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Sample records for hydroxylase genes lysine

  1. Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Uzawa, K.; Grzesik, W. J.; Nishiura, T.; Kuznetsov, S. A.; Robey, P. G.; Brenner, D. A.; Yamauchi, M.

    1999-01-01

    The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

  2. Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.

    PubMed Central

    Thariath, A; Socha, D; Valvano, M A; Viswanatha, T

    1993-01-01

    The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8423134

  3. Pitx2 Regulates Procollagen Lysyl Hydroxylase (Plod) Gene Expression

    PubMed Central

    Hjalt, Tord A.; Amendt, Brad A.; Murray, Jeffrey C.

    2001-01-01

    The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1–luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI. PMID:11157981

  4. Association between Tryptophan Hydroxylase 2 Gene Polymorphism and Completed Suicide

    ERIC Educational Resources Information Center

    Fudalej, Sylwia; Ilgen, Mark; Fudalej, Marcin; Kostrzewa, Grazyna; Barry, Kristen; Wojnar, Marcin; Krajewski, Pawel; Blow, Frederic; Ploski, Rafal

    2010-01-01

    The association between suicide and a single nucleotide polymorphism (rs1386483) was examined in the recently identified tryptophan hydroxylase 2 (TPH2) gene. Blood samples of 143 suicide victims and 162 age- and sex-matched controls were examined. The frequency of the TT genotype in the TPH2 polymorphism was higher in suicide victims than in…

  5. Examining the Impact of Gene Variants on Histone Lysine Methylation

    PubMed Central

    Van Rechem, Capucine; Whetstine, Johnathan R.

    2015-01-01

    In recent years, there has been a boom in the amount of genome-wide sequencing data that has uncovered important and unappreciated links between certain genes, families of genes and enzymatic processes and diseases such as cancer. Such studies have highlighted the impact that chromatin modifying enzymes could have in cancer and other genetic diseases. In this review, we summarize characterized mutations and single nucleotide polymorphisms (SNPs) in histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and histones. We primarily focus on variants with strong disease correlations and discuss how they could impact histone lysine methylation dynamics and gene regulation. PMID:24859469

  6. Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds.

    PubMed

    Lourenco-Jaramillo, Diana Lelidett; Sifuentes-Rincón, Ana María; Parra-Bracamonte, Gaspar Manuel; de la Rosa-Reyna, Xochitl Fabiola; Segura-Cabrera, Aldo; Arellano-Vera, Williams

    2012-04-01

    DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds. PMID:22888292

  7. Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds

    PubMed Central

    Lourenco-Jaramillo, Diana Lelidett; Sifuentes-Rincón, Ana María; Parra-Bracamonte, Gaspar Manuel; de la Rosa-Reyna, Xochitl Fabiola; Segura-Cabrera, Aldo; Arellano-Vera, Williams

    2012-01-01

    DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds. PMID:22888292

  8. Diverse alkane hydroxylase genes in microorganisms and environments

    PubMed Central

    Nie, Yong; Chi, Chang-Qiao; Fang, Hui; Liang, Jie-Liang; Lu, She-Lian; Lai, Guo-Li; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-01-01

    AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature. PMID:24829093

  9. Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes

    SciTech Connect

    Woo, S.L.C.; Dilella, A.G.

    1990-10-23

    This patent describes a method of detecting a mutation in a phenylalanine hydroxylase gene of human genomic DNA. Also described is an automated method of detecting PKU affected, PKU helerozgotes and normals in fetal to adult human samples.

  10. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains.

    PubMed

    Smits, T H; Röthlisberger, M; Witholt, B; van Beilen, J B

    1999-08-01

    We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C6-C11) or long-chain n-alkanes (C12-C16), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n-alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases. PMID:11207749

  11. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  12. Structure and expression of the human Lysyl hydroxylase gene (PLOD): Introns 9 and 16 contain Alu sequences at the sites of recombination in Ehlers-Danlos syndrome type VI patients

    SciTech Connect

    Heikkinen, J.; Hautala, T.; Kivirikko, K.I.

    1994-12-01

    Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5{prime} flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients. 21 refs., 2 figs., 2 tabs.

  13. Glucocorticoid status affects antidepressant regulation of locus coeruleus tyrosine hydroxylase and dorsal raphé tryptophan hydroxylase gene expression

    PubMed Central

    Heydendael, Willem; Jacobson, Lauren

    2009-01-01

    Brainstem monoaminergic nuclei express glucocorticoid receptors (GR), and glucocorticoids have been shown to inhibit expression of enzymes involved in monoamine synthesis. Monoamine deficits have been implicated in depression pathology. However, it is unknown if antidepressants regulate brainstem GR, and if glucocorticoids might influence antidepressant effects on monoamine-synthesizing enzymes. Our lab has found opposing effects of the monoamine oxidase inhibitor phenelzine and the tricyclic antidepressant imipramine on HPA activity and forebrain GR expression. We therefore hypothesized that phenelzine and imipramine would also affect brainstem GR gene expression differentially, and that antidepressant-induced changes in GR expression would correlate with effects on monoamine-synthesizing enzyme expression. Using in situ hybridization, we measured effects of chronic antidepressant treatment on brainstem GR, locus coeruleus and ventral tegmental area (VTA) tyrosine hydroxylase (TH), and dorsal raphé tryptophan hydroxylase (TPH2) gene expression in male C57BL/6 mice that were adrenalectomized and replaced with defined levels of corticosterone. GR expression was decreased by phenelzine in the locus coeruleus and decreased by imipramine in the dorsal raphé. Phenelzine increased locus coeruleus TH and imipramine increased dorsal raphé TPH2 gene expression in a glucocorticoid-dependent manner, suggesting that increases in these enzymes were due to relief of inhibitory glucocorticoid signaling. We did not find antidepressant effects on GR or TH expression in the VTA or on MR expression in any of the nuclei examined. Our findings represent a potential mechanism through which antidepressants and glucocorticoids could alter both HPA activity and mood via effects on brainstem GR, norepinephrine, and serotonin. PMID:19577549

  14. Characteristics of hydrocarbon hydroxylase genes in a thermophilic aerobic biological system treating oily produced wastewater.

    PubMed

    Liu, Ruyin; Gao, Yingxin; Ji, Yifeng; Zhang, Yu; Yang, Min

    2015-01-01

    Alkane and aromatic hydroxylase genes in a full-scale aerobic system treating oily produced wastewater under thermophilic condition (45-50 °C) in the Jidong oilfield, China, were investigated using clone library and quantitative polymerase chain reaction methods. Rather than the normally encountered integral-membrane non-haem iron monooxygenase (alkB) genes, only CYP153-type P450 hydroxylase genes were detected for the alkane activation, indicating that the terminal oxidation of alkanes might be mainly mediated by the CYP153-type alkane hydroxylases in the thermophilic aerobic process. Most of the obtained CYP153 gene clones showed distant homology with the reference sequences, which might represent novel alkane hydroxylases. For the aromatic activation, the polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) gene was derived from Gram-negative PAH-degraders belonging to the Burkholderiales order, with a 0.72% relative abundance of PAH-RHD gene to 16S rRNA gene. This was consistent with the result of 16S rRNA gene analysis, indicating that Burkholderiales bacteria might play a key role in the full-scale process of thermophilic hydrocarbon degradation.

  15. Genes encoding p-coumarate 3-hydroxylase (C3H) and methods of use

    DOEpatents

    Chapple, Clinton C. S.; Franke, Rochus; Ruegger, Max O.

    2006-07-04

    The present invention is directed to a method for altering secondary metabolism in plants, specifically phenylpropanoid metabolism. The present invention is further directed to a mutant p-coumarate 3-hydroxylase gene, referred to herein as the ref8 gene, its protein product which can be used to prepare gene constructs and transgenic plants. The gene constructs and transgenic plants are further aspects of the present invention.

