    Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

  17. Polyunsaturated fatty acids reduce Fatty Acid Synthase and Hydroxy-Methyl-Glutaryl CoA-Reductase gene expression and promote apoptosis in HepG2 cell line

    Directory of Open Access Journals (Sweden)

    Miccolis Angelica

    2011-01-01

    Full Text Available Abstract Background n-3 and n-6 polyunsaturated fatty acids (PUFAs are the two major classes of PUFAs encountered in the diet, and both classes of fatty acids are required for normal human health. Moreover, PUFAs have effects on diverse pathological processes impacting chronic disease, such as cardiovascular and immune disease, neurological disease, and cancer. Aim To investigate the effects of eicosapentaenoic acid (EPA and arachidonic acid (ARA on the proliferation and apoptosis of human hepatoma cell line HepG2 after exposure to increasing concentrations of EPA or ARA for 48 h. Moreover, in the same cells the gene expression of Fatty Acid Synthase (FAS and 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase (HMG-CoAR was also investigated. Method Cell growth and apoptosis were assayed by MTT and ELISA test, respectively after cell exposure to increasing concentrations of EPA and ARA. Reverse-transcription and real-time PCR was used to detect FAS and HMG-CoAR mRNA levels in treated cells. Results Our findings show that EPA inhibits HepG2 cell growth in a dose-dependent manner, starting from 25 μM (P Conclusion Our results demonstrate that EPA and ARA inhibit HepG2 cell proliferation and induce apoptosis. The down-regulation of FAS and HMG-CoAR gene expression by EPA and ARA might be one of the mechanisms for the anti-proliferative properties of PUFAs in an in vitro model of hepatocellular carcinoma.

  18. Bile Acids Function Synergistically To Repress Invasion Gene Expression in Salmonella by Destabilizing the Invasion Regulator HilD.

    Science.gov (United States)

    Eade, Colleen R; Hung, Chien-Che; Bullard, Brian; Gonzalez-Escobedo, Geoffrey; Gunn, John S; Altier, Craig

    2016-08-01

    Salmonella spp. are carried by and can acutely infect agricultural animals and humans. After ingestion, salmonellae traverse the upper digestive tract and initiate tissue invasion of the distal ileum, a virulence process carried out by the type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). Salmonellae coordinate SPI-1 expression with anatomical location via environmental cues, one of which is bile, a complex digestive fluid that causes potent repression of SPI-1 genes. The individual components of bile responsible for SPI-1 repression have not been previously characterized, nor have the bacterial signaling processes that modulate their effects been determined. Here, we characterize the mechanism by which bile represses SPI-1 expression. Individual bile acids exhibit repressive activity on SPI-1-regulated genes that requires neither passive diffusion nor OmpF-mediated entry. By using genetic methods, the effects of bile and bile acids were shown to require the invasion gene transcriptional activator hilD and to function independently of known upstream signaling pathways. Protein analysis techniques showed that SPI-1 repression by bile acids is mediated by posttranslational destabilization of HilD. Finally, we found that bile acids function synergistically to achieve the overall repressive activity of bile. These studies demonstrate a common mechanism by which diverse environmental cues (e.g., certain short-chain fatty acids and bile acids) inhibit SPI-1 expression. These data provide information relevant to Salmonella pathogenesis during acute infection in the intestine and during chronic infection of the gallbladder and inform the basis for development of therapeutics to inhibit invasion as a means of repressing Salmonella pathogenicity.

  19. Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines.

    Science.gov (United States)

    Park, Youngki; Carr, Timothy P

    2013-02-01

    Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.

  20. BASIC AMINO ACID CARRIER 2 gene expression modulates arginine and urea content and stress recovery in Arabidopsis leaves.

    Directory of Open Access Journals (Sweden)

    Séverine ePlanchais

    2014-07-01

    Full Text Available In plants, basic amino acids are important for the synthesis of proteins and signaling molecules and for nitrogen recycling. The Arabidopsis nuclear gene BASIC AMINO ACID CARRIER 2 (BAC2 encodes a mitochondria-located carrier that transports basic amino acids in vitro. We present here an analysis of the physiological and genetic function of BAC2 in planta. When BAC2 is overexpressed in vivo, it triggers catabolism of arginine, a basic amino acid, leading to arginine depletion and urea accumulation in leaves. BAC2 expression was known to be strongly induced by stress. We found that compared to wild type plants, bac2 null mutants (bac2-1 recover poorly from hyperosmotic stress when restarting leaf expansion. The bac2-1 transcriptome differs from the wild-type transcriptome in control conditions and under hyperosmotic stress. The expression of genes encoding stress-related transcription factors, arginine metabolism enzymes, and transporters is particularly disturbed in bac2-1, and in control conditions, the bac2-1 transcriptome has some hallmarks of a wild-type stress transcriptome. The BAC2 carrier is therefore involved in controlling the balance of arginine and arginine-derived metabolites and its associated amino acid metabolism is physiologically important in equipping plants to respond to and recover from stress.

  1. Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

    Science.gov (United States)

    Liu, Xiang-Lei; Lin, Jun; Hu, Hai-Feng; Zhou, Bin; Zhu, Bao-Quan

    2016-04-01

    Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

  2. Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Valdiglesias Vanessa

    2012-01-01

    Full Text Available Abstract Background Okadaic acid (OA, a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature. Results In this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h. A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure, excepting for NEFM, but their expression was similar to the controls at 48 h. Conclusions From all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.

  3. Expression Analysis of Phenylalanine Ammonia Lyase Gene and Rosmarinic Acid Production in Salvia officinalis and Salvia virgata Shoots Under Salicylic Acid Elicitation.

    Science.gov (United States)

    Ejtahed, Roghayeh Sadat; Radjabian, Tayebeh; Hoseini Tafreshi, Sayed Ali

    2015-08-01

    Partial fragments of phenylalanine ammonia lyase (PAL) genes were cloned and characterized from Salvia officinalis (SoPAL) and Salvia virgata (SvPAL). Different concentrations (250 and 500 μM) of exogenous salicylic acid (SA) were used when correlation between PAL expression and rosmarinic acid (RA) accumulation was compared. The results showed that the deduced cDNA sequences of the partial genes had high similarities with those of known PAL gene from other plant species. Semi-quantitative reverse transcription PCR (RT-PCR) analysis revealed that exogenous application of SA led to up-regulating of the PAL expression. Further analysis showed that in S. virgata, at higher concentration of SA, higher accumulation of RA was achieved, while in S. officinalis, the higher RA accumulation was observed at lower concentration of SA. It was concluded that there was no positive correlation between the intensity of PAL transcription and the RA accumulation in the studied species. Therefore, despite of the increase in transcription rate of the PAL at the higher concentration of SA, the lower amounts of RA were accumulated in the case of S. officinalis. Consequently, the hypothesis that PAL is the rate-determining step in RA biosynthesis is not always valid and probably some other unknown factors participate in the synthesis of phenolics.

  4. Structure and gene cluster of the O-antigen of Escherichia coli O156 containing a pyruvic acid acetal.

    Science.gov (United States)

    Duan, Zhifeng; Senchenkova, Sof'ya N; Guo, Xi; Perepelov, Andrei V; Shashkov, Alexander S; Liu, Bin; Knirel, Yuriy A

    2016-07-22

    The lipopolysaccharide of Escherichia coli O156 was degraded under mild acidic and alkaline conditions and the resulting polysaccharides were studied by sugar analysis and (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: where Rpyr indicates R-configurated pyruvic acid acetal. Minor O-acetyl groups also were present and tentatively localized on the Gal residues. The gene cluster for biosynthesis of the O-antigen of E. coli O156 was analyzed and shown to be consistent with the O-polysaccharide structure.

  5. D-amino acid oxidase activator gene (DAOA) variation affects cerebrospinal fluid homovanillic acid concentrations in healthy Caucasians

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas;

    2012-01-01

    3918342 and rs1421292, were significantly associated with CSF HVA concentrations. Rs3918342 was found to be nominally associated with CSF 5-HIAA concentrations. None of the polymorphisms were significantly associated with MHPG concentrations. Our results indicate that DAOA gene variation affects dopamine...

  6. Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Peng Li

    2013-11-01

    Full Text Available Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL, carnitine palmitoyltransferase IA (CPT IA, long chain acyl-CoA dehydrogenase (ACADL, and acetyl-CoA carboxylase (ACC. Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

  7. Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids.

    Science.gov (United States)

    Montesinos, M L; Herrero, A; Flores, E

    1997-02-01

    The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.

  8. TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

    Science.gov (United States)

    Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

    2014-04-01

    Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.

  9. A nonsense mutation in the acid α-glucosidase gene causes Pompe disease in Finnish and Swedish Lapphunds.

    Science.gov (United States)

    Seppälä, Eija H; Reuser, Arnold J J; Lohi, Hannes

    2013-01-01

    Pompe disease is a recessively inherited and often fatal disorder caused by the deficiency of acid α-glucosidase, an enzyme encoded by the GAA gene and needed to break down glycogen in lysosomes. This glycogen storage disease type II has been reported also in Swedish Lapphund dogs. Here we describe the genetic defect in canine Pompe disease and show that three related breeds from Scandinavia carry the same mutation. The affected dogs are homozygous for the GAA c.2237G>A mutation leading to a premature stop codon at amino acid position 746. The corresponding mutation has previously been reported in humans and causes infantile Pompe disease in combination with a second fully deleterious mutation. The affected dogs from both the Finnish as well as the Swedish breed mimic infantile-onset Pompe disease genetically, but also clinico-pathologically. Therefore this canine model provides a valuable tool for preclinical studies aimed at the development of gene therapy in Pompe disease.

  10. Influence of dietary docosahexaenoic acid in combination with other long-chain polyunsaturated fatty acids on expression of biosynthesis genes and phospholipid fatty acid compositions in tissues of post-smolt Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Betancor, Mónica B; Howarth, Fraser J E; Glencross, Brett D; Tocher, Douglas R

    2014-01-01

    To investigate interactions of dietary LC-PUFA, a dose-response study with a range of docosahexaenoic acid (DHA; 22:6n-3) levels (1 g kg(-1), 5 g kg(-1), 10 g kg(-1), 15 g kg(-1) and 20 g kg(-1)) was performed with post-smolts (111 ± 2.6g; mean ± S.D.) over a nine-week feeding period. Additional diets included 10 g kg(-1) DHA in combination with 10 g kg(-1) of either eicosapentaenoic acid (EPA; 20:5n-3) or arachidonic acid (ARA; 20:4n-6), and a diet containing 5 g kg(-1) each of DHA and EPA. The liver, brain, head kidney and gill were collected at the conclusion of the trial, and lipid and fatty acid compositions were determined as well as expression of genes of LC-PUFA biosynthesis. Total lipid content and class composition were largely unaffected by changes in dietary LC-PUFA. However, phospholipid (PL) fatty acid compositions generally reflected that of the diet, although the response varied between tissues. The liver most strongly reflected diet, followed by the head kidney. In both tissues increasing dietary DHA led to significantly increased DHA in PL and inclusion of EPA or ARA led to higher levels of these fatty acids. The brain showed the most conserved composition and gene expression profile, with increased dietary LC-PUFA resulting in only minor changes in PL fatty acids. Dietary LC-PUFA significantly affected the expression of Δ6 and Δ5 desaturases, Elovl 2, 4 and 5, and SREBPs although this varied between tissues with greatest effects observed in the liver followed by the head kidney, similar to PL fatty acid compositions.

  11. Effects of rice bran on performance, egg quality, oxidative status, yolk fatty acid composition, and fatty acid metabolism-related gene expression in laying ducks.

    Science.gov (United States)

    Ruan, D; Lin, Y C; Chen, W; Wang, S; Xia, W G; Fouad, A M; Zheng, C T

    2015-12-01

    The study was designed to evaluate the effects of different dietary levels of rice bran (RB) in laying duck diets on performance, egg quality, oxidation status, egg yolk fatty acid composition, and hepatic expression of fatty acid metabolism-related genes. Longyan females (1080) with similar BW at 19 wk of age were randomly assigned to 6 dietary treatments, each consisting of 6 replicates of 30 birds. The basal diet (I) was a typical corn-soybean ration while the experimental diets (II to VI) substituted RB for corn and wheat bran and a small reduction of soybean meal. The level of substitution in diets (II to VI) was 6%, 12%, 18%, 24%, and 30%, respectively. The experiment lasted for 12 wks. Average egg weight and daily egg mass decreased linearly as the level of RB inclusion increased (Pegg yolk linearly decreased with increasing RB, and many of the key polyunsaturated fatty acids (PUFA), like C18:2 n-6 and C18:3 n-3, linearly increased (Pegg yolk cholesterol or triglyceride content (P>0.05). In conclusion, the current study suggests that ducks from 19 to 31 wk could be fed diets with up to about 18% RB without effect on the number of eggs produced, egg quality, and oxidative status. Increasing amounts of RB linearly increased egg yolk concentrations of key fatty acids like C18:2 n-6 and C18:3 n-3 and decreased the hepatic abundance of FAS and SREBP-1 transcripts.

  12. Effects of fatty acid regulation on visfatin gene expression in adipocytes

    Institute of Scientific and Technical Information of China (English)

    WEN Yu; WANG Hong-wei; WU Jing; LU Hui-ling; HU Xiu-fen; Katherine Cianflone

    2006-01-01

    Background The levels of long-term elevated serum or intracellular free fatty acid (FFA) induce insulin resistance associated with central obesity. The insulin-mimetic protein visfatin is preferentially produced by visceral adipose tissues and has been implicated in obesity and insulin resistance. To identify that FFA is capable of inducing insulin resistance and to clarify the role of FFA on visfatin, we examined the effect of monounsaturated FFA oleate (C18:1) and saturated FFA palmitate (C16:0) on glucose transport and visfatin gene expression in cultured 3T3-L1 adipocytes or preadipocytes.Methods FFA-free DMEM/F12, 0.125 mmol/L, 0.5 mmol/1 and 1.0 mmol/L oleate or palmitate was added to cultured 3T3-L1 adipocytes or preadipocytes and incubated overnight. Glucose transport was assessed as 3H-2-deoxy-glucose uptake. Total RNA was extracted and subjected to RT-PCR for the measurement of visfatin mRNA levels. Statistical comparisons between control group and other groups were performed with the two-tailed paired t test, and one-way ANOVA was used to compare the mean values among the groups.Results Insulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. Upregulation was evident from 15 minutes to 1 hour exposure to insulin. However, after 6-hour exposure to insulin, there was a downregulation in the response to insulin. Dose response studies demonstrated that 2-deoxy glucose transport was increased by 336% at 50 nmol/L insulin (P<0.01), and reached a maximal effect at 100 nmol/L insulin(P<0.01). Oleate and palmitate treatment did not influence basal glucose transport (without insulin stimulation),whereas insulin-stimulated glucose transport was inhibited after overnight oleate and palmitate treatment in preadipocytes and adipocytes. In 3T3-L1 preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P<0.05, respectively), and the inhibition was dose dependent. In adipocytes, the inhibition was noted at 0

  13. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  14. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  15. UV-C-Induced alleviation of transcriptional gene silencing through plant–plant communication: Key roles of jasmonic acid and salicylic acid pathways

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Wei; Wang, Ting [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Xu, Shaoxin [School of physics and materials science, Anhui University, Hefei, Anhui, 230601 (China); Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China); Bian, Po, E-mail: bianpo@ipp.ac.cn [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Hefei, Anhui, 230031 (China)

    2016-08-15

    Highlights: • Transcriptional gene silencing (TGS) in plants can be epigenetically alleviated by volatile signals from UV-C- irradiated neighboring plants. • Alleviation of TGS can be induced by UV-C irradiation through plant–plant–plant communication. • JA and SA signals take part in interplant communication for alleviation of TGS. - Abstract: Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant–plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant–plant and plant–plant–plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant–plant and plant–plant–plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant–plant–plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA.