  16. Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this research was to clone and characterize the expression pattern of a kenaf (Hibiscus cannabinus L.) F5H gene that encodes ferulate 5-hydroxylase in the phenylpropanoid pathway. Kenaf is well known as a fast growing dicotyledonous plant, which makes it a valuable biomass plant. The ...

  17. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

    PubMed Central

    Gibello, A; Ferrer, E; Sanz, J; Martin, M

    1995-01-01

    The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

  18. Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism

    SciTech Connect

    Nielsen, D.A.; Goldman, D. ); Dean, M. )

    1992-12-01

    The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformation polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase. 37 refs., 2 figs., 1 tab.

  19. Mapping and genotypic analysis of NK-lysin gene in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Antimicrobial peptides (AMP) are important elements of the first line of defence against pathogens in animals. NK-lysin is a cationic AMP that plays a critical role in innate immunity. The chicken NK-lysin gene has been cloned and its antimicrobial and anticancer activity has been descri...

  20. Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation.

    PubMed Central

    Kim, Y; Ayoubi, P; Harker, A R

    1996-01-01

    Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker and Y. Kim, Appl. Environ. Microbiol. 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis. Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities. Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment. Complementation with a cosmid-based gene bank constructed from A. eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment. Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities. Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii. The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction. Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions. PMID:8795212

  1. Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation.

    PubMed

    Kim, Y; Ayoubi, P; Harker, A R

    1996-09-01

    Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker and Y. Kim, Appl. Environ. Microbiol. 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis. Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities. Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment. Complementation with a cosmid-based gene bank constructed from A. eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment. Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities. Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii. The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction. Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions. PMID:8795212

  2. Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7a-hydroxylase gene variation

    SciTech Connect

    Lin, J.P.; Hanis, C.L.; Boerwinkle, E.

    1994-09-01

    Among Mexican Americans the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this study no formal genetic analysis of gallbladder disease had been carried out nor had any contributing gene been identified. The results of complex segregation analysis in a sample of 232 Mexican Americans with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes {open_quotes}AA{close_quotes}, {open_quotes}Aa{close_quotes}, and {open_quotes}aa{close_quotes}, respectively, and 0.68, 0.30, and 0.00 for males, respectively. Human cholesterol 7a-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7a-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. For loci in the 5{prime}-end of the cholesterol 7a-hydroxylase gene, the frequency of the susceptibility alleles was twice as high in gallbladder disease patients compared to controls. The results of a linkage analysis provide evidence that the cholesterol 7a-hydroxylase gene and the inferred gallbladder disease gene are genetically linked.

  3. Plant fatty acid hydroxylases

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  4. Seed-specific expression of the lysine-rich protein gene sb401 significantly increases both lysine and total protein content in maize seeds.

    PubMed

    Yu, Jingjuan; Peng, Peng; Zhang, Xiujun; Zhao, Qian; Zhu, Dengyun; Sun, Xuehui; Liu, Junqi; Ao, Guangming

    2005-12-01

    The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants, and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli by microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis, and its expression was confirmed by Western blot analysis. Quantification of the lysine and protein contents in R1 maize seeds showed that, compared with the nontransgenic maize control, the lysine content increased by 16.1% to 54.8% and the total protein content increased by 11.6% to 39.0%. There were no visible morphological changes in the vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programs aimed at improving the nutritional value of maize.

  5. Mapping and genotypic analysis of the NK-lysin gene in chicken

    PubMed Central

    2014-01-01

    Background Antimicrobial peptides (AMP) are important elements of the first line of defence against pathogens in animals. NK-lysin is a cationic AMP that plays a critical role in innate immunity. The chicken NK-lysin gene has been cloned and its antimicrobial and anticancer activity has been described but its location in the chicken genome remains unknown. Here, we mapped the NK-lysin gene and examined the distribution of a functionally significant single nucleotide polymorphism (SNP) among different chicken inbred lines and heritage breeds. Results A 6000 rad radiation hybrid panel (ChickRH6) was used to map the NK-lysin gene to the distal end of chromosome 22. Two additional genes, the adipocyte enhancer-binding protein 1-like gene (AEBP1) and the DNA polymerase delta subunit 2-like (POLD2) gene, are located in the same NW_003779909 contig as NK-lysin, and were thus indirectly mapped to chromosome 22 as well. Previously, we reported a functionally significant SNP at position 271 of the NK-lysin coding sequence in two different chicken breeds. Here, we examined this SNP and found that the A allele appears to be more common than the G allele in these heritage breeds and inbred lines. Conclusions The chicken NK-lysin gene mapped to the distal end of chromosome 22. Two additional genes, AEBP1 and POLD2, were indirectly mapped to chromosome 22 also. SNP analyses revealed that the A allele, which encodes a peptide with a higher antimicrobial activity, is more common than the G allele in our tested inbred lines and heritage breeds. PMID:25001618

  6. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    PubMed Central

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

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

    SciTech Connect

    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.

  8. [Unique steroid 21-hydroxylase gene CYP21A2 polymorphism in patients with hyperandrogenism signs].

    PubMed

    Barannik, A P; Lavrova, N V; Shilov, I A; Koltunova, A A; Ozolinia, L A; Patrushev, L I

    2012-01-01

    Hyperandrogenism is a medical condition characterized by excessive production of male sex hormones (androgens) in woman organism. One of the major causes of hyperandrogenism is the autosomal-recessive disorder--congenital adrenal hyperplasia (CAH). The mutational defects in the steroid 21-hydroxylase CYP21A2 gene causing steroid 21-hydroxylase deficiency account for over 90% of CAH cases. Our paper describes the sequencing results of entire CYP21A2 gene from 15 patients with hyperandrogenism signs, which had not nine most prevalent mutations associated with nonclassic CAH as it was previously established. 26 polymorphisms were found by sequencing among which 25 were known previously and 23 of them are referred to "normal" gene variants which do not associated with CAH. At the same time the gene of every patient had unique its own distinctive combination of polymorphisms. New SNP represents synonymous substitution C --> T in 3' part of exon 8. All detected SNPs are not regularly distributed but are clustered along the gene. Notably, they were found in the neighborhood of initiation and termination codons and near the intron-exon boundaries of introns 2, 6 and 8. We hypothesize that "normal" clinically insignificant per se SNPs in unique combinations may influence spatial structure of CYP21A2 mRNA or its pre-mRNA splicing efficiency and decrease gene expression level. This assumption may explain the mechanism of pathological phenotype development in our patients.

  9. Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats.

    PubMed

    Kim, J; Lee, K S; Kwon, D-H; Bong, J J; Jeong, J Y; Nam, Y S; Lee, M S; Liu, X; Baik, M

    2014-02-01

    Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77 days for 25 days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p < 0.05) food intake, but retarded (p < 0.05) growth, increased (p < 0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p < 0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p < 0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p > 0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism. PMID:23441935

  10. Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens

    PubMed Central

    Chen, Junfeng; Yang, Chingyuan; Tizioto, Polyana C.; Huang, Huan; Lee, Mi O. K.; Payne, Harold R.; Lawhon, Sara D.; Schroeder, Friedhelm; Taylor, Jeremy F.; Womack, James E.

    2016-01-01

    Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease. PMID:27409794

  11. Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens.

    PubMed

    Chen, Junfeng; Yang, Chingyuan; Tizioto, Polyana C; Huang, Huan; Lee, Mi O K; Payne, Harold R; Lawhon, Sara D; Schroeder, Friedhelm; Taylor, Jeremy F; Womack, James E

    2016-01-01

    Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease. PMID:27409794

  12. Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

    PubMed Central

    Garrad, R C; Bhattacharjee, J K

    1992-01-01

    The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1

  13. Premature termination codon at the sterol 27-hydroxylase gene causes cerebrotendinous xanthomatosis in a French family.

    PubMed

    Segev, H; Reshef, A; Clavey, V; Delbart, C; Routier, G; Leitersdorf, E

    1995-02-01

    Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.

  14. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene.

    PubMed

    Chang, Shu; Berman, Judit; Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.

  15. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

    PubMed Central

    Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

  16. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene.

    PubMed

    Chang, Shu; Berman, Judit; Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

  17. Transcriptome Analysis Reveals Key Flavonoid 3'-Hydroxylase and Flavonoid 3',5'-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis.

    PubMed

    Wei, Kang; Wang, Liyuan; Zhang, Chengcai; Wu, Liyun; Li, Hailin; Zhang, Fen; Cheng, Hao

    2015-01-01

    The ratio of dihydroxylated to trihydroxylated catechins (RDTC) is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp) data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3) were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

  18. Structure and Mechanisms of Lysine Methylation Recognition by the Chromodomain in Gene Transcription†

    PubMed Central

    Yap, Kyoko L.; Zhou, Ming-Ming

    2011-01-01

    Histone methylation recognition is accomplished by a number of evolutionarily conserved protein domains, including those belonging to the methylated lysine-binding Royal family of structural folds. One well-known member of the Royal family, the chromodomain, is found in the HP1/Chromobox and CHD subfamilies of proteins, in addition to a small number of other proteins that are involved in chromatin remodeling and gene transcriptional silencing. Here we discuss the structure and function of the chromodomain within these proteins as histone methylated lysine binders, and how the functions of these chromodomains can be modulated by additional post-translational modifications or binding to nucleic acids. PMID:21288002

  19. Sequence variation at the phenylalanine hydroxylase gene in the British Isles

    SciTech Connect

    Tyfield, L.A. |; Stephenson, A.; Cockburn, F.

    1997-02-01

    Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations. 38 refs., 2 tabs.

  20. Regulation of tyrosine hydroxylase gene expression during differentiation of neuroblastoma cells.

    PubMed

    Summerhill, E M; Wood, K; Fishman, M C

    1987-07-01

    Differentiation of N1E-115 neuroblastoma cells into neuron-like cells, with extension of neurites and acquisition of excitable membranes, can be induced by dimethyl sulfoxide (DMSO). We have found this differentiation to be accompanied by an increase in tyrosine hydroxylase (TH) mRNA, an increase disproportionate to changes in mRNAs for other measured, non-neuron-specific genes. The mRNA increases slowly over several days and falls gradually after removal of DMSO. Nuclear run-on studies suggest that a change in the rate of transcription cannot explain the increase in steady-state mRNA levels. TH mRNA half-life does, however, increase. This suggests that regulation is exerted in this case not at the level of transcription but rather at that of mRNA stability. PMID:2887236

  1. Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms.

    PubMed Central

    Tusié-Luna, M T; White, P C

    1995-01-01

    Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by mutations in the steroid 21-hydroxylase gene (CYP21). Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed "gene conversions," although the mechanism by which they are generated is not well understood. To assess the frequency at which these different recombination events occur, we have used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal individuals. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were never detected in the matching leukocyte DNA. Gene conversions in the same region occur in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggest that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, gene conversions must occur by a mechanism distinct from unequal crossing-over. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479886

  2. Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...

  3. Identification and cell type specificity of the tyrosine hydroxylase gene promoter.

    PubMed Central

    Harrington, C A; Lewis, E J; Krzemien, D; Chikaraishi, D M

    1987-01-01

    Genomic DNA encoding the rat tyrosine hydroxylase (TH) gene was isolated from a lambda phage library using a nick-translated fragment from a cDNA clone for rat TH. We have determined the initiation site for TH RNA synthesis and have sequenced 1100 bases of the primary transcript and 5' flanking region. The 5' end of the transcript is the same in several rat tissues in which TH is expressed as well as in rat pheochromocytoma cells (PC). RNA prepared from PC cells that had been stimulated with dexamethasone also mapped to the same transcription start site. Sequence upstream from the initiation site contains the canonical TATA box, but no apparent CAAT box. When a portion of the 5' flanking region of the TH gene (-773 to + 27) is fused to the chloramphenicol acetyltransferase (CAT) gene, it promotes expression of CAT in pheochromocytoma cells and GH4 cells, but not in two neural tumour lines, RT4-D and B103, nor in several non neural cell lines. This suggests that this region of the TH gene has features that confer tissue-restricted expression on the TH promoter. Images PMID:2882469

  4. Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco.

    PubMed

    Meng, Chen; Zhang, Song; Deng, Yong-Sheng; Wang, Guo-Dong; Kong, Fan-Ying

    2015-11-01

    Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3-hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was up-regulated under chilling (4 °C), heat (42 °C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O2(·-) levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress. PMID:26372946

  5. Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree.

    PubMed

    Kumar, Santosh; Omer, Sumita; Patel, Krunal; Khan, Bashir M

    2013-02-01

    Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age. PMID:23070917

  6. Prolyl 4-hydroxylase activity-responsive transcription factors: From hydroxylation to gene expression and neuroprotection

    PubMed Central

    Siddiq, Ambreena; Aminova, Leila R; Ratan, Rajiv R

    2008-01-01

    Most homeostatic processes including gene transcription occur as a result of deviations in physiological tone that threatens the survival of the organism. A prototypical homeostatic stress response includes changes in gene expression following alterations in oxygen, iron or 2-oxoglutarate levels. Each of these cofactors plays an important role in cellular metabolism. Accordingly, a family of enzymes known as the Prolyl 4-hydroxylase (PHD) enzymes are a group of dioxygenases that have evolved to sense changes in 2-oxoglutarate, oxygen and iron via changes in enzyme activity. Indeed, PHDs are a part of an established oxygen sensor system that regulates transcriptional regulation of hypoxia/stress-regulated genes and thus are an important component of events leading to cellular rescue from oxygen, iron or 2-oxoglutarate deprivations. The ability of PHD activity to regulate homeostatic responses to oxygen, iron or 2-oxoglutarate metabolism has led to the development of small molecule inhibitors of the PHDs as a strategy for activating or augmenting cellular stress responses. These small molecules are proving effective in preclinical models of stroke and Parkinson's disease. However the precise protective pathways engaged by PHD inhibition are only beginning to be defined. In the current review, we summarize the role of iron, 2-oxoglutarate and oxygen in the PHD catalyzed hydroxylation reaction and provide a brief discussion of some of the transcription factors that play an effective role in neuroprotection against oxidative stress as a result of changes in PHD activity. PMID:17981760

  7. Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alpha-hydroxylase gene expression in the proximal renal tubule.

    PubMed

    Zhang, Martin Y H; Wang, Xuemei; Wang, Jonathan T; Compagnone, Nathalie A; Mellon, Synthia H; Olson, Jean L; Tenenhouse, Harriet S; Miller, Walter L; Portale, Anthony A

    2002-02-01

    Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.