  16. Dietary energy source largely affects tissue fatty acid composition but has minor influence on gene transcription in Iberian pigs.

    Science.gov (United States)

    Óvilo, C; Benítez, R; Fernández, A; Isabel, B; Núñez, Y; Fernández, A I; Rodríguez, C; Daza, A; Silió, L; López-Bote, C

    2014-03-01

    A trial was performed to compare the effects of different dietary sources of MUFA on the fatty acid (FA) composition, lipid metabolism, and gene transcription in different tissues of Iberian pigs. Twenty-seven Iberian male pigs of 28 kg live weight (LW) were divided in 2 groups and fed with 1 of 2 isocaloric diets: a standard diet with carbohydrates as energy source (CH) and a diet enriched with high-oleic sunflower oil (HO). Ham adipose tissue was sampled by biopsy at 44 and 70 kg LW. At 110 kg LW pigs were slaughtered and backfat, loin, and liver tissues were sampled. Animals of the HO group showed higher MUFA content and lower SFA in all the analyzed tissues (P dietary groups (PP < 0.01), 37 genes were considered differentially expressed (DE). Gene ontology allowed relating them with several biological functions including lipid metabolic processes. Quantitative PCR confirmed several DE genes in adipose tissue (RXRG, LEP, and ME1; P < 0.0001, P < 0.05, and P < 0.0001, respectively), but no DE gene was found in loin or liver tissues. Joint results agree with a metabolic adjustment of adipose tissue FA levels by the subtle effect of the diet on the regulation of several lipid metabolism pathways, mainly FA oxidation and prostanoid synthesis, with LEP, RXRG, and PTGS2 genes playing mayor roles.

  17. A novel plasmid for delivering genes into mammalian cells with noninvasive food and commensal lactic acid bacteria.

    Science.gov (United States)

    Tao, Lin; Pavlova, Sylvia I; Ji, Xin; Jin, Ling; Spear, Gregory

    2011-01-01

    Using food and commensal lactic acid bacteria (LAB) as vehicles for DNA delivery into epithelial cells is a new strategy for vaccine delivery or gene therapy. However, present methods for DNA delivery with LAB have suffered low efficiency. Our goal was to develop a new system to deliver DNA into epithelial cells with high efficiency using food and commensal LAB. An Escherichia coli-LAB shuttle plasmid, pLKV1, for DNA delivery into eukaryotic cells was constructed. Two reporter plasmids with green and red fluorescent protein genes were also constructed to monitor the uptake of protein and DNA, respectively. Bacteria delivering these reporter plasmids into Caco-2 cells were monitored by fluorescence microscopy. Several methods that weaken the bacterial cell wall prior to co-culture with Caco-2 cells were evaluated for their role in the improvement of gene transfer efficiency. Treating Streptococcus gordonii with penicillin and lysozyme greatly increased its rate of gene delivery to mammalian cells compared to untreated control bacteria, while glycine pretreatment promoted the highest gene transfer rate for Lactococcus lactis. Uptake of green fluorescent bacteria by Caco-2 cells showed that the cell wall-weakening treatment promoted the internalization of the noninvasive bacteria into Caco-2 cells. In conclusion, we have developed a noninvasive system using LAB as a vehicle for vaccine delivery or gene therapy, and tested this system in vitro with Caco-2 cells.

  18. Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes.

    Science.gov (United States)

    Lee, Sunhee; Flores-Encarnación, M; Contreras-Zentella, M; Garcia-Flores, L; Escamilla, J E; Kennedy, Christina

    2004-08-01

    Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only approximately 6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA(-) phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.

  19. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    Energy Technology Data Exchange (ETDEWEB)

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  20. Adipose tissue transcriptional response of lipid metabolism genes in growing Iberian pigs fed oleic acid v. carbohydrate enriched diets.

    Science.gov (United States)

    Benítez, R; Núñez, Y; Fernández, A; Isabel, B; Rodríguez, C; Daza, A; López-Bote, C; Silió, L; Óvilo, C

    2016-06-01

    Diet influences animal body and tissue composition due to direct deposition and to the nutrients effects on metabolism. The influence of specific nutrients on the molecular regulation of lipogenesis is not well characterized and is known to be influenced by many factors including timing and physiological status. A trial was performed to study the effects of different dietary energy sources on lipogenic genes transcription in ham adipose tissue of Iberian pigs, at different growth periods and on feeding/fasting situations. A total of 27 Iberian male pigs of 28 kg BW were allocated to two separate groups and fed with different isocaloric feeding regimens: standard diet with carbohydrates as energy source (CH) or diet enriched with high oleic sunflower oil (HO). Ham subcutaneous adipose tissue was sampled by biopsy at growing (44 kg mean BW) and finishing (100 kg mean BW) periods. The first sampling was performed on fasted animals, while the last sampling was performed twice, with animals fasted overnight and 3 h after refeeding. Effects of diet, growth period and feeding/fasting status on gene expression were explored quantifying the expression of a panel of key genes implicated in lipogenesis and lipid metabolism processes. Quantitative PCR revealed several differentially expressed genes according to diet, with similar results at both timings: RXRG, LEP and FABP5 genes were upregulated in HO group while ME1, FASN, ACACA and ELOVL6 were upregulated in CH. The diet effect on ME1 gene expression was conditional on feeding/fasting status, with the higher ME1 gene expression in CH than HO groups, observed only in fasting samples. Results are compatible with a higher de novo endogenous synthesis of fatty acids (FA) in the carbohydrate-supplemented group and a higher FA transport in the oleic acid-supplemented group. Growth period significantly affected the expression of most of the studied genes, with all but PPARG showing higher expression in finishing pigs according to

  1. Self-assembled ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates for targeted gene delivery.

    Science.gov (United States)

    Liang, Kun; Bae, Ki Hyun; Lee, Fan; Xu, Keming; Chung, Joo Eun; Gao, Shu Jun; Kurisawa, Motoichi

    2016-03-28

    Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.

  2. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    Science.gov (United States)

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress.

  3. Chromosomal localization of a novel retinoic acid induced gene RA28 and the protein distribution of its encoded protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Gene RA28 is a retinoic acid induced novel gene isolated in our laboratory previously. All-trans retinoic acid (ATRA) was used to induce lung adenocarcinoma cell line GLC-82, and RA28 was obtained by subtractive hybridization. Green fluorescent protein (GFP) has emerged as a unique tool for examining introcellular phenomena in living cells. GFP possesses an intrinsic fluorescence at 488 nm that does not require other co-factors. In this report, an eukaryotic expression plasmid pEGFP-C1-RA28 was constructed and transfected with parental cell line GLC-82 to analyze protein expression and its distribution in living cells. Moreover, radiation hybrid (RH) technique was used to localize RA28 to the chromosome. The results show that gene RA28 is mapped to the chromosome 19q13.1 region, its encoded protein is distributed on cell membrane. All the results further demonstrate that GFP and RH techniques are accurate, fast, repetitive, and will be powerful methods for investigating the gene and protein localization.

  4. A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI.

    Science.gov (United States)

    Takala, T M; Saris, P E J

    2002-08-01

    A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.

  5. Expression of genes associated with the biosynthetic pathways of abscisic acid, gibberellin, and ethylene during the germination of lettuce seeds.

    Science.gov (United States)

    Clemente, A C S; Guimarães, R M; Martins, D C; Gomes, L A A; Caixeta, F; Reis, R G E; Rosa, S D V F

    2015-01-01

    Seed germination and dormancy are complex phenomena that are controlled by many genes and environmental factors. Such genes are indicated by phytohormones that interact with each other, and may cause dormancy or promote seed germination. The objective of this study was to investigate gene expression associated with the biosynthetic pathways of abscisic acid (ABA), gibberellic acid (GA), and ethylene (ET) in dormant and germinated lettuce seeds. The expressions of LsNCED, LsGA3ox1, and ACO-B were evaluated in germinating and dormant seeds from the cultivars Everglades, Babá de Verão, Verônica, Salinas, Colorado, and Regina 71. The expressions of LsNCED, LsGA3ox1, and ACO-B were related to the biosynthesis of ABA, GA, and ET, respectively; therefore, the presence of these substances depends on genotype. LsNCED expression only occurred in dormant seeds, and was connected to dormancy. LsGA3ox1expression only occurred in germinated seeds, and was connected to germination. The ACO-B gene was involved in ET biosynthesis, and was expressed differently in germinated and dormant seeds, depending on the genotype, indicating different functions for different characteristics. Furthermore, sensitivity to phytohormones appeared to be more important than the expression levels of LsNCED, LsGA3ox1, or ACO-B.

  6. Dysbindin and d-amino-acid-oxidase gene polymorphisms associated with positive and negative symptoms in schizophrenia

    DEFF Research Database (Denmark)

    Wirgenes, Katrine V; Djurovic, Srdjan; Agartz, Ingrid;

    2009-01-01

    BACKGROUND: Schizophrenia is a genetically complex disorder with an unknown pathophysiology. Several genes implicated in glutamate metabolism have been associated with the disorder. Recent studies of polymorphisms in the dystrobrevin-binding protein 1 gene (DTNBP1; dysbindin) and D......-amino-acid-oxidase (DAO) gene, both involved in glutamate receptor function, reported associations with negative symptoms and with anxiety and depression, respectively, when measured with the Positive and Negative Syndrome Scale (PANSS). METHODS: In the present study, the suggested association between dysbindin and DAO...... single nucleotide polymorphisms (SNPs) and PANSS scores was analyzed in 155 Norwegian schizophrenia patients. RESULTS: There was a significant association between the dysbindin SNP rs3213207 and severity of both negative symptoms and total symptom load, as well as between the DAO SNP rs2070587 and total...

  7. Genetic variation in toll-like receptors and retinoic acid-inducible gene I and outcome of hepatitis C virus infection: a candidate gene association study

    DEFF Research Database (Denmark)

    Clausen Nygaard, Louise; Ladelund, S; Weis, N;

    2014-01-01

    with resolution in the discovery cohort were genotyped in a validation cohort. Multivariate logistic regression adjusted for sex, hepatitis B surface antigen, HIV infection and the interleukin-28B rs12979860 SNP was performed in the combined cohort. Haplotype reconstruction and linkage disequilibrium analysis......We evaluated the effects of genetic variation in toll-like receptors (TLR), retinoic acid-inducible gene I (RIG-I) and their signalling pathways on spontaneous hepatitis C virus (HCV) resolution. We screened 95 single-nucleotide polymorphisms (SNPs) in 22 genes. SNPs significantly associated...... were performed. srs2233437, rs730775 and rs28362857 in Inhibitor of NF-kB ε (IkBε) and rs352140 in TLR9 were associated with spontaneous HCV resolution (P ≤ 0.05) in the discovery cohort (n = 308). In the validation cohort (n = 216), we replicated a significant association with HCV resolution for two...

  8. 5-lipoxygenase and 5-lipoxygenase-activating protein gene polymorphisms, dietary linoleic acid, and risk for breast cancer.

    Science.gov (United States)

    Wang, Jun; John, Esther M; Ingles, Sue Ann

    2008-10-01

    The n-6 polyunsaturated fatty acid 5-lipoxygenase pathway has been shown to play a role in the carcinogenesis of breast cancer. We conducted a population-based case-control study among Latina, African-American, and White women from the San Francisco Bay area to examine the association of the 5-lipoxygenase gene (ALOX5) and 5-lipoxygenase-activating protein gene (ALOX5AP) with breast cancer risk. Three ALOX5AP polymorphisms [poly(A) microsatellite, -4900 A>G (rs4076128), and -3472 A>G (rs4073259)] and three ALOX5 polymorphisms [Sp1-binding site (-GGGCGG-) variable number of tandem repeat polymorphism, -1279 G>T (rs6593482), and 760 G>A (rs2228065)] were genotyped in 802 cases and 888 controls. We did not find significant main effects of ALOX5 and ALOX5AP genotypes on breast cancer risk that were consistent across race or ethnicity; however, there was a significant interaction between the ALOX5AP -4900 A>G polymorphism and dietary linoleic acid intake (P=0.03). Among women consuming a diet high in linoleic acid (top quartile of intake, >17.4 g/d), carrying the AA genotype was associated with higher breast cancer risk (age- and race-adjusted odds ratio, 1.8; 95% confidence interval, 1.2-2.9) compared with carrying genotypes AG or GG. Among women consuming acid, ALOX5AP -4900 genotype was not associated with breast cancer risk (age- and race-adjusted odds ratio, 0.9; 95% confidence interval, 0.7-1.2). These results support a role for n-6 polyunsaturated fatty acids in breast carcinogenesis and suggest that epidemiologic studies on dietary fat and breast cancer should take into account genetic predisposition related to n-6 polyunsaturated fatty acid metabolism.

  9. Hexanoic acid and polyethylene glycol double grafted amphiphilic chitosan for enhanced gene delivery: influence of hydrophobic and hydrophilic substitution degree.

    Science.gov (United States)

    Layek, Buddhadev; Haldar, Manas K; Sharma, Gitanjali; Lipp, Lindsey; Mallik, Sanku; Singh, Jagdish

    2014-03-03

    Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. To overcome this difficulty a series of hexanoic acid (HA) and monomethoxy poly(ethylene glycol) (mPEG) double grafted chitosan-based (HPC) nanomicelles were developed as nonviral gene carrier. HPC polymers with various HA and mPEG substitution degrees were synthesized, and their chemical structures were confirmed by (1)H NMR spectroscopy. HPC nanomicelles exhibited excellent blood compatibility and cell viability, as demonstrated by in vitro hemolysis and MTT assay, respectively. The cationic HPC nanomicelles retained the plasmid DNA (pDNA) binding capacity of chitosan and formed stable HPC/pDNA polyplexes with diameters below 200 nm. Both hydrophobic and hydrophilic substitution resulted in suppressed nonspecific protein adsorption on HPC/pDNA polyplexes and increased pDNA dissociation. However, resistance against DNase I degradation was enhanced by HA conjugation while being inhibited by mPEG substitution. Amphiphilic modification resulted in 3-4.5-fold higher cellular uptake in human embryonic kidney 293 cells (HEK 293) mainly through clathrin-mediated pathway. The optimal HPC/pDNA polyplexes displayed 50-fold and 1.2-fold higher gene transfection compared to unmodified chitosan and Fugene, respectively, in HEK 293 cells. Moreover, both the cellular uptake and in vitro transfection study suggested a clear dependence of gene expression on the extent of HA and mPEG substitution. These findings demonstrate that amphiphilic HPC nanomicelles with the proper combination of HA and mPEG substitution could be used as a promising gene carrier for efficient gene therapy.