  8. New cyclodextrin derivative containing poly(L-lysine) dendrons for gene and drug co-delivery.

    PubMed

    Ma, Dong; Zhang, Hong-Bin; Chen, Yu-Yun; Lin, Jian-Tao; Zhang, Li-Ming

    2013-09-01

    To develop a multifunctional polymeric carrier for gene and drug co-delivery, a new cyclodextrin derivative containing poly(L-lysine) dendrons was prepared by the click conjugation of per-6-azido-β-cyclodextrin with propargyl focal point poly(L-lysine) dendron of third generation and then characterized by FTIR, (1)H NMR, and GPC analyses. It was found that such a conjugate could form colloidally stable nanocomplexes with plasmid DNA in aqueous system and exhibited high gene transfection efficiency. Moreover, it could load efficiently methotrexate drug with anticancer activity and showed a sustained release behavior. Different from commonly used amphiphilic copolymers with cationic character, the as obtained cyclodextrin derivative may be used directly for the combinatorial delivery of nucleic acid and lipophilic anticancer drugs without a complicated micellization process. PMID:23769303

  9. The role of tyrosine hydroxylase gene variants in suicide attempt in schizophrenia.

    PubMed

    Hu, Jiayi; Chan, Lai Fong; Souza, Renan P; Tampakeras, Maria; Kennedy, James L; Zai, Clement; De Luca, Vincenzo

    2014-01-24

    Evidence has shown that attempted suicide in psychiatric disorders is a complex interplay of genes and environment. Noradrenergic dysfunction due to abnormalities in the tyrosine hydroxylase (TH) gene has been implicated in the pathogenesis of suicidal behavior in mood disorders. However, suicide is a leading cause of mortality in schizophrenia too. Recent evidence suggests that TH gene variants may also increase the risk of suicide attempts in schizophrenia patients, although the interaction with established clinical risk factors is unclear. This study aimed to identify TH gene variants conferring risk for suicide attempt in schizophrenia while accounting for the interaction between this gene and clinical risk factors. We performed analysis on four TH SNPs (rs11564717, rs11042950, rs2070762, rs689) and the common TCAT repeat (UniSTS:240639) for 234 schizophrenia patients (51 suicide attempters and 183 non-attempters). Clinical risk factors and ethnic stratification were included as covariates. Single marker analysis identified the SNP rs11564717 (p=0.042) and the TCAT(6) (p=0.004) as risk variants for suicide attempt. We also identified the haplotype A-A-A-G as a risk factor for suicide attempt (p=0.0025). In conclusion, our findings suggest that TH polymorphisms may contribute to the risk of attempted suicide in schizophrenia even after accounting for established clinical risk factors and ethnic stratification. Further larger scale studies are needed to confirm these findings and to understand the mechanisms underlying the role of TH gene variants in suicide attempt in schizophrenia. PMID:24275212

  10. Clinical phenotype and mutation spectrum of the CYP21A2 gene in patients with steroid 21-hydroxylase deficiency.

    PubMed

    Choi, J-H; Jin, H-Y; Lee, B H; Ko, J M; Lee, J-J; Kim, G-H; Jung, C-W; Lee, J; Yoo, H-W

    2012-01-01

    Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.

  11. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    PubMed

    Norris, M L; Millhorn, D E

    1995-10-01

    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  12. Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja.

    PubMed

    Guo, Yong; Qiu, Li-Juan

    2013-06-01

    Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC1.14.3.21) and flavonoid 3',5'-hydroxylase (F3'5'H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.

  13. Histone H4 lysine 20 acetylation is associated with gene repression in human cells

    PubMed Central

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  14. Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

    PubMed

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  15. A new process for obtaining hydroxytyrosol using transformed Escherichia coli whole cells with phenol hydroxylase gene from Geobacillus thermoglucosidasius.

    PubMed

    Orenes-Piñero, Esteban; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2013-08-15

    Phenol hydroxylase gene cloning from the thermophilic bacteria Geobacillus thermoglucosidasius was used to develop an effective method to convert tyrosol into the high-added-value compound hydroxytyrosol by hydroxylation. Phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2 genes and strictly dependent on NADH and FAD. These two genes were subcloned together as a 2 kb fragment into Escherichia coli Rosetta cells, and the transformants were able to grow and effectively transform up to 5 mM of phenol and tyrosol using IPTG (isopropyl-β-D-thiogalactopyranoside) as inducer. In addition, when a new fragment with a 340 pb upstream pheA1 gene was subcloned, a similar biotransformation rate was attained without IPTG, confirming that this fragment encodes for a phenol hydroxylase promoter that can be recognised by E. coli. Both transformants brought about the total bioconversion of monophenols at a high concentration (5 mM), which represents an increase, both in concentration and in yield, compared with that previously described in the bibliography. The use of the transformant with its constitutive promoter was more interesting from a biotechnological point of view, since it is not necessary to use IPTG. It also gave rise to greater operational stability.

  16. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    SciTech Connect

    Day, D.J.; Barany, F.; Speiser, P.W.

    1995-09-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.

  17. Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.

    PubMed

    Millhorn, D E; Raymond, R; Conforti, L; Zhu, W; Beitner-Johnson, D; Filisko, T; Genter, M B; Kobayashi, S; Peng, M

    1997-02-01

    Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension. PMID:9027733

  18. Regulation of tyrosine hydroxylase gene expression during hypoxia: role of Ca2+ and PKC.

    PubMed

    Raymond, R; Millhorn, D

    1997-02-01

    Gene expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by reductions in oxygen tension (hypoxia). Hypoxia-induced regulation of the TH gene is due to the binding of specific transcription factors to specific sites on the 5' flanking region of the gene. The purpose of this study was to identify the second messenger system(s) responsible for regulation of the TH gene during hypoxia. Fura-2 fluorescence imaging of rat pheochromocytoma (PC12) cells, an O2-sensitive cell line, revealed that there is an increase in cytosolic calcium (Ca2+) associated with exposure to hypoxia. Based on the evidence that the transcription factors that bind to the TH promoter during hypoxia can also be induced by elevations in cytosolic Ca2+, the role of Ca2+ in the hypoxic regulation of the TH gene was explored. To assay the effect of hypoxia on TH gene expression, Northern blot analyses of total RNA were performed on PC12 cells exposed to hypoxia in the presence or absence of specific inhibitors. The addition of the L-type calcium channel blockers nifedipine or verapamil caused partial inhibition of the hypoxia-induced increase in TH mRNA. The increase in cytosolic Ca2+ during hypoxia was also only partially inhibited by addition of nifedipine. Importantly, chelation of extracellular Ca2+ completely inhibited the increase in TH mRNA by hypoxia. Pretreatment of PC12 cells with BAPTA/AM, an intracellular Ca2+ chelator, inhibited the hypoxic induction of TH gene expression in a dose-dependent manner. Addition of chelerythrine chloride (CHL), a protein kinase C inhibitor, to the media before exposure to hypoxia also resulted in an inhibition of TH induction by hypoxia. These results suggest that hypoxia regulates TH gene expression by a mechanism that is dependent on influx of calcium from the extracellular stores, partially but not exclusively through the L-type calcium channels. These results further suggest that a member of the

  19. Evolutionary conservation of an atypical glucocorticoid-responsive element in the human tyrosine hydroxylase gene.

    PubMed

    Sheela Rani, C S; Soto-Pina, Alexandra; Iacovitti, Lorraine; Strong, Randy

    2013-07-01

    The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at -7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein-1 (AP-1)-like motif in the rat TH gene. We cloned this hTH-CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH-CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5'-TGACTAA at -7243 bp completely abolished the Dex-stimulated Luc activity of hTH-CRII construct. The AP-1 agonist, tetradeconoyl-12,13-phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP-1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid-responsive element in a 7 bp AP-1-like motif in the promoter region at -7.24 kb of the human TH gene.

  20. Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

    PubMed

    Sun, Run-Ze; Pan, Qiu-Hong; Duan, Chang-Qing; Wang, Jun

    2015-12-01

    Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. PMID:26433636

  1. Serotonin Transporter and Tryptophan Hydroxylase Gene Variations Mediate Working Memory Deficits of Cocaine Users.

    PubMed

    Havranek, Michael M; Vonmoos, Matthias; Müller, Christian P; Büetiger, Jessica R; Tasiudi, Eve; Hulka, Lea M; Preller, Katrin H; Mössner, Rainald; Grünblatt, Edna; Seifritz, Erich; Quednow, Boris B

    2015-12-01

    Cocaine users consistently develop working memory (WM) impairments but the mediating molecular mechanisms are unknown so far. Recent evidence suggests that the serotonin (5-HT) system is altered by chronic cocaine use, while also being involved in WM processing. Thus, we investigated the effects of genetic variations impacting 5-HT activity and of peripheral 5-HT transporter (5-HTT) mRNA expression on WM performance in cocaine users and stimulant naive controls. Two hundred twenty participants (126 cocaine users, 94 controls) were assessed with visuospatial, spatial, and verbal WM tasks, genotyped for the length polymorphism in the promoter region of the 5-HTT (5-HTTLPR), the variable number of tandem repeats in the second intron of the 5-HTT (VNTR In2), two single-nucleotide polymorphisms (rs4570625 and rs1386497) in the tryptophan hydroxylase-2 (TPH2) gene and quantified for peripheral 5-HTT mRNA expression in whole-blood samples. Several significant gene × environment interactions between 5-HT genotypes and cocaine use on WM emerged: in cocaine users, the long/long (5-HTTLPR), 9+10/9+10 (VNTR In2) and C/C (TPH2 rs1386497) genotypes were risk alleles for WM impairments, whereas in healthy controls these polymorphisms were associated with improved WM performance. Analogously, high 5-HTT mRNA levels were associated with worse executive WM performance in cocaine users but with increased performance in controls. These gene × environment interactions suggest that the 5-HT system has an important role in the development of cognitive deficits in chronic cocaine users. Hence, pharmacological compounds targeting 5-HT neurotransmission might be promising for the treatment of cognitive deficits in cocaine dependence. PMID:26013962

  2. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    NASA Technical Reports Server (NTRS)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  3. Regulation of tyrosine hydroxylase gene expression in the rat carotid body by hypoxia.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Lawson, E E; Millhorn, D E

    1992-04-01

    The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells. PMID:1347783

  4. Modification of flower colour by suppressing β-ring carotene hydroxylase genes in Oncidium.

    PubMed

    Wang, H-M; To, K-Y; Lai, H-M; Jeng, S-T

    2016-03-01

    Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow. PMID:26404515

  5. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  6. Mutations of the phenylalanine hydroxylase gene in Iranian patients with phenylketonuria.

    PubMed

    Biglari, Alireza; Saffari, Fatemeh; Rashvand, Zahra; Alizadeh, Safarali; Najafipour, Reza; Sahmani, Mehdi

    2015-01-01

    Phenylketonuria (PKU) is an autosomal recessive disease which results from mutations in the phenylalanine hydroxylase (PAH) gene. The aim of this study was the identification of sixteen different mutations in Iranian patients with hyperphenylalaninemia. The mutations were detected during the characterization of PAH genotypes of 39 PKU patients from Qazvin and Zanjan provinces of Iran. PAH mutations have been analyzed by PCR and direct sequencing of PCR products of the promoter region and all 13 exons of PAH gene, including the splicing sites. A mutation detection rate of 74.3 % was realized. Two mutations were found at high frequencies: R176X (10.25 %) and p.P281L (10.25 %). The frequencies of the other mutations were: IVS2+5G>A (2.56 %), IVS2+5G>C (2.56 %), p.L48S (2.56 %), p.R243Q (2.56 %), p.R252Q (5.12 %), p.R261Q (7.69 %), p.R261X (5.12 %), p.E280K (2.56 %), p.I283N (2.56 %), IVS9+5G>A (2.56 %), IVS9+1G>A (1.28 %), IVS11+1G>C (1.28 %), p.C357R (1.28 %), c.632delC (2.56 %). The present results confirm the high heterogeneity of the PAH locus and contribute to information about the distribution and frequency of PKU mutations in the Iranian population. PMID:26413448

  7. Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

    SciTech Connect

    Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. ); Konecki, D.S.; Lichter-Konecki, U.

    1992-09-01

    The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.

  8. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    SciTech Connect

    Guldberg, P.; Henriksen, K.F.; Guettler, F.

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  9. Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

    PubMed Central

    2011-01-01

    Background Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2). We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH. Methods A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay. Results Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6%) chromosomes. The most frequent mutation was I2 splice (27%) followed by Ile173Asn (26%), Arg 357 Trp (19%), Gln319stop, 16% and Leu308InsT (12%), whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state. Conclusion Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined. PMID:21329531

  10. Modification of flower colour by suppressing β-ring carotene hydroxylase genes in Oncidium.

    PubMed

    Wang, H-M; To, K-Y; Lai, H-M; Jeng, S-T

    2016-03-01

    Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow.

  11. Isoform of castor oleate hydroxylase

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2005-12-13

    The present invention relates to oleate hydroxylase genes, proteins, and methods of their use. The present invention also relates to methods of using the oleate hydroxylase genes and proteins, including in their expression in transgenic organisms and in the production of hydroxylated fatty acids.

  12. Cloning and Characterization of a Flavonoid 3'-Hydroxylase Gene from Tea Plant (Camellia sinensis).

    PubMed

    Zhou, Tian-Shan; Zhou, Rui; Yu, You-Ben; Xiao, Yao; Li, Dong-Hua; Xiao, Bin; Yu, Oliver; Yang, Ya-Jun

    2016-02-22

    Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.

  13. Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis)

    PubMed Central

    Zhou, Tian-Shan; Zhou, Rui; Yu, You-Ben; Xiao, Yao; Li, Dong-Hua; Xiao, Bin; Yu, Oliver; Yang, Ya-Jun

    2016-01-01

    Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves. PMID:26907264

  14. Population genetics of a functional variant of the dopamine beta-hydroxylase gene (DBH).

    PubMed

    Cubells, J F; Kobayashi, K; Nagatsu, T; Kidd, K K; Kidd, J R; Calafell, F; Kranzler, H R; Ichinose, H; Gelernter, J

    1997-07-25

    Dopamine beta-hydroxylase (E.C. 1.14.17.1; protein abbreviation: DbetaH) catalyzes conversion of dopamine to norepinephrine. Previous work identified two expressed alleles of the gene encoding DbetaH (locus symbol DBH), containing either G or T at nucleotide position 910, resulting in specification by codon 304 of alanine (DBH*304A) or serine (DBH*304S), respectively. The current study employed denaturing gradient gel electrophoresis to identify these alleles, and after developing a PCR RFLP for rapid genotyping, estimated the frequencies of the alleles in African-Americans, European-Americans, and in several geographically dispersed populations (Mbuti, Danes, Adygei, Chinese, Japanese, Surui, Maya, and Nasioi). DBH*304A was the most common allele in all populations tested, with allele frequencies greater than 0.80 in each case. There was significant heterogeneity in allele frequency across population groups. The DBH*304S allele was most common in subjects of African descent, and least common in East Asians and individuals from indigenous populations of North and South America. The frequency of DBH*304S was significantly higher in African-Americans (0.16) than in European-Americans (0.06; P < 0.004). Of the four DBH*304S homozygotes observed, all were Europeans and three of the four were Danes. Based on empirical P-values generated by computer simulation, the observed proportions of DBH*304S homozygotes did not differ significantly from Hardy-Weinberg expectations in any of the populations after Bonferroni correction for multiple comparisons. The observation of significant heterogeneity in DBH*304S allele frequency across different population samples demonstrates the importance of controlling for population stratification in future studies testing for associations between DBH*304S and clinical phenotypes. PMID:9259372

  15. Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

    PubMed

    Paisse, Sandrine; Duran, Robert; Coulon, Frédéric; Goñi-Urriza, Marisol

    2011-11-01

    The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination. PMID:21660544

  16. Biodegradable nanoparticles composed of dendrigraft poly-L-lysine for gene delivery.

    PubMed

    Kodama, Yukinobu; Nakamura, Tadahiro; Kurosaki, Tomoaki; Egashira, Kanoko; Mine, Toyoharu; Nakagawa, Hiroo; Muro, Takahiro; Kitahara, Takashi; Higuchi, Norihide; Sasaki, Hitoshi

    2014-08-01

    We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy.