  10. Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    MI Haifeng; MAI Kangsen; ZHANG Wenbing; WU Chenglong; CAI Yinghua

    2011-01-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish.The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus).These genes are trypsinogen 1 (poTRY),elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY).Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00,0.40,0.80 and 1.50%),4 levels of PA (0.00,0.20,0.40 and 0.80),respectively.Japanese flounder (initial weight 2.45 g±0.01 g)were fed with these diets for 10 weeks with three replications per treatment.At the end of 10 weeks,supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression ofpoTRY and poCTRY (P<0.05).The same level of dietary SBS significantly decreased the gene expression of poEL.In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%)significantly decreased the gene expression ofpoTRY,poCTRY and poEL (P<0.05).However,excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05).These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder,and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  11. Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle.

    Science.gov (United States)

    Slaninova, Vera; Krafcikova, Michaela; Perez-Gomez, Raquel; Steffal, Pavel; Trantirek, Lukas; Bray, Sarah J; Krejci, Alena

    2016-02-01

    Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues.

  12. Transcriptional profiling of the PDR gene family in rice roots in response to plant growth regulators, redox perturbations and weak organic acid stresses.

    Science.gov (United States)

    Moons, Ann

    2008-12-01

    The role of plant pleiotropic drug resistance (PDR) type ATP-binding cassette (ABC) transporters remains poorly understood. We characterized the expression of the rice pleiotropic drug resistance (PDR) gene family in roots, where PDR transporters are believed to have major functions. A prototypical oligonucleotide array was developed containing 70-mers chosen in the gene-specific 3' untranslated regions of the rice PDR genes, other full-molecule rice ABC transporter genes and relevant marker genes. Jasmonates, which are involved in plant defense and secondary metabolism, proved major inducers of PDR gene expression. Over half of the PDR genes were JA-induced in roots of rice; OsPDR9 to the highest level. Salicylic acid, involved in plant pathogen defense, markedly induced the expression of OsPDR20. OsPDR20 was cDNA cloned and characterized. Abscisic acid, typically involved in water deficit responses, particularly induced OsPDR3 in roots and shoot and OsPDR6 in rice leaves. OsPDR9 and OsPDR20 were furthermore up-regulated in response to dithiothreitol- or glutathione-induced redox perturbations. Exogenous application of the weak organic acids lactic acid, malic acid, and citric acid differentially induced the expression of OsPDR3, OsPDR8, OsPDR9 and OsPDR20 in rice seedling roots. This transcriptional survey represents a guide for the further functional analysis of individual PDR transporters in roots of rice.

  13. Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

    OpenAIRE

    Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.; Scannapieco, Frank A.

    2012-01-01

    Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were di...

  14. Association analyses between brain-expressed fatty-acid binding protein (FABP) genes and schizophrenia and bipolar disorder.

    Science.gov (United States)

    Iwayama, Yoshimi; Hattori, Eiji; Maekawa, Motoko; Yamada, Kazuo; Toyota, Tomoko; Ohnishi, Tetsuo; Iwata, Yasuhide; Tsuchiya, Kenji J; Sugihara, Genichi; Kikuchi, Mitsuru; Hashimoto, Kenji; Iyo, Masaomi; Inada, Toshiya; Kunugi, Hiroshi; Ozaki, Norio; Iwata, Nakao; Nanko, Shinichiro; Iwamoto, Kazuya; Okazaki, Yuji; Kato, Tadafumi; Yoshikawa, Takeo

    2010-03-05

    Deficits in prepulse inhibition (PPI) are a biological marker for psychiatric illnesses such as schizophrenia and bipolar disorder. To unravel PPI-controlling mechanisms, we previously performed quantitative trait loci (QTL) analysis in mice, and identified Fabp7, that encodes a brain-type fatty acid binding protein (Fabp), as a causative gene. In that study, human FABP7 showed genetic association with schizophrenia. FABPs constitute a gene family, of which members FABP5 and FABP3 are also expressed in the brain. These FABP proteins are molecular chaperons for polyunsaturated fatty acids (PUFAs) such as arachidonic and docosahexaenoic acids. Additionally, the involvement of PUFAs has been documented in the pathophysiology of schizophrenia and mood disorders. Therefore in this study, we examined the genetic roles of FABP5 and 3 in schizophrenia (N = 1,900 in combination with controls) and FABP7, 5, and 3 in bipolar disorder (N = 1,762 in the case-control set). Three single nucleotide polymorphisms (SNPs) from FABP7 showed nominal association with bipolar disorder, and haplotypes of the same gene showed empirical associations with bipolar disorder even after correction of multiple testing. We could not perform association studies on FABP5, due to the lack of informative SNPs. FABP3 displayed no association with either disease. Each FABP is relatively small and it is assumed that there are multiple regulatory elements that control gene expression. Therefore, future identification of unknown regulatory elements will be necessary to make a more detailed analysis of their genetic contribution to mental illnesses.

  15. Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus, PTCC 1709

    Directory of Open Access Journals (Sweden)

    Hasan Korbekandi

    2011-01-01

    Full Text Available Objective: The clavulanic acid regulatory gene (claR is in the clavulanic acid biosyntheticgene cluster that encodes ClaR. This protein is a putative regulator of the late steps ofclavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR,isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus.Materials and Methods: In this experimental study, two different strains of S. clavuligeruswere used (PTCC 1705 and DSM 738, of which there is no claR sequence record forstrain PTCC 1705 in all three main gene banks. The specific designed primers were subjectedto a few base modifications for introduction of the recognition sites of BamHI andClaI. The claR gene was amplified by polymerase chain reaction (PCR using DNA isolatedfrom S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism(PCR-RFLP, and sequencing were used for molecular analysis of the claR gene.The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claRwas ligated into a pBluescript (pBs vector and transformed into E. coli.Results: The entire sequence of the isolated claR (Iranian strain was identified. Thepresence of the recombinant vector in the transformed colonies was confirmed by thecolony-PCR procedure. The correct structure of the recombinant vector, isolated from thetransformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestionwith restriction enzymes.Conclusion: The constructed recombinant cassette, named pZSclaR, can be regardedas an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claRhas been cloned accompanied with its precisely selected promoter so it could be used inexpression vectors. Hence the ClaR is known as a putative regulatory protein. The overproducedprotein could also be used for other related investigations, such as a mobilityshift assay.

  16. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    Science.gov (United States)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  17. Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesis-related genes during salicylic acid signaling.

    Directory of Open Access Journals (Sweden)

    Modhumita Ghosh Dasgupta

    Full Text Available Withania somnifera (L. Dunal is a valued medicinal plant with pharmaceutical applications. The present study was undertaken to analyze the salicylic acid induced leaf transcriptome of W. somnifera. A total of 45.6 million reads were generated and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp. A total of 71,062 transcripts were annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR families were mined from the transcriptome data and their pattern of expression post 17 and 36 hours of salicylic acid treatment was documented. The analysis revealed significant up-regulation of all families of PR genes by 36 hours post treatment except WsPR10. The relative fold expression of transcripts ranged from 1 fold to 6,532 fold. The two families of peroxidases including the lignin-forming anionic peroxidase (WsL-PRX and suberization-associated anionic peroxidase (WsS-PRX recorded maximum expression of 377 fold and 6532 fold respectively, while the expression of WsPR10 was down-regulated by 14 fold. Additionally, the most stable reference gene for normalization of qRT-PCR data was also identified. The effect of SA on the accumulation of major secondary metabolites of W. somnifera including withanoside V, withaferin A and withanolide A was also analyzed and an increase in content of all the three metabolites were detected. This is the first report on expression patterns of PR genes during salicylic acid signaling in W. somnifera.

  18. Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesis-related genes during salicylic acid signaling.

    Science.gov (United States)

    Ghosh Dasgupta, Modhumita; George, Blessan Santhosh; Bhatia, Anil; Sidhu, Om Prakash

    2014-01-01

    Withania somnifera (L.) Dunal is a valued medicinal plant with pharmaceutical applications. The present study was undertaken to analyze the salicylic acid induced leaf transcriptome of W. somnifera. A total of 45.6 million reads were generated and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp. A total of 71,062 transcripts were annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR) families were mined from the transcriptome data and their pattern of expression post 17 and 36 hours of salicylic acid treatment was documented. The analysis revealed significant up-regulation of all families of PR genes by 36 hours post treatment except WsPR10. The relative fold expression of transcripts ranged from 1 fold to 6,532 fold. The two families of peroxidases including the lignin-forming anionic peroxidase (WsL-PRX) and suberization-associated anionic peroxidase (WsS-PRX) recorded maximum expression of 377 fold and 6532 fold respectively, while the expression of WsPR10 was down-regulated by 14 fold. Additionally, the most stable reference gene for normalization of qRT-PCR data was also identified. The effect of SA on the accumulation of major secondary metabolites of W. somnifera including withanoside V, withaferin A and withanolide A was also analyzed and an increase in content of all the three metabolites were detected. This is the first report on expression patterns of PR genes during salicylic acid signaling in W. somnifera.

  19. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    Science.gov (United States)

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  20. Effects of dietary fat saturation on fatty acid composition and gene transcription in different tissues of Iberian pigs.

    Science.gov (United States)

    Benítez, R; Núñez, Y; Fernández, A; Isabel, B; Fernández, A I; Rodríguez, C; Barragán, C; Martín-Palomino, P; López-Bote, C; Silió, L; Óvilo, C

    2015-04-01

    The effect of two diets, respectively enriched with SFA (S) and PUFA (P), on FA tissue composition and gene expression was studied in fattened Iberian pigs. The FA composition of adipose, muscular and liver tissues was affected by dietary treatment. S group showed higher MUFA and MUFA/SFA ratio and lower PUFA and n-6/n-3 ratio than P group in all analyzed tissues. In muscle and liver the extracted lipids were separated into neutral lipids and polar lipid fractions which showed significantly different responses to the dietary treatment, especially in liver where no significant effect of diet was observed in NL fraction. The expression of six candidate genes related to lipogenesis and FA oxidation was analyzed by qPCR. In liver, stearoyl CoA desaturase (SCD), acetyl CoA carboxylase alpha (ACACA) and malic enzyme 1 (ME1) genes showed higher expression in S group. SCD, ACACA, ME1, and fatty acid synthase (FASN) gene expression levels showed a wide variation across the tested tissues, with much higher expression levels observed in adipose tissue than other tissues. Tissue FA profile and gene expression results support the deposition of dietary FA, the lipogenic effect of dietary saturated fat in liver and the employment of saturated dietary fat for endogenous synthesis of MUFA in all the analyzed tissues.

  1. Effects of dietary soybean stachyose and phytic acid on gene expressions of serine proteases in Japanese flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Mi, Haifeng; Mai, Kangsen; Zhang, Wenbing; Wu, Chenglong; Cai, Yinghua

    2011-09-01

    Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder ( Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively. Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY ( P0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes' expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

  2. Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion.

    Science.gov (United States)

    Camilleri, Michael; Carlson, Paula; Acosta, Andres; Busciglio, Irene

    2015-07-01

    The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT(2) PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥ 2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion.

  3. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+-Abscisic Acid Producing Ascomycete Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Zhong-Tao Ding

    2015-05-01

    Full Text Available The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

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

    Directory of Open Access Journals (Sweden)

    Sofía Arriarán

    2015-11-01

    Full Text Available 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.

  5. Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    XU Yunjian (徐云剑); GU Xuesong (顾雪松); LI Jun (李珺); LI Qing (李 晴); Peter J. Davies; ZHU Yuxian (朱玉贤)

    2003-01-01

    The AAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficient E. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with the AAIR expression vector. Further study revealed that overexpression of the pea AAIR cDNA in acetohydroxy acid isomeroreductase deficient E. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of the AAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulate AAIR gene expression.

  6. In Ovo Administration of Silver Nanoparticles and/or Amino Acids Influence Metabolism and Immune Gene Expression in Chicken Embryos

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    Subrat K. Bhanja

    2015-04-01

    Full Text Available Due to their physicochemical and biological properties, silver nanoparticles (NanoAg have a wide range of applications. In the present study, their roles as a carrier of nutrients and an immunomodulator were tested in chicken embryos. Cysteine (Cys+NanoAg injected embryos had smaller livers but heavier breasts on the 19th day of embryogenesis. Cys injected embryos had lower oxygen consumption compared to threonine (Thr or NanoAg injected embryos. The energy expenditure in Thr+NanoAg, or NanoAg injected embryos was higher than Cys or Cys+NanoAg but was not different from uninjected control embryos. Relative expression of the hepatic insulin-like growth factor-I (IGF-I gene was higher in Cys or NanoAg injected embryos after lipopolysaccharide (LPS induction. The gene expression of hepatic tumour necrosis factor-alpha (TNF-α and interleukin-6 (IL-6 did not differ among amino acids, NanoAg and uninjected controls in the non-LPS groups, but increased by many folds in the LPS treated NanoAg, Cys and Cys+NanoAg groups. In LPS treated spleens, TNF-α expression was also up-regulated by NanoAg, amino acids and their combinations, but interleukin-10 (IL-10 expression was down-regulated in Thr, Cys or Thr+NanoAg injected embryos. Toll like receptor-2 (TLR2 expression did not differ in NanoAg or amino acids injected embryos; however, toll like receptor-4 (TLR4 expression was higher in all treated embryos, except for Cys+NanoAg, than in uninjected control embryos. We concluded that NanoAg either alone or in combination with amino acids did not affect embryonic growth but improved immunocompetence, indicating that NanoAg and amino acid complexes can act as potential agents for the enhancement of innate and adaptive immunity in chicken.