  17. Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

    NASA Astrophysics Data System (ADS)

    Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

    2010-07-01

    Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

  18. Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17-20-lyase deficiency in two french canadian patients

    SciTech Connect

    LaFlamme, N.; Leblanc, J.F.; Mailloux, J.

    1996-01-01

    Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21{alpha}-hydroxylase and 11{beta}-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17{alpha}-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17{alpha}-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg{sup 96} (CGG) into a Trp (TGG) in exon 1. Both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17{alpha}-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 {alpha}-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17 enzyme. 31 refs., 4 figs., 1 tab.

  19. Transcript encoded on the opposite strand of the human steroid 21-hydroxylase/complement component C4 gene locus.

    PubMed Central

    Morel, Y; Bristow, J; Gitelman, S E; Miller, W L

    1989-01-01

    The gene encoding human adrenal steroid 21-hydroxylase (P450c21) and its highly similar pseudogene are duplicated in tandem with the two genes encoding the fourth component of human serum hemolytic complement (C4). This 60-kilobase gene complex, which lies within the major histocompatibility complex on the short arm of human chromosome 6, has been studied in considerable detail because genetic disorders in steroid 21-hydroxylation and in C4 are common. We have cloned a cDNA encoded by a previously unidentified gene in this region. This gene lies on the strand of DNA opposite from the strand containing the P450c21 and C4 genes, and it overlaps the last exon of P450c21. The newly identified gene encodes mRNAs of 3.5 and 1.8 kilobases that are expressed in the adrenal and in a Leydig cell tumor but are not expressed in nonsteroidogenic tissues. The sequence of the longest cDNA (2.7 kilobases) shows no similarity to known sequences available in two computerized data bases. The 5' end of this sequence is characterized by three repeats, each encoding about 100 amino acids flanked by potential sites for proteolytic cleavage. Although numerous studies have shown that gene deletions causing congenital adrenal hyperplasia occur in this region, none of these gene deletions extends into this newly identified gene, suggesting that it encodes an essential function. Images PMID:2475872

  20. Dietary lysine affected the expression of genes related to lipid metabolism in skeletal muscle of finishing pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been reported that some amino acids can function as signaling molecules to regulate skeletal muscle growth in mammals. This study was conducted to identify those genes that may be regulated by amino acid lysine and responsible for muscle growth and meat quality of pigs. Nine crossbred barrows...

  1. Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro.

    PubMed

    Wang, Xuemei; Zhang, Martin Y H; Miller, Walter L; Portale, Anthony A

    2002-06-01

    The rate-limiting, hormonally regulated step in the biological activation of vitamin D is its 1alpha-hydroxylation to 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] in the kidney, catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1alpha. We previously cloned the human P450c1alpha cDNA and gene, and identified 14 different mutations, including 7 missense, in 19 patients with 1alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1. None of the missense mutations encoded a protein with detectable enzymatic activity in vitro. Although there is phenotypic variation among such patients, the molecular basis of this variation is unknown. We analyzed 6 additional patients with clinical and radiographic features of rickets; in 4 patients the laboratory abnormalities were typical of 1alpha-hydroxylase deficiency, but in 2 they were unusually mild [mild hypocalcemia and normal serum 1,25-(OH)(2)D concentration]. Direct sequencing revealed that all patients had P450c1alpha mutations on both alleles. Five new and 2 known mutations were identified. The new mutations included a 5-bp deletion with a 6-bp novel insertion causing a frameshift in exon 2, and a G to A change at +1 of intron 2; a minigene experiment proved that this intronic mutation prevented proper splicing. Three new missense mutations were found and tested by expressing the mutant cDNA in mouse Leydig MA-10 cells. The R389G mutant was totally inactive, but mutant L343F retained 2.3% of wild-type activity, and mutant E189G retained 22% of wild-type activity. The two mutations that confer partial enzyme activity in vitro were found in the 2 patents with mild laboratory abnormalities, suggesting that such mutations contribute to the phenotypic variation observed in patients with 1alpha-hydroxylase deficiency.

  2. Disordered regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase gene expression by phosphorus in X-linked hypophosphatemic (hyp) mice.

    PubMed

    Azam, Nasreen; Zhang, Martin Y H; Wang, Xuemei; Tenenhouse, Harriet S; Portale, Anthony A

    2003-08-01

    X-linked hypophosphatemic (Hyp) mice exhibit hypophosphatemia, impaired renal phosphate reabsorption, defective skeletal mineralization, and disordered regulation of vitamin D metabolism: In Hyp mice, restriction of dietary phosphorus induces a decrease in serum concentration of 1,25-dihydroxyvitamin D and renal activity of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), and induces an increase in renal activity of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). In contrast, in wild-type mice, phosphorus restriction stimulates renal 1alpha-hydroxylase gene expression and suppresses that of 24-hydroxylase. To determine the molecular basis for the disordered regulation of vitamin D metabolism in Hyp mice, we determined renal mitochondrial 1alpha-hydroxylase activity and the renal abundance of p450c1alpha and p450c24 mRNA in wild-type and Hyp mice fed either control, low-, or high-phosphorus diets for 5 d. In wild-type mice, phosphorus restriction increased 1alpha-hydroxylase activity and p450c1alpha mRNA expression by 6-fold and 3-fold, respectively, whereas in the Hyp strain the same diet induced changes of similar magnitude but opposite in direction. Phosphorus supplementation was without effect in wild-type mice, whereas in Hyp mice the same diet induced 3-fold and 2-fold increases, respectively, in enzyme activity and p450c1alpha mRNA abundance. In wild-type mice, both renal 1alpha-hydroxylase activity and p450c1alpha mRNA abundance varied inversely and significantly with serum phosphorus concentrations, whereas in Hyp mice the relationship between both renal parameters and serum phosphorus concentration was direct. In Hyp mice, phosphorus restriction induced a significant increase in renal p450c24 mRNA abundance, in contrast to the lack of effect observed in wild-type mice. The present findings demonstrate that regulation of both the p450c1alpha and p45024 genes by phosphorus is disordered in Hyp mice at the level of renal 1alpha-hydroxylase

  3. Gene delivery to neuroblastoma cells by poly (l-lysine)-grafted low molecular weight polyethylenimine copolymers.

    PubMed

    Askarian, Saeedeh; Abnous, Khalil; Darroudi, Majid; Oskuee, Reza Kazemi; Ramezani, Mohammad

    2016-07-01

    Polyethylenimine (PEI) and poly (l-lysine) (PLL) are among the most investigated non-viral gene carriers. However, both polymers contain deficiencies that restrict their applications. In the present study, we synthesized PLL-alkyl-PEI conjugates via 6-carbon alkyl linker and investigated their possible advantages in gene delivery. Four PLL copolymers were synthesized with different molecular weights and ratios of PEI. The physiochemical properties of synthesized conjugates such as size, zeta potential, DNA condensation ability, buffering capacity and cytotoxicity were investigated. Renilla luciferase assay was employed to evaluate the gene transfection efficiency of pDNA-polymer to Neuro2A cell line. DNA condensation and particle size measurements showed that new PLL-PEI conjugates could form polyplexes in nano-scale size in the range of 99-122 nm and were able to condense DNA at low concentration. While cytotoxicity reduced in some groups, the transfection efficiency increased about 2.8 and 4 fold as compared to the unmodified PEI 1.8 kDa and 10 kDa, respectively. The results of the present study showed that the chemical modifications of PEI with PLL could significantly improve transfection efficiency and PLP10-10% shows the most promise as a new gene carrier. PMID:27118207

  4. Distribution of the C1473G polymorphism in tryptophan hydroxylase 2 gene in laboratory and wild mice.

    PubMed

    Osipova, D V; Kulikov, A V; Mekada, K; Yoshiki, A; Moshkin, M P; Kotenkova, E V; Popova, N K

    2010-07-01

    The neurotransmitter serotonin is implicated in the regulation of various forms of behavior, including aggression, sexual behavior and stress response. The rate of brain serotonin synthesis is determined by the activity of neuronal-specific enzyme tryptophan hydroxylase 2. The missense C1473G substitution in mouse tryptophan hydroxylase 2 gene has been shown to lower the enzyme activity and brain serotonin level. Here, the C1473G polymorphism was investigated in 84 common laboratory inbred strains, 39 inbred and semi-inbred strains derived from wild ancestors (mostly from Eurasia) and in 75 wild mice trapped in different locations in Russia and Armenia. Among all the classical inbred strains studied, only substrains of BALB/c, A and DBA, as well as the IITES/Nga and NZW/NSlc strains were homozygous for the 1473G allele. In contrast to laboratory strains, the 1473G allele was not present in any of the samples from wild and wild-derived mice, although the wild mice varied substantially in the C1477T neutral substitution closely linked to the C1473G polymorphism. According to these results, the frequency of the 1473G allele in natural populations does not exceed 0.5%, and the C1473G polymorphism is in fact a rare mutation that is possibly eliminated by the forces of natural selection.