  7. Abscisic acid enhances tolerance of wheat seedlings to drought and regulates transcript levels of genes encoding ascorbate-glutathione biosynthesis

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    Wei eLiting

    2015-06-01

    Full Text Available Glutathione (GSH and ascorbate (ASA are associated with the abscisic acid (ABA-induced abiotic tolerance in higher plant, however, its molecular mechanism remains obscure. In this study, exogenous application (10 μM of ABA significantly increased the tolerance of seedlings of common wheat (Triticum aestivum L. suffering from 5 days of 15% polyethylene glycol (PEG-stimulated drought stress, as demonstrated by increased shoot lengths and shoot and root dry weights, while showing decreased contents of hydrogen peroxide (H2O2 and malondialdehyde (MDA. Under drought stress conditions, ABA markedly increased contents of GSH and ASA in both leaves and roots of ABA-treated plants. Temporal and spatial expression patterns of eight genes encoding ASA and GSH synthesis-related enzymes were measured using quantitative real-time reverse transcription polymerase chain reaction (qPCR. The results showed that ABA temporally regulated the transcript levels of genes encoding ASA-GSH cycle enzymes. Moreover, these genes exhibited differential expression patterns between the root and leaf organs of ABA-treated wheat seedlings during drought stress. These results implied that exogenous ABA increased the levels of GSH and ASA in drought-stressed wheat seedlings in time- and organ-specific manners. Moreover, the transcriptional profiles of ASA-GSH synthesis-related enzyme genes in the leaf tissue were compared between ABA- and salicylic acid (SA-treated wheat seedlings under PEG-stimulated drought stress, suggesting that they increased the contents of ASA and GSH by differentially regulating expression levels of ASA-GSH synthesis enzyme genes. Our results increase our understanding of the molecular mechanism of ABA-induced drought tolerance in higher plants

  8. Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation.

    Science.gov (United States)

    Zhu, Ying; Liu, Xiaoguang; Yang, Shang-Tian

    2005-04-20

    Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.

  9. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    Science.gov (United States)

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  10. The action of exogenous abscisic and gibberellic acids on gene expression in germinating castor beans.

    Science.gov (United States)

    Dommes, J; Northcote, D H

    1985-09-01

    Exogenously applied abscisic acid inhibits isocitrate-lyase activity of the endosperm during germination of castor-bean seeds. Amounts of isocitrate-lyase mRNA have been estimated by immunoprecipitation of in-vitro-translated polypeptide products. Exogenous abscisic acid leads to an inhibition of isocitrate lyase-mRNA accumulation. A large proportion of this effect of the growth factor may be accounted for by its action in inhibiting the overall accumulation of ribosomal RNA and total mRNA. However, the effect of abscisic acid on protein synthesis is not general, as the production of some mRNAs was stimulated. The major mRNA stored in the dry seed, coding for a 25600-Mr polypeptide that normally disappears within the first 12 h of germination, exhibited high levels in abscisic-acid-treated endosperms throughout the germination period. Three complementary DNA clones, of which two clones are complementary to isocitrate lyase, have been used to measure levels of transcripts during seed germination. The accumulation of both transcripts was inhibited by exogenous abscisic acid. The data strongly indicate that the action of abscisic acid on isocitrate lyase synthesis is either to inhibit the transcription, or to increase the transcript turnover. Exogenous gibberellic acid is able to counteract the inhibitory effects of abscisic acid.

  11. Fatty acid desaturase gene variants, cardiovascular risk factors, and myocardial infarction in the costa rica study

    Science.gov (United States)

    Genetic variation in fatty acid desaturases (FADS) has previously been linked to long-chain polyunsaturated fatty acids (PUFAs) in adipose tissue and cardiovascular risk. The goal of our study was to test associations between six common FADS polymorphisms (rs174556, rs3834458, rs174570, rs2524299, r...

  12. Aspergillus flavus Blast2GO gene ontology database: elevated growth temperature alters amino acid metabolism

    Science.gov (United States)

    The availability of a representative gene ontology (GO) database is a prerequisite for a successful functional genomics study. Using online Blast2GO resources we constructed a GO database of Aspergillus flavus. Of the predicted total 13,485 A. flavus genes 8,987 were annotated with GO terms. The mea...

  13. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    Science.gov (United States)

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  14. Identification of genes involved in indole-3-acetic acid biosynthesis by Gluconacetobacter diazotrophicus PAL5 strain using transposon mutagenesis

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    ELISETE PAINS RODRIGUES

    2016-10-01

    Full Text Available Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA; however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01, which exhibited 95% less indolic compounds that the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA and indole-3-lactate (ILA. In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO. GDI2456 (lao gene forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao, cccA and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with

  15. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    Science.gov (United States)

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  16. Investigation of Gamma-aminobutyric acid (GABA A receptors genes and migraine susceptibility

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    Ciccodicola Alfredo

    2008-12-01

    Full Text Available Abstract Background Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24–28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ, which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24–28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type ε (GABRE and type θ (GABRQ genes and their involvement in migraine. Methods We have performed an association analysis in a large population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls examining a set of 3 single nucleotide polymorphisms (SNPs in the coding region (exons 3, 5 and 9 of the GABRE gene and also the I478F coding variant of the GABRQ gene. Results Our study did not show any association between the examined SNPs in our test population (P > 0.05. Conclusion Although these particular GABA receptor genes did not show positive association, further studies are necessary to consider the role of other GABA receptor genes in migraine susceptibility.

  17. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

    Science.gov (United States)

    Mandal, Shantanu; Upadhyay, Shivangi; Wajid, Saima; Ram, Mauji; Jain, Dharam Chand; Singh, Ved Pal; Abdin, Malik Zainul; Kapoor, Rupam

    2015-07-01

    It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

  18. Diversity and Distribution of Arsenic-Related Genes Along a Pollution Gradient in a River Affected by Acid Mine Drainage.

    Science.gov (United States)

    Desoeuvre, Angélique; Casiot, Corinne; Héry, Marina

    2016-04-01

    Some microorganisms have the capacity to interact with arsenic through resistance or metabolic processes. Their activities contribute to the fate of arsenic in contaminated ecosystems. To investigate the genetic potential involved in these interactions in a zone of confluence between a pristine river and an arsenic-rich acid mine drainage, we explored the diversity of marker genes for arsenic resistance (arsB, acr3.1, acr3.2), methylation (arsM), and respiration (arrA) in waters characterized by contrasted concentrations of metallic elements (including arsenic) and pH. While arsB-carrying bacteria were representative of pristine waters, Acr3 proteins may confer to generalist bacteria the capacity to cope with an increase of contamination. arsM showed an unexpected wide distribution, suggesting biomethylation may impact arsenic fate in contaminated aquatic ecosystems. arrA gene survey suggested that only specialist microorganisms (adapted to moderately or extremely contaminated environments) have the capacity to respire arsenate. Their distribution, modulated by water chemistry, attested the specialist nature of the arsenate respirers. This is the first report of the impact of an acid mine drainage on the diversity and distribution of arsenic (As)-related genes in river waters. The fate of arsenic in this ecosystem is probably under the influence of the abundance and activity of specific microbial populations involved in different As biotransformations.

  19. Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery.

    Science.gov (United States)

    Zhang, Lei; Yu, Min; Wang, Jun; Tang, Rupei; Yan, Guoqing; Yao, Weijing; Wang, Xin

    2016-08-01

    A series of novel pH-sensitive gene delivery vectors (POEI 1, 2, and 3) are synthesized through Michael addition from low molecular weight PEI (LMW PEI) via acid-labile ortho ester linkage with terminal acrylates (OEAc) by various feed molar ratios. The obtained POEI 1 and POEI 2 can efficiently condense plasmid DNA into nanoparticles with size range of 200-300 nm and zeta-potentials of about +15 mV while protecting DNA from enzymatic digestion compared with POEI 3. Significantly, ortho ester groups of POEI main-chains can make an instantaneous degradation-response to acidic endosomal pH (≈5.0), resulting in accelerated disruption of polyplexes and intracellular DNA release. MTT assay reveals that all POEIs exhibit much lower cytotoxicity in different cells than branched PEI (25 KDa). As expected, POEI 1 and POEI 2 perform improved gene transfection in vitro, suggesting that such polycations might be promising gene vectors based on overcoming toxicity-efficiency contradiction.

  20. Effects of intramuscular administration of folic acid and vitamin B12 on granulosa cells gene expression in postpartum dairy cows.

    Science.gov (United States)

    Gagnon, A; Khan, D R; Sirard, M-A; Girard, C L; Laforest, J-P; Richard, F J

    2015-11-01

    The fertility of dairy cows is challenged during early lactation, and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improve energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg of folic acid and 10 mg of vitamin B12 (treated group) beginning 24 (standard deviation=4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the precalving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the 2 groups. Plasma concentrations of folates and vitamin B12 were increased in treated animals. Daily dry matter intake was not significantly different between the 2 groups before [13.5 kg; standard error (SE)=0.5] and after (23.6 kg; SE=0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE=1.4) and 38.1 (SE=1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of β-hydroxybutyrate tended to be lower in cows injected with the vitamin supplement, 0.47 (SE=0.04) versus 0.55 (SE=0.03) for treated and control cows, respectively. The ovarian follicle ≥12 mm in diameter was collected by ovum pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the effect of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation

  1. Grape Seed Procyanidins and Cholestyramine Differentially Alter Bile Acid and Cholesterol Homeostatic Gene Expression in Mouse Intestine and Liver.

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    Rebecca M Heidker

    Full Text Available Bile acid (BA sequestrants, lipid-lowering agents, may be prescribed as a monotherapy or combination therapy to reduce the risk of coronary artery disease. Over 33% of adults in the United States use complementary and alternative medicine strategies, and we recently reported that grape seed procyanidin extract (GSPE reduces enterohepatic BA recirculation as a means to reduce serum triglyceride (TG levels. The current study was therefore designed to assess the effects on BA, cholesterol and TG homeostatic gene expression following co-administration with GSPE and the BA sequestrant, cholestyramine (CHY. Eight-week old male C57BL/6 mice were treated for 4 weeks with either a control or 2% CHY-supplemented diet, after which, they were administered vehicle or GSPE for 14 hours. Liver and intestines were harvested and gene expression was analyzed. BA, cholesterol, non-esterified fatty acid and TG levels were also analyzed in serum and feces. Results reveal that GSPE treatment alone, and co-administration with CHY, regulates BA, cholesterol and TG metabolism differently than CHY administration alone. Notably, GSPE decreased intestinal apical sodium-dependent bile acid transporter (Asbt gene expression, while CHY significantly induced expression. Administration with GSPE or CHY robustly induced hepatic BA biosynthetic gene expression, especially cholesterol 7α-hydroxylase (Cyp7a1, compared to control, while co-administration further enhanced expression. Treatment with CHY induced both intestinal and hepatic cholesterologenic gene expression, while co-administration with GSPE attenuated the CHY-induced increase in the liver but not intestine. CHY also induced hepatic lipogenic gene expression, which was attenuated by co-administration with GSPE. Consequently, a 25% decrease in serum TG levels was observed in the CHY+GSPE group, compared to the CHY group. Collectively, this study presents novel evidence demonstrating that GSPE provides additive and

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

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    Zhiliang Yu

    2015-01-01

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

  3. Construction of a multiplex promoter reporter platform to monitor Staphylococcus aureus virulence gene expression and the identification of usnic acid as a potent suppressor of psm gene expression

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    Peng GAO

    2016-08-01

    Full Text Available As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  4. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

    Science.gov (United States)

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  5. Fruit load induces changes in global gene expression and in abscisic acid (ABA) and indole acetic acid (IAA) homeostasis in citrus buds.

    Science.gov (United States)

    Shalom, Liron; Samuels, Sivan; Zur, Naftali; Shlizerman, Lyudmila; Doron-Faigenboim, Adi; Blumwald, Eduardo; Sadka, Avi

    2014-07-01

    Many fruit trees undergo cycles of heavy fruit load (ON-Crop) in one year, followed by low fruit load (OFF-Crop) the following year, a phenomenon known as alternate bearing (AB). The mechanism by which fruit load affects flowering induction during the following year (return bloom) is still unclear. Although not proven, it is commonly accepted that the fruit or an organ which senses fruit presence generates an inhibitory signal that moves into the bud and inhibits apical meristem transition. Indeed, fruit removal from ON-Crop trees (de-fruiting) induces return bloom. Identification of regulatory or metabolic processes modified in the bud in association with altered fruit load might shed light on the nature of the AB signalling process. The bud transcriptome of de-fruited citrus trees was compared with those of ON- and OFF-Crop trees. Fruit removal resulted in relatively rapid changes in global gene expression, including induction of photosynthetic genes and proteins. Altered regulatory mechanisms included abscisic acid (ABA) metabolism and auxin polar transport. Genes of ABA biosynthesis were induced; however, hormone analyses showed that the ABA level was reduced in OFF-Crop buds and in buds shortly following fruit removal. Additionally, genes associated with Ca(2+)-dependent auxin polar transport were remarkably induced in buds of OFF-Crop and de-fruited trees. Hormone analyses showed that auxin levels were reduced in these buds as compared with ON-Crop buds. In view of the auxin transport autoinhibition theory, the possibility that auxin distribution plays a role in determining bud fate is discussed.

  6. Fatty acid desaturase gene variants, cardiovascular risk factors, and myocardial infarction in the Costa Rica Study

    Directory of Open Access Journals (Sweden)

    Stella eAslibekyan

    2012-05-01

    Full Text Available Genetic variation in fatty acid desaturases (FADS has previously been linked to long-chain polyunsaturated fatty acids (PUFAs in adipose tissue and cardiovascular risk. The goal of our study was to test associations between six common FADS polymorphisms (rs174556, rs3834458, rs174570, rs2524299, rs174589, rs174627, adipose tissue fatty acids, intermediate cardiovascular risk factors, and nonfatal myocardial infarction (MI in a matched population based case-control study of Costa Rican adults (n=1756. Generalized linear models and multiple conditional logistic regression models were used to assess the associations of interest. Analyses involving intermediate cardiovascular risk factors and MI were also conducted in two replication cohorts, The Nurses’ Health Study (n=1200 and The Health Professionals Follow-Up Study (n=1295. In the Costa Rica Study, genetic variation in the FADS cluster was associated with a robust linear decrease in adipose gamma-linolenic, arachidonic, and eicosapentaenoic fatty acids, and significant or borderline significant increases in the eicosadienoic, eicosatrienoic, and dihomo-gamma-linolenic fatty acids. However, the associations with adipose tissue fatty acids did not translate into changes in inflammatory biomarkers, blood lipids, or the risk of MI in the discovery or the replication cohorts. In conclusion, fatty acid desaturase polymorphisms impact long-chain PUFA biosynthesis, but their overall effect on cardiovascular health likely involves multiple pathways and merits further investigation.

  7. Ascorbic acid-dependent gene expression in Streptococcus pneumoniae and the activator function of the transcriptional regulator UlaR2

    NARCIS (Netherlands)

    Afzal, Muhammad; Shafeeq, Sulman; Kuipers, Oscar P

    2015-01-01

    In this study, we have explored the impact of ascorbic acid on the transcriptome of Streptococcus pneumoniae D39. The expression of several genes and operons, including the ula operon (which has been previously shown to be involved in ascorbic acid utilization), the AdcR regulon (which has been prev

  8. Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101.

    Science.gov (United States)

    Cheng, Rubin; Ge, Yuqing; Yang, Bo; Zhong, Xiaoming; Lin, Xiangzhi; Huang, Zhen

    2013-06-01

    Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.

  9. Cloning and Characterization of a Novel Purple Acid Phosphatase Gene (MtPAP1) from Medicago truncatula Barrel Medic

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A novel purple acid phosphatase gene (MtPAP1) was isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp in length with an open reading frame (ORF) of 1 398 bp capable of encoding an N-terminal signal peptide of 23 amino acids. The transcripts of MtPAP1 were mainly detected in leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced in leaves and increased in roots, with the strongest hybridization signal detected in roots. A chimeric gene construct fusing MtPAP1 and GFPwas made in which the fusion was driven by the CaMV35S promoter. Transgenlc Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal microscopic analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptide at the N-terminal. The coding region of MtPAP1 without signal peptide was inserted into the prokaryotic expression vector pET-30a (+) and overexpressed in Escherlchia coll BL21(DE3). The acid phosphatase (APase) proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An enzyme activity assay demonstrated that the APase activity in the transformed bacteria was 3.16-fold higher than that of control. The results imply that MtPAP1 functions to improve phosphorus acquisition in plants under conditions of phosphorus (P) stress.

  10. Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid

    DEFF Research Database (Denmark)

    Westergaard, M; Henningsen, J; Svendsen, M L

    2001-01-01

    using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression...... nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order...... a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease...

  11. Gene Therapy for Advanced Melanoma: Selective Targeting and Therapeutic Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Joana R. Viola

    2013-01-01

    Full Text Available Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed.

  12. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

    Energy Technology Data Exchange (ETDEWEB)

    Lowden, Michael J.; Skorupski, Karen; Pellegrini, Maria; Chiorazzo, Michael G.; Taylor, Ronald K.; Kull, F. Jon (Dartmouth)

    2010-03-04

    Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 {angstrom} resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

  13. Hyaluronic Acid Conjugates as Vectors for the Active Targeting of Drugs, Genes and Nanocomposites in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Silvia Arpicco

    2014-03-01

    Full Text Available Hyaluronic acid (HA is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

  14. Association of polymorphisms in genes involved in the dopaminergic pathway with blood pressure and uric acid levels in Chinese females.

    Science.gov (United States)

    Yeh, Ting-Kuang; Yeh, Ting-Chi; Weng, Chi-Feng; Shih, Bing-Fu; Tsao, Hsueh-Jen; Hsiao, Chien-Hua; Chuang, Fu-Tai; Hu, Chung-Yi; Chang, Chun-Yen

    2010-12-01

    Since the high degree of heritability of physiological traits was demonstrated by twin and adoption studies, contemporary researchers in the fields of clinical medicine, behavioral science, and genetics have acknowledged the crucial role of genetic factors in human physiology. The study described herein explores the association between physiological parameters and the dopaminergic system using molecular genetic techniques. A total of 558 Taiwanese female volunteers, ranging from 16 to 17 years, were recruited. Single nucleotide polymorphisms in genes involved in the dopaminergic pathway were selected for analysis. Systolic blood pressure and diastolic blood pressure were associated significantly with the catechol-O-methyltransferase (COMT) Val158Met polymorphism and the dopamine β-hydroxylase (DBH) C1021T polymorphism. Furthermore, plasma uric acid was associated significantly with the COMT Val158Met polymorphism. Our study suggests the possible involvement of genetic polymorphisms in COMT and DBH in the regulation of blood pressure and plasma uric acid.

  15. Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress.

    Science.gov (United States)

    Zhang, Luan; Xiong, Zhi-ting; Xu, Zhong-rui; Liu, Chen; Cai, Shen-wen

    2014-06-01

    The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3µM Cu(2+) (control) and 10µM Cu(2+) for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation.

  16. Mutations in the fatty acid 2-hydroxylase gene are associated with leukodystrophy with spastic paraparesis and dystonia.

    Science.gov (United States)

    Edvardson, Simon; Hama, Hiroko; Shaag, Avraham; Gomori, John Moshe; Berger, Itai; Soffer, Dov; Korman, Stanley H; Taustein, Ilana; Saada, Ann; Elpeleg, Orly

    2008-11-01

    Myelination is a complex, developmentally regulated process whereby myelin proteins and lipids are coordinately expressed by myelinating glial cells. Homozygosity mapping in nine patients with childhood onset spasticity, dystonia, cognitive dysfunction, and periventricular white matter disease revealed inactivating mutations in the FA2H gene. FA2H encodes the enzyme fatty acid 2-hydroxylase that catalyzes the 2-hydroxylation of myelin galactolipids, galactosylceramide, and its sulfated form, sulfatide. To our knowledge, this is the first identified deficiency of a lipid component of myelin and the clinical phenotype underscores the importance of the 2-hydroxylation of galactolipids for myelin maturation. In patients with autosomal-recessive unclassified leukodystrophy or complex spastic paraparesis, sequence analysis of the FA2H gene is warranted.

  17. A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

    Science.gov (United States)

    Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Cruz de Carvalho, Maria H

    2013-11-15

    A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.

  18. Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis.

    Directory of Open Access Journals (Sweden)

    Lizhi Wu

    Full Text Available UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA, the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.

  19. Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rachid Lahlali

    Full Text Available An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc, suppressed clubroot (Plasmodiophora brassicae -Pb on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001 with the severity of clubroot at 5 weeks after treatment at a low (2×10(5 spores pot(-1 but not high (2×10(5 spores pot(-1 dose of pathogen inoculum. Transcript levels of nine B. napus (Bn genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL. These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2, ethylene (BnACO, auxin (BnAAO1, and PR-2 protein (BnPR-2 biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte.

  20. Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis.

    Science.gov (United States)

    Wu, Lizhi; Chaudhary, Sandeep C; Atigadda, Venkatram R; Belyaeva, Olga V; Harville, Steven R; Elmets, Craig A; Muccio, Donald D; Athar, Mohammad; Kedishvili, Natalia Y

    2016-01-01

    UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.

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

    DEFF Research Database (Denmark)

    Campa, Daniele; McKay, James; Sinilnikova, Olga

    2009-01-01

    -binding protein), which is induced by glucose, and SREBP-1 (sterol response element-binding protein-1), which is stimulated by insulin through the PI3K/Akt signal transduction pathway. We investigated whether the genetic variability of the genes encoding for ChREBP, SREBP and FAS (respectively, MLXIPL, SREBF1...... and FASN) is related to breast cancer risk and body-mass index (BMI) by studying 1,294 breast cancer cases and 2,452 controls from the European Prospective Investigation on Cancer (EPIC). We resequenced the FAS gene and combined information of SNPs found by resequencing and SNPs from public databases....... Using a tagging approach and selecting 20 SNPs, we covered all the common genetic variation of these genes. In this study we were not able to find any statistically significant association between the SNPs in the FAS, ChREBP and SREPB-1 genes and an increased risk of breast cancer overall...

  2. Hepatic phenotype of liver fatty acid binding protein gene-ablated mice

    OpenAIRE

    Martin, Gregory G.; Atshaves, Barbara P.; Huang, Huan; McIntosh, Avery L.; Williams, Brad J.; Pai, Pei-Jing; Russell, David H.; Kier, Ann B.; Schroeder, Friedhelm

    2009-01-01

    Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these L-FABP−/− mice was attributable to genetic inhomogeneity, these L-FABP−/− mice were fu...

  3. Pharmacologic doses of ascorbic acid repress specificity protein (Sp) transcription factors and Sp-regulated genes in colon cancer cells.

    Science.gov (United States)

    Pathi, Satya S; Lei, Ping; Sreevalsan, Sandeep; Chadalapaka, Gayathri; Jutooru, Indira; Safe, Stephen

    2011-01-01

    Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.

  4. Gene expression, serum amino acid levels, and growth performance of pigs fed dietary leucine and lysine at different ratios.

    Science.gov (United States)

    García, H; Morales, A; Araiza, A; Htoo, J K; Cervantes, M

    2015-03-06

    We examined 96 pigs (28.1 ± 0.83 kg) to analyze the effect of Leu:Lys ratios on expression of the cationic amino acid transporters b(0,+) and CAT-1 in the jejunum and liver as well as myosin expression in 2 muscles to estimate the optimum standardized ileal digestible (SID) Leu:Lys ratio for growth rate and efficiency. A wheat-and wheat bran-based diets were formulated to meet the requirements of SID amino acids other than Leu (0.70%) and Lys (0.80%). L-Leu was added to the basal diet in 5 SID Leu:Lys ratios (88, 100, 120, 140, and 160% in diets 1-5). Tissue samples were collected from 8 pigs with ratios of 88, 120, and 160%. Relative expression of b(0,+), CAT-1, and myosin was analyzed. b(0,+) expression in the jejunum was higher but lower in the liver of pigs with the 120% ratio compared to those with the 88 or 160% ratio; myosin expression in longissimus dorsi was also higher in pigs with the 120% ratio (P pigs with 120 or 160% ratios than in pigs with 88%. Serum concentration of nearly all amino acids decreased with excess dietary Leu (P dietary Leu:Lys ratio affects the expression of genes coding for amino acid transporters and myosin, the availability of Lys, and the growth rate and efficiency in pigs.

  5. Down-regulation of the Caffeic acid O-methyltransferase Gene in Switchgrass Reveals a Novel Monolignol Analog

    Energy Technology Data Exchange (ETDEWEB)

    Tschaplinski, Timothy J [ORNL; Standaert, Robert F [ORNL; Engle, Nancy L [ORNL; Martin, Madhavi Z [ORNL; Sangha, Amandeep K [ORNL; Parks, Jerry M [ORNL; Smith, Jeremy C [ORNL; Samuel, Reichel [ORNL; Pu, Yunqiao [ORNL; Ragauskas, A J [Georgia Institute of Technology; Hamilton, Choo Yieng [ORNL; Fu, Chunxiang [Noble Foundation; Wang, Zeng-Yu [Noble Foundation; Davison, Brian H [ORNL; Dixon, Richard A [Noble Foundation; Mielenz, Jonathan R [ORNL

    2012-01-01

    Down-regulation of the caffeic acid 3-O-methyltransferase (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors, confirming the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. Although there was no indication that iso-sinapyl alcohol was integrated into the cell wall, diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth.

  6. Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells.

    Science.gov (United States)

    Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua; Berggren-Söderlund, Maria; Nilsson-Ehle, Peter; Zhang, Xiaoying; Xu, Ning

    2014-02-28

    It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPARβ/δ pathway.

  7. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    Energy Technology Data Exchange (ETDEWEB)

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  8. A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

    Science.gov (United States)

    Zhang, Xiqing; Zhang, Shuli; Shi, Yan; Shen, Fadi; Wang, Haikuan

    2014-07-01

    Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.

  9. Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    XU; Yunjian; (

    2003-01-01

    [1]Zhu, Y. X., Zhang, Y. F., Li, H. Y., Molecular cloning of GA suppressed G2 pea genes by cDNA RDA, Science in China, Ser. C, 1997, 40(4): 379-383.[2]Xu, H., Xu, Y., Gu, X. et al., Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea, Chinese Science Bulletin, 2001, 46(21): 1808-1811.[3]Dumas, R., Joyard, J., Douce, R., Purification and characterization of acetohydroxy acid reductoisomerase from spinach chloroplasts, Biochem. J., 1989, 262: 971-976.[4]Dumas, R., Butikofer, M. C., Job, D. et al., Evidence for two catalytically different magnesium binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis, Biochemistry, 1995, 34: 6026-6036.[5]Singh, B. K., Biosynthesis of valine, leucine, and isoleucine, in Plant Amino Acids-Biochemistry and Biotechnology (ed. Singh, B. K.), New York: Marcel Dekker Inc., 1999, 227-247.[6]Reynolds, T. L., Effect of chlorsulfuron valine and isoleucine on division and tracheary element differentiation in cell suspension cultures of Solanum carolinense, J. Plant Physiol., 1986, 125(1-2): 179-184.[7]Spackman, V. M. T., Cobb, A. H., Cell cycle inhibition of potato root tips treated with Imazethapyr, Annals of Applied Biology, 1999, 135(3): 585-587.[8]Zelenaya-Troitskaya, O., Perlman, P. S., Butow, R. A., An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability, EMBO J., 1995, 14: 3268-3276.[9]MacAlpine, D. M., Perlman, P. S., Butow, R. A., The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway, EMBO J., 2000, 19(4): 767-775.[10]Kaufman, B. A., Newman, S. M., Hallberg, R. L. et al., In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins, Proc. Natl. Acad. Sci. USA, 2000, 97: 7772-7777.[11]Mazliak, P., Plant membrane

  10. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    Science.gov (United States)

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  11. Increased expression and secretion of recombinant hIFNγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Razaghi Ali

    2015-12-01

    Full Text Available Transcriptional co-regulation of adjacent genes has been observed for prokaryotic and eukaryotic organisms, alike. High levels of gene adjacency were also found in a wide variety of yeast species with a high frequency of co-regulated gene sets. The aim of this research was to study how selective pressure on the Histidinol dehydrogenase gene (HIS4, using amino acid starvation, affects the level of expression and secretion of the adjacent human interferon gamma gene (hIFNγ in the recombinant Pichia pastoris GS115 strain, a histidine-deficient mutant. hIFNγ was cloned into the pPIC9 vector adjacent to the HIS4 gene, a gene essential for histidine biosynthesis, which was then transformed into P. pastoris. The transformed P. pastoris was cultured under continuous amino acid starvation in amino acid-free minimal medium for ten days, with five inoculations into unspent medium every second day. Under these conditions, only successfully transformed cells (hIFNγ -HIS4+ are able to synthesise histidine and therefore thrive. As shown by ELISA, amino acid starvation-induced selective pressure on HIS4 improved expression and secretion of the adjacent hIFNγ by 55% compared to unchallenged cells. RT-qPCR showed that there was also a positive correlation between duration of amino acid starvation and increased levels of the hIFNγ RNA transcripts. According to these results, it is suggested that these adjacent genes (hIFNγ and HIS4 in the transformed P. pastoris are transcriptionally co-regulated and their expression is synchronised. To the best of the knowledge of the authors; this is the first study demonstrating that amino acid starvationinduced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.

  12. Single nucleotide polymorphism analysis on chicken extra-celluar fatty acid binding protein gene and its associations with fattiness trait

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Fattiness is an important parameter to estimate meat quality, which has high heritability. In this experiment, F2 chickens derived from Broilers crossing to Silky were used to study the effect of extracellular fatty acid binding protein (EX-FABP) gene on abdominal fat accumulation. 1.6 kb of the 5′ region of the gene was amplified by six pairs of primers, and then single nucleotide poly-morphisms (SNPs) were detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. There were four nucleotides variations found, A-G at -1807, C-A at -1805, T-C at -1011 and a C insertion at -1000 respectively. The result of least square analysis suggests that the birds with BB genotype defined by the second pair of primer have a higher abdominal fat weight and abdominal fat percentage than the birds with the other genotypes (AA and AB). It implied that EX-FABP gene could be a candidate locus or linked to a major gene to significantly affect abdominal fat traits in chicken.