  5. A patient with Ehlers-Danlos syndrome type VI is a compound heterozygote for mutations in the lysyl hydroxylase gene.

    PubMed Central

    Ha, V T; Marshall, M K; Elsas, L J; Pinnell, S R; Yeowell, H N

    1994-01-01

    In the present study, we have isolated and sequenced the complementary DNAs of two mutant alleles for lysyl hydroxylase (LH) in fibroblasts from one patient (AT750) with Ehlers-Danlos syndrome type VI (EDS VI). We have identified a putative mutation in each allele which may be responsible for the patient's decreased LH (normalized to prolyl hydroxylase) activity (24% of normal). Intermediate levels of LH activity were measured in the patient's parents, who are clinically normal (father 52%; mother 86%). After the cloning of cDNAs and amplification by PCR, sequence analysis revealed two equally distributed populations of cDNAs for LH in the AT750 cell line. Each allele revealed different but significant changes from the normal sequence. In one allele (allele 1), the most striking change was a triple base deletion that would result in the loss of residue Glu532. The most significant difference in the other allele (allele 2) was a G-->A change which would produce a Gly678-->Arg codon change in a highly conserved region of the enzyme. Restriction analysis identified that allele 1 was inherited from the proband's mother and allele 2 from the father. This study represents the first example of compound heterozygosity for the LH gene in an EDS VI patient, and it appears that there is an additive effect of each mutant allele on clinical expression in this patient. Images PMID:8163671

  6. Characterization of the grappa Gene, the Drosophila Histone H3 Lysine 79 Methyltransferase

    PubMed Central

    Shanower, Gregory A.; Muller, Martin; Blanton, Jason L.; Honti, Viktor; Gyurkovics, Henrik; Schedl, Paul

    2005-01-01

    We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development. PMID:15371351

  7. Regulation of Lipogenic Gene Expression by Lysine-specific Histone Demethylase-1 (LSD1)*

    PubMed Central

    Abdulla, Arian; Zhang, Yi; Hsu, Fu-Ning; Xiaoli, Alus M.; Zhao, Xiaoping; Yang, Ellen S. T.; Ji, Jun-Yuan; Yang, Fajun

    2014-01-01

    Dysregulation of lipid homeostasis is a common feature of several major human diseases, including type 2 diabetes and cardiovascular disease. However, because of the complex nature of lipid metabolism, the regulatory mechanisms remain poorly defined at the molecular level. As the key transcriptional activators of lipogenic genes, such as fatty acid synthase (FAS), sterol regulatory element-binding proteins (SREBPs) play a pivotal role in stimulating lipid biosynthesis. Several studies have shown that SREBPs are regulated by the NAD+-dependent histone deacetylase SIRT1, which forms a complex with the lysine-specific histone demethylase LSD1. Here, we show that LSD1 plays a role in regulating SREBP1-mediated gene expression. Multiple lines of evidence suggest that LSD1 is required for SREBP1-dependent activation of the FAS promoter in mammalian cells. LSD1 knockdown decreases SREBP-1a at the transcription level. Although LSD1 affects nuclear SREBP-1 abundance indirectly through SIRT1, it is also required for SREBP1 binding to the FAS promoter. As a result, LSD1 knockdown decreases triglyceride levels in hepatocytes. Taken together, these results show that LSD1 plays a role in regulating lipogenic gene expression, suggesting LSD1 as a potential target for treating dysregulation of lipid metabolism. PMID:25190802

  8. Serotonin and Early Cognitive Development: Variation in the Tryptophan Hydroxylase 2 Gene Is Associated with Visual Attention in 7-Month-Old Infants

    ERIC Educational Resources Information Center

    Leppanen, Jukka M.; Peltola, Mikko J.; Puura, Kaija; Mantymaa, Mirjami; Mononen, Nina; Lehtimaki, Terho

    2011-01-01

    Background: Allelic variation in the promoter region of a gene that encodes tryptophan hydroxylase isoform 2 (TPH2), a rate-limiting enzyme of serotonin synthesis in the central nervous system, has been associated with variations in cognitive function and vulnerability to affective spectrum disorders. Little is known about the effects of this gene…

  9. Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

    NASA Technical Reports Server (NTRS)

    Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

    2004-01-01

    Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

  10. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

    PubMed

    Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  11. Effect of tryptophan hydroxylase-2 gene polymorphism G-703 T on personality in a population representative sample.

    PubMed

    Lehto, Kelli; Vaht, Mariliis; Mäestu, Jarek; Veidebaum, Toomas; Harro, Jaanus

    2015-03-01

    The tryptophan hydroxylase-2 gene (TPH2) is coding for the key enzyme of serotonin (5-HT) synthesis in the brain and has been associated with a number of psychiatric conditions. A functional variation in the TPH2 gene (G-703T, rs4570625) has been found to affect anxiety-related personality; however, information is very limited regarding the five factor model (FFM) personality traits. We have examined the association of the TPH2 G-703T polymorphism with FFM personality traits, and the possible modulation by the functional variation in the serotonin transporter gene (5-HTTLPR) in a large longitudinal population representative sample. The FFM personality traits were assessed in both birth cohorts of the Estonian Children Personality Behaviour and Health Study at ages 15 (n=742) and 18 (n=834). Significant association of the TPH2 genotype with Neuroticism and Conscientiousness was found at age 15, and with Extraversion and Conscientiousness at age 18. Participants with the T/T genotype scored significantly lower on Neuroticism and higher on Conscientiousness and Extraversion scales. In addition, a gene×gene interaction effect on Conscientiousness was revealed: the TPH2 genotype effect was evident only in the 5-HTTLPR S-allele carriers. These results provide further evidence on the possible role of genetic variations in 5-HT neurotransmission on development of personality traits, and suggest a functional interaction between two key proteins in the 5-HT-ergic system.

  12. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

    PubMed Central

    MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  13. Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

    PubMed

    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-08-01

    Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification. PMID:24880475

  14. Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

    PubMed

    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-08-01

    Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

  15. Characterization and expression profile of complete functional domain of granulysin/NK-lysin homologue (buffalo-lysin) gene of water buffalo (Bubalus bubalis).

    PubMed

    Kandasamy, Sukumar; Mitra, Abhijit

    2009-04-15

    Granulysin (GNLY)/NK-lysin (NKL) is an effector antimicrobial cationic peptide expressed in the cytotoxic and natural killer lymphocytes. We report here cDNA sequence (405bp) encoding the complete functional domain of buffalo-lysin (bu-lysin), and its expression profile in the various tissues. The nucleotide sequence of bu-lysin exhibited >85% identity with the bovine lysin. Comparison of the deduced amino acid sequence of bu-lysin with those of GNLY/NKL of different species revealed the conservation of six cysteine (Cys) residues and five alpha helices. Unlike the homologues in other species, bu-lysin composed of 11 positively charged Lys residues as in equine. The expression of bu-lysin mRNA in the in vitro cultured lymphocytes was inducible and increased markedly (p<0.05) in a dose dependant manner when incubated with Concanavalin A (ConA). The expression of bu-lysin mRNA in the different tissues was variable: comparatively higher in the spleen and lymph node, moderate in the uterine endometrium and low in the liver and kidney. These results indicate the existence and active expression of GNLY/NKL homologue in water buffalo having a significant influence in immune response.

  16. The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color.

    PubMed

    Moreau, Carol; Ambrose, Mike J; Turner, Lynda; Hill, Lionel; Ellis, T H Noel; Hofer, Julie M I

    2012-06-01

    The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

  17. Identification of hepatic nuclear factor 1 binding sites in the 5′ flanking region of the human phenylalanine hydroxylase gene: Implication of a dual function of phenylalanine hydroxylase stimulator in the phenylalanine hydroxylation system

    PubMed Central

    Lei, Xiang-Dong; Kaufman, Seymour

    1998-01-01

    Phenylalanine hydroxylase stimulator (PHS) is a component of the phenylalanine hydroxylation system that is involved in the regeneration of the cofactor tetrahydrobiopterin. It is also identical to the dimerization cofactor of hepatocyte nuclear factor 1 (HNF1) (DCoH) that is able to enhance the transcriptional activity of HNF1. Moreover, it has the structural potential for binding macromolecules such as proteins and nucleic acids, consistent with its involvement in gene expression. We investigated whether PHS/DCoH could enhance the expression of phenylalanine hydroxylase (PAH). Cotransfection assays showed that DCoH itself could not transactivate the 9-kb human PAH 5′ flanking fragment. However, this 9-kb fragment was transactivated by HNF1 in a dose-dependent manner with a maximum of nearly 8-fold activation; DCoH potentiated this transactivation by another 1.6-fold. The HNF1 binding sites were located at −3.5 kb in a region that is 77.5% identical to the mouse liver-specific hormone-inducible PAH gene enhancer. This study suggests a possible dual function of PHS in vivo in the human phenylalanine hydroxylation system: it is involved in the regeneration of the cofactor tetrahydrobiopterin and can also enhance the expression of the human PAH gene. PMID:9465044

  18. Association between A218C polymorphism of the tryptophan-hydroxylase-1 gene, harm avoidance and binge eating behavior in bulimia nervosa.

    PubMed

    Monteleone, Palmiero; Tortorella, Alfonso; Martiadis, Vassilis; Serino, Ismene; Di Filippo, Carmela; Maj, Mario

    2007-06-21

    Genes involved in serotonin transmission are likely involved in the biological predisposition to bulimia nervosa. We investigated whether the A218C polymorphism of the tryptophan-hydroxylase-1 gene was associated to bulimia nervosa and/or to some phenotypic aspects of the disorder. One hundred eighty Caucasian women (91 patients with bulimia nervosa and 89 healthy controls) were enrolled into the study. They underwent a blood sample collection for A218C polymorphism of the tryptophan-hydroxylase-1 genotyping and a clinical evaluation assessing comorbidity for Axis I and II psychiatric disorders, harm avoidance personality dimension and bulimic symptoms. The distribution of both tryptophan-hydroxylase-1 A218C genotypes and alleles did not significantly differ between patients and controls. Bulimic women with the AA genotype exhibited a more severe binge eating behavior and higher harm avoidance scores than those with CC genotype. These findings support the idea that tryptophan-hydroxylase-1 A218C polymorphism does not play a part in the genetic susceptibility to bulimia nervosa, but it seems to be involved in predisposing bulimic patients to a more disturbed eating behavior and higher harm avoidance.

  19. Histone Lysine Methylation in TGF-β1 Mediated p21 Gene Expression in Rat Mesangial Cells

    PubMed Central

    Guo, Qiaoyan; Li, Xiaoxia; Han, Hongbo; Li, Chaoyuan; Liu, Shujun; Gao, Wenhui

    2016-01-01

    Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases. PMID:27247942

  20. Muscle-directed gene therapy for phenylketonuria (PKU): Development of transgenic mice with muscle-specific phenylalanine hydroxylase expression

    SciTech Connect

    Harding, C.O.; Messing, A.; Wolff, J.A.

    1994-09-01

    Phenylketonuria (PKU) is an attractive target for gene therapy because of shortcomings in current therapy including lifelong commitment to a difficult and expensive diet, persistent mild cognitive deficits in some children despite adequate dietary therapy, and maternal PKU syndrome. Phenylalanine hydroxylase (PAH) is normally expressed only in liver, but we propose to treat PKU by introducing the gene for PAH into muscle. In order to evaluate both the safety and efficacy of this approach, we have a developed a trangenic mouse which expresses PAH in both cardiac and skeletal muscle. The transgene includes promoter and enhancer sequences from the mouse muscle creatine kinase (MCK) gene fused to the mouse liver PAH cDNA. Mice which have inherited the transgene are healthy, active, and do not exhibit any signs of muscle weakness or wasting. Ectopic PAH expression in muscle is not detrimental to the health, neurologic function, or reproduction of the mice. Pah{sup enu2} hyperphenylalaninemic mice, a model of human PAH deficiency, bred to carry the transgene have substantial PAH expression in cardiac and skeletal muscle but none in liver. Muscle PAH expression alone does not complement the hyperphenylalaninemic phenotype of Pah{sup enu2} mice. However, administration of reduced tetrahydrobiopterin to transgenic Pah{sup enu2} mice is associated with a 25% mean decrease in serum phenylalanine levels. We predict that ectopic expression of PAH in muscle along with adequate muscle supplies of reduced biopterin cofactor will decrease hyperphenylalaninemia in PKU.

  1. Impaired executive control is associated with a variation in the promoter region of the tryptophan hydroxylase 2 gene.

    PubMed

    Reuter, Martin; Ott, Ulrich; Vaitl, Dieter; Hennig, Jürgen

    2007-03-01

    Current models of attention describe attention not as a homogenous entity but as a set of neural networks whose measurement yields a set of three endophenotypes-alerting, orienting, and executive control. Previous findings revealed different neuroanatomical regions for these subsystems, and data from twin studies indicate differences in their heritability. The present study investigated the molecular genetic basis of attention in a sample of 100 healthy subjects. Attention performance was assessed with the attention network test that distinguishes alerting, orienting, and executive control (conflict) using a simple reaction time paradigm with different cues and congruent and incongruent flankers. Two gene loci on candidate genes for cognitive functioning, the functional catechol-O-methyltransferase (COMT) VAL158MET and the tryptophan hydroxylase 2 (TPH2) -703 G/T promoter polymorphism, were tested for possible associations with attention. COMT is involved in the catabolism of dopamine, and TPH is the rate-limiting enzyme for serotonin synthesis. Results showed no effect of the COMT polymorphism on attention performance. However, the TT genotype of TPH2 -03 G/T was significantly associated with more errors (a possible indicator of impaired impulse control; p = .001) and with decreased performance in executive control (p = .001). This single-nucleotide polymorphism on the TPH2 gene explained more than 10% of the variance in both indicators of attention stressing the role of the serotonergic system for cognitive functions.

  2. Structure of a bovine gene for P-450c21 (steroid 21-hydroxylase) defines a novel cytochrome P-450 gene family.

    PubMed Central

    Chung, B C; Matteson, K J; Miller, W L

    1986-01-01

    P-450c21, a cytochrome P-450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P-450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21-hydroxylated steroids. Images PMID:3487086

  3. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

    PubMed

    Jansen, G A; Ofman, R; Ferdinandusse, S; Ijlst, L; Muijsers, A O; Skjeldal, O H; Stokke, O; Jakobs, C; Besley, G T; Wraith, J E; Wanders, R J

    1997-10-01

    Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

  4