  13. Molecular Characterization and Functional Analysis of a New Acid Phosphatase Gene (Ha-acp1) from Heterodera avenae

    Institute of Scientific and Technical Information of China (English)

    LIU Yan-ke; HUANG Wen-kun; LONG Hai-bo; PENG Huan; HE Wen-ting; PENG De-liang

    2014-01-01

    For sedentary endo-parasitic nematodes, parasitism genes encoding secretory protein expressed in the subventral glands cells always play an important role during the early parasitic process. A new acid phosphatase gene (Ha-acp1) expressed in the subventral glands of the cereal cyst nematode (Heterodera avenae) was cloned and the characteristics of the gene were analyzed. Results showed that the gene had a putative signal peptide for secretion and in situ hybridization showed that the transcripts of Ha-acp1 accumulated speciifcally in the subventral gland cells of H. avenae. Southern blot analysis suggested that Ha-acp1 belonged to a multigene family. RT-PCR analysis indicated that this transcription was strong at the pre-parasitic juveniles. Knocking down Ha-acp1 using RNA interference technology could reduce nematode infectivity by 50%, and suppress the development of cyst. Results indicated that Ha-acp1 could play an important role in destroying the defense system of host plants.

  14. Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus.

    Science.gov (United States)

    Hong, Eun Jin; Kim, Na Kyeong; Lee, Doyup; Kim, Won Gon; Lee, Inhyung

    2015-11-01

    To explore novel bioactive compounds produced via activation of secondary metabolite (SM) gene clusters, we overexpressed an ortholog of laeA, a gene that encodes a global positive regulator of secondary metabolism in Aspergillus fumisynnematus F746. Overexpression of the laeA gene under the alcA promoter resulted in the production of less pigment, shorter conidial head chains, and fewer conidia. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis revealed that SM production in OE::laeA was significantly increased, and included new metabolites that were not detected in the wild type. Among them, a compound named F1 was selected on the basis of its high production levels and antibacterial effects. F1 was purified by column chromatography and preparative TLC and identified as cyclopiazonic acid (CPA) by LC/MS, which had been previously known as mycotoxin. As A. fumisynnematus was not known to produce CPA, these results suggest that overexpression of the laeA gene can be used to explore the synthesis of useful bioactive compounds, even in a fungus for which the genome sequence is unavailable.

  15. Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids.

    Science.gov (United States)

    Schleifman, Erica B; Bindra, Ranjit; Leif, Jean; del Campo, Jacob; Rogers, Faye A; Uchil, Pradeep; Kutsch, Olaf; Shultz, Leonard D; Kumar, Priti; Greiner, Dale L; Glazer, Peter M

    2011-09-23

    Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells, and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA, and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34(+) hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months posttransplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function.

  16. Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2 with schizophrenia

    Directory of Open Access Journals (Sweden)

    Iwata Nakao

    2004-08-01

    Full Text Available Abstract Background The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 with schizophrenia. Methods We genotyped 100 Japanese schizophrenics and 100 controls recruited from the Kyushu area for 11 single nucleotide polymorphism (SNP markers distributed in the SLC1A2 region using the direct sequencing and pyrosequencing methods, and examined allele, genotype and haplotype association with schizophrenia.The positive finding observed in the Kyushu samples was re-examined using 100 Japanese schizophrenics and 100 controls recruited from the Aichi area. Results We found significant differences in genotype and allele frequencies of SNP2 between cases and controls (P = 0.013 and 0.008, respectively. After Bonferroni corrections, the two significant differences disappeared. We tested haplotype associations for all possible combinations of SNP pairs. SNP2 showed significant haplotype associations with the disease (P = 9.4 × 10-5, P = 0.0052 with Bonferroni correction, at the lowest in 8 combinations. Moreover, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis of our two sample sets. Conclusion We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby SLC1A2 in the Japanese population.

  17. The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: a gene expression approach.

    Science.gov (United States)

    Schlotz, Nina; Sørensen, Jesper Givskov; Martin-Creuzburg, Dominik

    2012-08-01

    Nutritional ecology of the aquatic model genus Daphnia has received much attention in past years in particular with regard to dietary polyunsaturated fatty acids (PUFAs) which are crucial for growth and reproduction. Besides their significant role as membrane components, C20 PUFAs serve as precursors for eicosanoids, hormone-like mediators of reproduction, immunity and ion transport physiology. In the present study we investigate transcriptomic changes in Daphnia magna in response to different algal food organisms substantially differing in their PUFA composition using quantitative real-time PCR and relate them to concomitantly documented life history data. The selection of target genes includes representatives that have previously been shown to be responsive to the eicosanoid biosynthesis inhibitor ibuprofen. The beneficial effect of C20 PUFA-rich food on reproduction and population growth rates was accompanied by an increased vitellogenin (DmagVtg1) gene expression in D. magna. Additionally, genes involved in eicosanoid signaling were particularly influenced by dietary C20 PUFA availability. For example, the cyclooxygenase gene (Cox), coding for a central enzyme in the eicosanoid pathway, was highly responsive to the food treatments. Our results suggest that dietary PUFAs are fundamental in D. magna physiology as substrate for eicosanoid synthesis and that these eicosanoids are important for D. magna reproduction.

  18. Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.

    Science.gov (United States)

    Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

    2013-08-30

    We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

  19. Analysis of the transcriptome of Erigeron breviscapus uncovers putative scutellarin and chlorogenic acids biosynthetic genes and genetic markers.

    Directory of Open Access Journals (Sweden)

    Ni-Hao Jiang

    Full Text Available Erigeron breviscapus (Vant. Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable.Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37% were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40% primer pairs were successfully amplified and 19 (52.78% primer pairs exhibited polymorphisms.Using next generation sequencing (NGS technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

  20. Antiproliferative effect of ascorbic acid is associated with the inhibition of genes necessary to cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Sophie Belin

    Full Text Available BACKGROUND: Ascorbic acid (AA, or Vitamin C, is most well known as a nutritional supplement with antioxidant properties. Recently, we demonstrated that high concentrations of AA act on PMP22 gene expression and partially correct the Charcot-Marie-Tooth disease phenotype in a mouse model. This is due to the capacity of AA, but not other antioxidants, to down-modulate cAMP intracellular concentration by a competitive inhibition of the adenylate cyclase enzymatic activity. Because of the critical role of cAMP in intracellular signalling, we decided to explore the possibility that ascorbic acid could modulate the expression of other genes. METHODS AND FINDINGS: Using human pangenomic microarrays, we found that AA inhibited the expression of two categories of genes necessary for cell cycle progression, tRNA synthetases and translation initiation factor subunits. In in vitro assays, we demonstrated that AA induced the S-phase arrest of proliferative normal and tumor cells. Highest concentrations of AA leaded to necrotic cell death. However, quiescent cells were not susceptible to AA toxicity, suggesting the blockage of protein synthesis was mainly detrimental in metabolically-active cells. Using animal models, we found that high concentrations of AA inhibited tumor progression in nude mice grafted with HT29 cells (derived from human colon carcinoma. Consistently, expression of tRNA synthetases and ieF2 appeared to be specifically decreased in tumors upon AA treatment. CONCLUSIONS: AA has an antiproliferative activity, at elevated concentration that could be obtained using IV injection. This activity has been observed in vitro as well in vivo and likely results from the inhibition of expression of genes involved in protein synthesis. Implications for a clinical use in anticancer therapies will be discussed.

  1. Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis

    Indian Academy of Sciences (India)

    Song Yu; Chen Ligang; Zhang Liping; Yu Diqiu

    2010-09-01

    Through activating specific transcriptional programmes, plants can launch resistance mechanisms to stressful environments and acquire a new equilibrium between development and defence. To screen the rice WRKY transcription factor which functions in abiotic stress tolerance and modulates the abscisic acid (ABA) response, we generated a whole array of 35S-OsWRKY transgenic Arabidopsis. In this study, we report that 35S-OsWRKY72 transgenic Arabidopsis, whose seed germination was retarded under normal conditions, emerged more sensitive to mannitol, NaCl, ABA stresses and sugar starvation than vector plants. Meanwhile, 35S-OsWRKY72 transgenic Arabidopsis displayed early flowering, reduced apical dominance, lost high temperature-induced hypocotyl elongation response, and enhanced gravitropism response, which were similar to the auxin-related gene mutants aux1, axr1 and bud1. Further, semi-quantitative RT-PCR showed that the expression patterns of three auxin-related genes AUX1, AXR1 and BUD1 were significantly altered in rosette leaves and inflorescences of 35S-OsWRKY72 plants compared with control Arabidopsis, and two ABA-related genes ABA2 and ABI4 were induced in 35S-OsWRKY72 seedlings. In addition, northern blot analysis indicated that, in rice, OsWRKY72 was inducible by polyethylene glycol (PEG), NaCl, naphthalene acetic acid (NAA), ABA and 42°C, similar to its orthologue AtWRKY75 in Arabidopsis, implying that these two WRKY genes might be required for multiple physiological processes in their plants. Together, these results suggest that OsWRKY72 interferes in the signal cross-talk between the ABA signal and auxin transport pathway in transgenic Arabidopsis.

  2. Molecular Analysis of the Clavulanic Acid Regulatory Gene Isolated from an Iranian Strain of Streptomyces Clavuligerus , PTCC 1709

    Science.gov (United States)

    Hojati, Zohreh; Salehi, Zahra; Motovali-Bashi, Majid; Korbekandi, Hasan; Jami, Saeed

    2011-01-01

    Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay. PMID:23508694

  3. Dietary sunflower oil modulates milk fatty acid composition without major changes in adipose and mammary tissue fatty acid profile or related gene mRNA abundance in sheep.

    Science.gov (United States)

    Castro-Carrera, T; Frutos, P; Leroux, C; Chilliard, Y; Hervás, G; Belenguer, A; Bernard, L; Toral, P G

    2015-04-01

    There are very few studies in ruminants characterizing mammary and adipose tissue (AT) expression of genes and gene networks for diets causing variations in milk fatty acid (FA) composition without altering milk fat secretion, and even less complementing this information with data on tissue FA profiles. This work was conducted in sheep in order to investigate the response of the mammary gland and the subcutaneous and perirenal AT, in terms of FA profile and mRNA abundance of genes involved in lipid metabolism, to a diet known to modify milk FA composition. Ten lactating Assaf ewes were randomly assigned to two treatments consisting of a total mixed ration based on alfalfa hay and a concentrate (60 : 40) supplemented with 0 (control diet) or 25 (SO diet) g of sunflower oil/kg of diet dry matter for 7 weeks. Milk composition, including FA profile, was analysed after 48 days on treatments. On day 49, the animals were euthanized and tissue samples were collected to analyse FA and mRNA abundance of 16 candidate genes. Feeding SO did not affect animal performance but modified milk FA composition. Major changes included decreases in the concentration of FA derived from de novo synthesis (e.g. 12:0, 14:0 and 16:0) and increases in that of long-chain FA (e.g. 18:0, c9-18:1, trans-18:1 isomers and c9,t11-CLA); however, they were not accompanied by significant variations in the mRNA abundance of the studied lipogenic genes (i.e. ACACA, FASN, LPL, CD36, FABP3, SCD1 and SCD5) and transcription factors (SREBF1 and PPARG), or in the constituent FA of mammary tissue. Regarding the FA composition of AT, the little influence of SO did not appear to be linked to changes in gene mRNA abundance (decreases of GPAM and SREBF1 in both tissues, and of PPARG in the subcutaneous depot). Similarly, the great variation between AT (higher contents of saturated FA and trans-18:1 isomers in the perirenal, and of cis-18:1, c9,t11-CLA and n-3 PUFA in the subcutaneous AT) could not be related to

  4. The Modulatory Effect of Ellagic Acid and Rosmarinic Acid on Ultraviolet-B-Induced Cytokine/Chemokine Gene Expression in Skin Keratinocyte (HaCaT Cells

    Directory of Open Access Journals (Sweden)

    Serena Lembo

    2014-01-01

    Full Text Available Ultraviolet radiation (UV induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA and rosmarinic acid (RA are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α gene expression in HaCaT cells after UVB irradiation. Cells were treated with UVB (100 mJ/cm2 and simultaneously with EA (5 μM in 0.1% DMSO or RA (2.7 μM in 0.5% DMSO. Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function.

  5. Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

    Institute of Scientific and Technical Information of China (English)

    DONG Jia; WEI Li-bin; HU Yan; GUO Wang-zhen

    2013-01-01

    Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACX and GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue-and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis (DPA), however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate (MeJA), cold, and abscisic acid (ABA) treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

  6. Structural Basis for Ether-a-go-go-Related Gene K+ Channel Subtype-Dependent Activation by Niflumic Acid[S

    Science.gov (United States)

    Fernandez, David; Sargent, John; Sachse, Frank B.; Sanguinetti, Michael C.

    2008-01-01

    Niflumic acid [2-((3-(trifluoromethyl)phenyl)amino)-3-pyridin-ecarboxylic acid, NFA] is a nonsteroidal anti-inflammatory drug that also blocks or modulates the gating of a wide spectrum of ion channels. Here we investigated the mechanism of channel activation by NFA on ether-a-go-go-related gene (ERG) K+ channel subtypes expressed in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. NFA acted from the extracellular side of the membrane to differentially enhance ERG channel currents independent of channel state. At 1 mM, NFA shifted the half-point for activation by −6, −18, and −11 mV for ERG1, ERG2, and ERG3 channels, respectively. The half-point for channel inactivation was shifted by +5 to +9 mV by NFA. The structural basis for the ERG subtype-specific response to NFA was explored with chimeric channels and site-directed mutagenesis. The molecular determinants of enhanced sensitivity of ERG2 channels to NFA were isolated to an Arg and a Thr triplet in the extracellular S3-S4 linker. PMID:18218980

  7. Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

    Science.gov (United States)

    Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

    2011-09-01

    A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

  8. A new low linolenic acid allele of GmFAD3A gene in soybean PE1690

    Science.gov (United States)

    Relative fatty acid content of soybean oil is about 12 % palmitic acid, 4 % stearic acid, 23 % oleic acid, 54 % linoleic acid, and 8 % linolenic acid. To improve oxidative stability and quality of oil, breeding programs have mainly focused on reducing saturated fatty acids, increasing oleic acid, an...

  9. An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems.

    Science.gov (United States)

    Lin, Song; Du, Fusheng; Wang, Yang; Ji, Shouping; Liang, Dehai; Yu, Lei; Li, Zichen

    2008-01-01

    Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.

  10. Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids

    OpenAIRE

    Liu, Weitian; Jiao, Hongmei; Murray, Nancy C.; O'Connor, Marion; Roelofs, Wendell L.

    2002-01-01

    Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, that makes E11 isomers of monounsaturated (E11-16 and E11-14) fatty acids and a diunsaturated (E9,E11-14) fatty acid. In the pheromone gland, the monoene pr...

  11. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600

    Directory of Open Access Journals (Sweden)

    Biaosheng Lin

    2016-01-01

    Full Text Available To clone and express the γ-polyglutamic acid (γ-PGA synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.

  12. Widespread Inter- and Intra-Domain Horizontal Gene Transfer of d-Amino Acid Metabolism Enzymes in Eukaryotes

    Science.gov (United States)

    Naranjo-Ortíz, Miguel A.; Brock, Matthias; Brunke, Sascha; Hube, Bernhard; Marcet-Houben, Marina; Gabaldón, Toni

    2016-01-01

    Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that d-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether d-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role. PMID:28066338

  13. Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expression.

    Science.gov (United States)

    Karabudak, T; Bor, M; Özdemir, F; Türkan, İ

    2014-03-01

    Cold stress is among the environmental stressors limiting productivity, yield and quality of agricultural plants. Tolerance to cold stress is associated with the increased unsaturated fatty acids ratio in the plant membranes which are also known to be substrates of octadecanoid pathway for jasmonate and other oxylipins biosynthesis. Accumulation of osmoprotectant, glycine betaine (GB) is well known to be effective in the protecting membranes and mitigating cold stress effects but, the mode of action is poorly understood. We studied the role of GB in cold stress responses of two tomato cultivated varieties; Gerry (cold stress sensitive) and T47657 (moderately cold stress tolerant) and compared the differences in lypoxygenase-13 (TomLOXF) and fatty acid desaturase 7 (FAD7) gene expression profiles and physiological parameters including relative growth rates, relative water content, osmotic potential, photosynthetic efficiency, membrane leakage, lipid peroxidation levels. Our results indicated that GB might have a role in inducing FAD7 and LOX expressions for providing protection against cold stress in tomato plants which could be related to the desaturation process of lipids leading to increased membrane stability and/or induction of other genes related to stress defense mechanisms via octadecanoid pathway or lipid peroxidation products.

  14. A nonsense mutation in the acid α-glucosidase gene causes Pompe disease in Finnish and Swedish Lapphunds.

    Directory of Open Access Journals (Sweden)

    Eija H Seppälä

    Full Text Available Pompe disease is a recessively inherited and often fatal disorder caused by the deficiency of acid α-glucosidase, an enzyme encoded by the GAA gene and needed to break down glycogen in lysosomes. This glycogen storage disease type II has been reported also in Swedish Lapphund dogs. Here we describe the genetic defect in canine Pompe disease and show that three related breeds from Scandinavia carry the same mutation. The affected dogs are homozygous for the GAA c.2237G>A mutation leading to a premature stop codon at amino acid position 746. The corresponding mutation has previously been reported in humans and causes infantile Pompe disease in combination with a second fully deleterious mutation. The affected dogs from both the Finnish as well as the Swedish breed mimic infantile-onset Pompe disease genetically, but also clinico-pathologically. Therefore this canine model provides a valuable tool for preclinical studies aimed at the development of gene therapy in Pompe disease.

  15. The maize (Zea mays ssp. mays var. B73 genome encodes 33 members of the purple acid phosphatase gene family

    Directory of Open Access Journals (Sweden)

    Eliécer eGonzález Muñoz

    2015-05-01

    Full Text Available Purple acid phosphatases (PAPs play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73 reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members.

  16. Next-generation re-sequencing of genes involved in increased platelet reactivity in diabetic patients on acetylsalicylic acid.

    Science.gov (United States)

    Postula, Marek; Janicki, Piotr K; Eyileten, Ceren; Rosiak, Marek; Kaplon-Cieslicka, Agnieszka; Sugino, Shigekazu; Wilimski, Radosław; Kosior, Dariusz A; Opolski, Grzegorz; Filipiak, Krzysztof J; Mirowska-Guzel, Dagmara

    2016-06-01

    The objective of this study was to investigate whether rare missense genetic variants in several genes related to platelet functions and acetylsalicylic acid (ASA) response are associated with the platelet reactivity in patients with diabetes type 2 (T2D) on ASA therapy. Fifty eight exons and corresponding introns of eight selected genes, including PTGS1, PTGS2, TXBAS1, PTGIS, ADRA2A, ADRA2B, TXBA2R, and P2RY1 were re-sequenced in 230 DNA samples from T2D patients by using a pooled PCR amplification and next-generation sequencing by Illumina HiSeq2000. The observed non-synonymous variants were confirmed by individual genotyping of 384 DNA samples comprising of the individuals from the original discovery pools and additional verification cohort of 154 ASA-treated T2DM patients. The association between investigated phenotypes (ASA induced changes in platelets reactivity by PFA-100, VerifyNow and serum thromboxane B2 level [sTxB2]), and accumulation of rare missense variants (genetic burden) in investigated genes was tested using statistical collapsing tests. We identified a total of 35 exonic variants, including 3 common missense variants, 15 rare missense variants, and 17 synonymous variants in 8 investigated genes. The rare missense variants exhibited statistically significant difference in the accumulation pattern between a group of patients with increased and normal platelet reactivity based on PFA-100 assay. Our study suggests that genetic burden of the rare functional variants in eight genes may contribute to differences in the platelet reactivity measured with the PFA-100 assay in the T2DM patients treated with ASA.

  17. Dietary conjugated linoleic acid modify gene expression in liver, muscles, and fat tissues of finishing pigs

    DEFF Research Database (Denmark)

    Tous, Nuria; Theil, Peter Kappel; Lauridsen, Charlotte

    2012-01-01

    in these tissues, except for SM intermuscular fat. Dietary CLA increased PPARγ in LT muscle (P tissues studied (P fat. Transcription of genes related to FA synthesis was reduced by CLA in SM muscle and liver (SREBP1......, both P fat. Saturated FA were increased in all studied tissues (P ....01), while monosaturated and polyunsaturated FA were reduced in a tissue-specific way by CLA. It was concluded that dietary CLA affected transcription of genes and fat metabolism in a tissue-specific manner....

  18. Mutations in soybean KASIIa gene are correlated with high levels of seed palmitic acid

    Science.gov (United States)

    A complete understanding of the biosynthetic pathways involved in the formation of soybean seed oils is required to develop lines with useful oil profiles. In particular, modification of the content of saturated fatty acids using genetics has been a target for soybean breeders for many years. One st...

  19. ADS genes for reducing saturated fatty acid levels in seed oils

    Science.gov (United States)

    Heilmann, Ingo H.; Shanklin, John

    2010-02-02

    The present invention relates to enzymes involved in lipid metabolism. In particular, the present invention provides coding sequences for Arabidopsis Desaturases (ADS), the encoded ADS polypeptides, and methods for using the sequences and encoded polypeptides, where such methods include decreasing and increasing saturated fatty acid content in plant seed oils.

  20. ENHANCED DISEASE SUSCEPTIBILITY 1 and SALICYLIC ACID act redundantly to regulate resistance gene-mediated signaling

    Science.gov (United States)

    Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), ...

  1. Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs.

    Directory of Open Access Journals (Sweden)

    Xiangfang Zeng

    Full Text Available Dietary modulation of the synthesis of endogenous host defense peptides (HDPs represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2, pBD3, epididymis protein 2 splicing variant C (pEP2C, and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.

  2. Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

    Science.gov (United States)

    Park, Duck Hwan; Mirabella, Rossana; Bronstein, Philip A; Preston, Gail M; Haring, Michel A; Lim, Chun Keun; Collmer, Alan; Schuurink, Robert C

    2010-10-01

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

  3. Submesoscale characteristics and transcription of a fatty acid elongase gene from a freshwater green microalgae, Myrmecia incisa Reisigl

    Science.gov (United States)

    Yu, Shuiyan; Liu, Shicheng; Li, Chunyang; Zhou, Zhigang

    2011-01-01

    Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4ω-6, δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chrétiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5'/3' rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5'-untranslated region (UTR) and a 431 bp 3'-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.

  4. Submesoscale characteristics and transcription of a fatty acid elongase gene from a freshwater green microalgae, Myrmecia incisa Reisigl

    Institute of Scientific and Technical Information of China (English)

    YU Shuiyan; LIU Shicheng; LI Chunyang; ZHOU Zhigang

    2011-01-01

    Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4ω-6,△,5,8, 11,14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chretiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5'/3' rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5'-untranslated region (UTR) and a 431 bp 3'-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.

  5. Acidic pH shock induces the expressions of a wide range of stress-response genes

    Directory of Open Access Journals (Sweden)

    Hong Soon-Kwang

    2008-12-01

    Full Text Available Abstract Background Environmental signals usually enhance secondary metabolite production in Streptomycetes by initiating complex signal transduction system. It is known that different sigma factors respond to different types of stresses, respectively in Streptomyces strains, which have a number of unique signal transduction mechanisms depending on the types of environmental shock. In this study, we wanted to know how a pH shock would affect the expression of various sigma factors and shock-related proteins in S. coelicolor A3(2. Results According to the results of transcriptional and proteomic analyses, the major number of sigma factor genes were upregulated by an acidic pH shock. Well-studied sigma factor genes of sigH (heat shock, sigR (oxidative stress, sigB (osmotic shock, and hrdD that play a major role in the secondary metabolism, were all strongly upregulated by the pH shock. A number of heat shock proteins including the DnaK family and chaperones such as GroEL2 were also observed to be upregulated by the pH shock, while their repressor of hspR was strongly downregulated. Oxidative stress-related proteins such as thioredoxin, catalase, superoxide dismutase, peroxidase, and osmotic shock-related protein such as vesicle synthases were also upregulated in overall. Conclusion From these observations, an acidic pH shock was considered to be one of the strongest stresses to influence a wide range of sigma factors and shock-related proteins including general stress response proteins. The upregulation of the sigma factors and shock proteins already found to be related to actinorhodin biosynthesis was considered to have contributed to enhanced actinorhodin productivity by mediating the pH shock signal to regulators or biosynthesis genes for actinorhodin production.

  6. Effects of receptor-selective retinoids on CYP26 gene expression and metabolism of all-trans-retinoic acid in intestinal cells.

    Science.gov (United States)

    Lampen, A; Meyer, S; Nau, H

    2001-05-01

    Retinoids mediate most of their function via interaction with retinoid receptors [retinoic acid receptors (RARs) and retinoid X receptors (RXRs)], which act as ligand-activated transcription factors controlling the expression of a number of target genes. The complex mechanistic pattern of retinoid-induced effects on gene expression of CYP26 and intestinal metabolism of all-trans-retinoic acid (RA) was investigated here by studying the effects of retinoid ligands with relative selectivity for binding and transactivation of the retinoid acid receptors, RARs and RXRs, in human intestinal Caco-2 cells. We show here that CYP26 is expressed in human duodenum and colon. In Caco-2 cells not only all-trans-RA but also synthetic agonists of the RAR induced intestinal CYP26 gene expression and all-trans-RA metabolism as well. The RARalpha ligand Am580 induced the CYP26 gene expression more than the RARbeta ligand CD2019 or the RARgamma ligand CD437 suggesting the highest specificity for RARalpha on intestinal CYP26 gene regulation. RXR ligands alone did not induce CYP26 gene expression or RA metabolism in Caco-2 cells at all. But together with the RARalpha ligand, Am580, there were enhanced effects on the induction of CYP26 gene expression and on the induction of the metabolism of all-trans-RA. We conclude that gene regulation of CYP26 and the metabolism of all-trans-RA in intestinal cells is regulated through RXR and RAR heterodimerization. When coadministered, RAR agonists showed the highest potency for CYP26 gene regulation. Receptor-selective retinoids showed enhanced effects on induction of CYP26 gene expression and all-trans-retinoic acid metabolism.

  7. Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the Functional tfdA Gene

    DEFF Research Database (Denmark)

    Bælum, Jacob; Prestat, Emmanuel; David, Maude M.

    2012-01-01

    Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five...... continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate...

  8. Intestinal short-chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA.

    Science.gov (United States)

    Lawhon, Sara D; Maurer, Russell; Suyemoto, Mitsu; Altier, Craig

    2002-12-01

    Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl-phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Delta(ackA-pta) mutation alone had no effect on virulence but, in combination with Delta(barA), it increased the oral LD50 24-fold. Thus, the combined loss of the BarA- and acetate-dependent pathways is required to reduce virulence. Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive

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

    Directory of Open Access Journals (Sweden)

    Honghao Chen

    2013-12-01

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

  10. Nano-polyplex based on oleoyl-carboxymethy-chitosan (OCMCS) and hyaluronic acid for oral gene vaccine delivery.

    Science.gov (United States)

    Liu, Ya; Wang, Fang-Qin; Shah, Zeana; Cheng, Xiao-Jie; Kong, Ming; Feng, Chao; Chen, Xi-Guang

    2016-09-01

    Here we described nano-polyplexes (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) as novel potential carriers for oral gene vaccines delivery. Aerolysin gene (aerA) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-HA/aerA (OHA) NPs. OHA NPs performed the optimal parameters, i.e. smallest (154.5±9.4nm), positive charged (+7.9±0.5mV) and monodispersed system with the N/P ratio of 5 and OCMCS/HA weight ratio of 4. Upon the introduction of HA, OHA NPs was beneficial for the DNA release in intestinal environments in comparison to OA NPs. The mean fluorescence intensity detected in Caco-2 cells incubated with OHA NPs was about 2.5-fold higher than that of OA NPs; however, it decreased significantly in the presence of excess free HA. The OHA NPs and OA NPs decreased the transepithelial electric resistance (TEER) of Caco-2 monolayers obviously and induced increasing the apparent permeability coefficient (Papp) of DNA by 5.45-6.09 folds compared with free DNA. Significantly higher (P<0.05) antigen-specific antibodies were detected in serum after orally immunized with OHA NPs than that immunized with OA NPs and DNA alone in carps. These results enable the OHA NPs might resolve challenges arising from gastrointestinal damage to gene antigens, and offer an approach applicable for oral vaccination.

  11. Overexpression of ubiquitin and amino acid permease genes in association with antimony resistance in Leishmania tropica field isolates.

    Science.gov (United States)

    Kazemi-Rad, Elham; Mohebali, Mehdi; Khadem-Erfan, Mohammad Bagher; Hajjaran, Homa; Hadighi, Ramtin; Khamesipour, Ali; Rezaie, Sassan; Saffari, Mojtaba; Raoofian, Reza; Heidari, Mansour

    2013-08-01

    The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

  12. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract

    Directory of Open Access Journals (Sweden)

    Sridar Chittur

    2011-01-01

    Full Text Available Chronic inflammation of the hair follicle (HF is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA. Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4 associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

  13. Retinoic acid regulates CD1d gene expression at the transcriptional level in human and rodent monocytic cells.

    Science.gov (United States)

    Chen, Qiuyan; Ross, A Catharine

    2007-04-01

    CD1d belongs to a group of nonclassical antigen-presenting molecules that present glycolipid antigens and thereby activate natural killer T (NKT) cells, a subset of bifunctional T cells. Little is known so far regarding the expression and physiologic regulation of CD1d. Here we show that all-trans-retinoic acid (RA), the active metabolite of vitamin A, rapidly (1 hr after treatment) increases CD1d mRNA in human and rodent monocytic cells at a physiologic dose (10 nM). The induction is RA specific and RA receptor (RAR) dependent-RA and an RARalpha agonist, Am580, both had a pronounced positive effect, whereas the addition of RARalpha antagonist partially blocked the increase in CD1d mRNA induced by RA and Am580. The induction was also completely blocked by the presence of actinomycin D. A putative RA-response element was identified in the distal 5' flanking region of the CD1d gene, which binds nuclear retinoid receptors and was responsive to RA in both gel mobility shift assay and transient transfection assay in THP-1 cells. These results further confirmed the transcriptional regulation of RA in CD1d gene expression. Moreover, RA significantly increased alpha-galactosylceramide-induced spleen cell proliferation. These studies together provide evidence for a previously unknown mechanism of CD1d gene expression regulation by RA and suggest that RA is a significant modulator of NKT cell activation.

  14. The HU regulon is composed of genes responding to anaerobiosis, acid stress, high osmolarity and SOS induction.

    Directory of Open Access Journals (Sweden)

    Jacques Oberto

    Full Text Available BACKGROUND: The Escherichia coli heterodimeric HU protein is a small DNA-bending protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking HU grow very poorly and display many phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the transcription profile of every Escherichia coli gene in the absence of one or both HU subunits. This genome-wide in silico transcriptomic approach, performed in parallel with in vivo genetic experimentation, defined the HU regulon. This large regulon, which comprises 8% of the genome, is composed of four biologically relevant gene classes whose regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS induction. CONCLUSIONS/SIGNIFICANCE: The regulation a large number of genes encoding enzymes involved in energy metabolism and catabolism pathways by HU explains the highly pleiotropic phenotype of HU-deficient cells. The uniform chromosomal distribution of the many operons regulated by HU strongly suggests that the transcriptional and nucleoid architectural functions of HU constitute two aspects of a unique protein-DNA interaction mechanism.

  15. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

    Science.gov (United States)

    Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

    2015-02-01

    Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

  16. Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model

    Institute of Scientific and Technical Information of China (English)

    Sailas BENJAMIN; Silke FLOTHO; Torsten B(O)RCHERS; Friedrich SPENER

    2013-01-01

    The purpose of this study was to examine the induction profiles(as judged by quantitative reverse transcription polymerase chain reaction(qRT-PCR))of peroxisome proliferator-activated receptor(PPAR)α,β,Y subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element(PPRE)in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid(9-CLA)or trans-10,cis-12-octadecadienoic acid (10-CLA)or their precursor fatty acids(FAs).HepG2 cells were treated with 100 μmol/L 9-CLA or 10-CLA or their precursor FAs,viz.,oleic,linoleic,and trans-11-vaccenic acids against bezafibrate control to evaluate the induction/expression profiles of PPAR α,β,Y subtypes and major PPAR-target genes bearing a functional PPRE,i.e.,fatty acid transporter(FAT),glucose transporter-2(GLUT-2),liver-type FA binding protein(L-FABP),acyl CoA oxidase-1 (ACOX-1),and peroxisomal bifunctional enzyme(PBE)with reference to β-actin as house keeping gene.Of the three housekeeping genes(glyceraldehyde 3-phosphate dehydrogenase(GAPDH),β-actin,and ubiquitin),β-actin was found to be stable.Dimethyl sulfoxide(DMSO),the common solubilizer of agonists,showed a significantly higher induction of genes analyzed.qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT,GLUT-2,and L-FABP(~0.5-2.0-fold).Compared to 10-CLA,9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE,while 10-CLA decreased the induction of PBE less than did ACOX-1.Both CLAs and precursor FAs upregulated PPRE-bearing genes,but with comparatively less or marginal activation of PPAR subtypes.This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation,thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE.To sum up,the expression profiles of these candidate genes showed that CLAs and their precursor FAs are involved in lipid

  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. Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation.

    Science.gov (United States)

    McDowell, J M; Cuzick, A; Can, C; Beynon, J; Dangl, J L; Holub, E B

    2000-06-01

    To better understand the genetic requirements for R gene-dependent defense activation in Arabidopsis, we tested the effect of several defense response mutants on resistance specified by eight RPP genes (for resistance to Peronospora parasitica) expressed in the Col-0 background. In most cases, resistance was not suppressed by a mutation in the SAR regulatory gene NPR1 or by expression of the NahG transgene. Thus, salicylic acid accumulation and NPR1 function are not necessary for resistance mediated by these RPP genes. In addition, resistance conferred by two of these genes, RPP7 and RPP8, was not significantly suppressed by mutations in either EDS1 or NDR1. RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1 which affect ethylene or jasmonic acid signaling. Double mutants were therefore tested. RPP7 and RPP8 were weakly suppressed in an eds1-2/ndr1-1 background, suggesting that these RPP genes operate additively through EDS1, NDR1 and as-yet-undefined signaling components. RPP7 was not compromised in coi1/npr1 or coi1/NahG backgrounds. These observations suggest that RPP7 initiates resistance through a novel signaling pathway that functions independently of salicylic acid accumulation or jasmonic acid response components.

  19. Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

    2014-01-01

    In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

  20. Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

    Science.gov (United States)

    Grigsby, Christopher L.; Ho, Yi-Ping; Lin, Chao; Engbersen, Johan F. J.; Leong, Kam W.

    2013-11-01

    As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

  1. Jinggangmycin increases fecundity of the brown planthopper, Nilaparvata lugens (Stål) via fatty acid synthase gene expression.

    Science.gov (United States)

    Li, Lei; Jiang, Yiping; Liu, Zongyu; You, Linlin; Wu, You; Xu, Bing; Ge, Linquan; Stanley, David; Song, Qisheng; Wu, Jincai

    2016-01-01

    The antibiotic jinggangmycin (JGM) is mainly used in controlling the rice sheath blight, Rhizoctonia solani, in China. JGM also enhances reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål). To date, however, molecular mechanisms of the enhancement are unclear. Our related report documented the influence of foliar JGM sprays on ovarian protein content. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) protocols to analyze ovarian proteins of BPH females following JGM spray (JGM-S) and topical application (JGM-T). We recorded changes in expression of 284 proteins (142↑ and 142↓) in JGM-S compared to the JGM-S control group (S-control); 267 proteins were differentially expressed (130↑ and 137↓) in JGM-T compared to the JGM-T control group (T-control), of which, 22 proteins were up-regulated in both groups. Comparing the JGM-S to the JGM-T group, 114 proteins were differentially expressed (62↑ and 52↓). Based on the biological significance of fatty acids, pathway annotation and enrichment analysis, we designed a dsRNA construct to silence a gene encoding fatty acid synthase (FAS). FAS was more highly expressed in JGM-S vs S-control and JGM-S vs JGM-T groups. The dsFAS treatment reduced fecundity by about 46% and reduced ovarian and fat body fatty acid concentrations in JGM-S-treated females relative to controls. We infer FAS provides critically needed fatty acids to support JGM-enhanced fecundity in BPH.

  2. Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment

    Science.gov (United States)

    Henriques, Ana C.; Azevedo, Rui M.S.

    2016-01-01

    Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea. PMID:27761315

  3. A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

    Science.gov (United States)

    Zeng, Xin; Pan, Shirong; Li, Jie; Wang, Chi; Wen, Yuting; Wu, Hongmei; Wang, Cuifeng; Wu, Chuanbin; Feng, Min

    2011-09-01

    Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

  4. Abscisic acid regulates pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol accumulation in developing flax (Linum usitatissimum L.) seeds.

    Science.gov (United States)

    Renouard, Sullivan; Corbin, Cyrielle; Lopez, Tatiana; Montguillon, Josiane; Gutierrez, Laurent; Lamblin, Frédéric; Lainé, Eric; Hano, Christophe

    2012-01-01

    Secoisolariciresinol diglucoside (SDG), the main phytoestrogenic lignan of Linum usitatissimum, is accumulated in the seed coat of flax during its development and pinoresinol-lariciresinol reductase (PLR) is a key enzyme in flax for its synthesis. The promoter of LuPLR1, a flax gene encoding a pinoresinol lariciresinol reductase, contains putative regulatory boxes related to transcription activation by abscisic acid (ABA). Gel mobility shift experiments evidenced an interaction of nuclear proteins extracted from immature flax seed coat with a putative cis-acting element involved in ABA response. As ABA regulates a number of physiological events during seed development and maturation we have investigated its involvement in the regulation of this lignan synthesis by different means. ABA and SDG accumulation time courses in the seed as well as LuPLR1 expression were first determined in natural conditions. These results showed that ABA timing and localization of accumulation in the flax seed coat could be correlated with the LuPLR1 gene expression and SDG biosynthesis. Experimental modulations of ABA levels were performed by exogenous application of ABA or fluridone, an inhibitor of ABA synthesis. When submitted to exogenous ABA, immature seeds synthesized 3-times more SDG, whereas synthesis of SDG was reduced in immature seeds treated with fluridone. Similarly, the expression of LuPLR1 gene in the seed coat was up-regulated by exogenous ABA and down-regulated when fluridone was applied. These results demonstrate that SDG biosynthesis in the flax seed coat is positively controlled by ABA through the transcriptional regulation of LuPLR1 gene.

  5. Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits.

    Directory of Open Access Journals (Sweden)

    Yu-Li Liu

    Full Text Available D-amino acid oxidase (DAO has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016. This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001 and replicated samples (corrected p = 0.0003. The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.

  6. A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

    Science.gov (United States)

    Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

    2009-05-01

    Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

  7. Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment.

    Science.gov (United States)

    Henriques, Ana C; Azevedo, Rui M S; De Marco, Paolo

    2016-01-01

    Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea.

  8. Plasma fatty acid ratios affect blood gene expression profiles--a cross-sectional study of the Norwegian Women and Cancer Post-Genome Cohort.

    Science.gov (United States)

    Olsen, Karina Standahl; Fenton, Christopher; Frøyland, Livar; Waaseth, Marit; Paulssen, Ruth H; Lund, Eiliv

    2013-01-01

    High blood concentrations of n-6 fatty acids (FAs) relative to n-3 FAs may lead to a "physiological switch" towards permanent low-grade inflammation, potentially influencing the onset of cardiovascular and inflammatory diseases, as well as cancer. To explore the potential effects of FA ratios prior to disease onset, we measured blood gene expression profiles and plasma FA ratios (linoleic acid/alpha-linolenic acid, LA/ALA; arachidonic acid/eicosapentaenoic acid, AA/EPA; and total n-6/n-3) in a cross-section of middle-aged Norwegian women (n = 227). After arranging samples from the highest values to the lowest for all three FA ratios (LA/ALA, AA/EPA and total n-6/n-3), the highest and lowest deciles of samples were compared. Differences in gene expression profiles were assessed by single-gene and pathway-level analyses. The LA/ALA ratio had the largest impact on gene expression profiles, with 135 differentially expressed genes, followed by the total n-6/n-3 ratio (125 genes) and the AA/EPA ratio (72 genes). All FA ratios were associated with genes related to immune processes, with a tendency for increased pro-inflammatory signaling in the highest FA ratio deciles. Lipid metabolism related to peroxisome proliferator-activated receptor γ (PPARγ) signaling was modified, with possible implications for foam cell formation and development of cardiovascular diseases. We identified higher expression levels of several autophagy marker genes, mainly in the lowest LA/ALA decile. This finding may point to the regulation of autophagy as a novel aspect of FA biology which warrants further study. Lastly, all FA ratios were associated with gene sets that included targets of specific microRNAs, and gene sets containing common promoter motifs that did not match any known transcription factors. We conclude that plasma FA ratios are associated with differences in blood gene expression profiles in this free-living population, and that affected genes and pathways may influence the

  9. Plasma fatty acid ratios affect blood gene expression profiles--a cross-sectional study of the Norwegian Women and Cancer Post-Genome Cohort.

    Directory of Open Access Journals (Sweden)

    Karina Standahl Olsen

    Full Text Available High blood concentrations of n-6 fatty acids (FAs relative to n-3 FAs may lead to a "physiological switch" towards permanent low-grade inflammation, potentially influencing the onset of cardiovascular and inflammatory diseases, as well as cancer. To explore the potential effects of FA ratios prior to disease onset, we measured blood gene expression profiles and plasma FA ratios (linoleic acid/alpha-linolenic acid, LA/ALA; arachidonic acid/eicosapentaenoic acid, AA/EPA; and total n-6/n-3 in a cross-section of middle-aged Norwegian women (n = 227. After arranging samples from the highest values to the lowest for all three FA ratios (LA/ALA, AA/EPA and total n-6/n-3, the highest and lowest deciles of samples were compared. Differences in gene expression profiles were assessed by single-gene and pathway-level analyses. The LA/ALA ratio had the largest impact on gene expression profiles, with 135 differentially expressed genes, followed by the total n-6/n-3 ratio (125 genes and the AA/EPA ratio (72 genes. All FA ratios were associated with genes related to immune processes, with a tendency for increased pro-inflammatory signaling in the highest FA ratio deciles. Lipid metabolism related to peroxisome proliferator-activated receptor γ (PPARγ signaling was modified, with possible implications for foam cell formation and development of cardiovascular diseases. We identified higher expression levels of several autophagy marker genes, mainly in the lowest LA/ALA decile. This finding may point to the regulation of autophagy as a novel aspect of FA biology which warrants further study. Lastly, all FA ratios were associated with gene sets that included targets of specific microRNAs, and gene sets containing common promoter motifs that did not match any known transcription factors. We conclude that plasma FA ratios are associated with differences in blood gene expression profiles in this free-living population, and that affected genes and pathways may

  10. Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

    Science.gov (United States)

    Tokuyama, S; Hatano, K

    1995-03-01

    The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    Science.gov (United States)

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  12. Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

    Directory of Open Access Journals (Sweden)

    Tschaplinski Timothy J

    2012-09-01

    Full Text Available Abstract Background Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para

  13. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    Directory of Open Access Journals (Sweden)

    Xiaojian Yang

    Full Text Available BACKGROUND: Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0 and high bile salt (0.3-1.5% and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7 CFU/chick or phosphate-buffered saline (PBS at 1 day of age followed by Salmonella challenge (10(4 CFU/chick next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1. These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10 in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. CONCLUSIONS/SIGNIFICANCE: The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in

  14. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

    OpenAIRE

    Creelman, R A; Tierney, M L; Mullet, J E

    1992-01-01

    Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to ...

  15. Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.

    Science.gov (United States)

    Jensen, Kristian K; Previs, Stephen F; Zhu, Lei; Herath, Kithsiri; Wang, Sheng-Ping; Bhat, Gowri; Hu, Guanghui; Miller, Paul L; McLaren, David G; Shin, Myung K; Vogt, Thomas F; Wang, Liangsu; Wong, Kenny K; Roddy, Thomas P; Johns, Douglas G; Hubbard, Brian K

    2012-01-15

    The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids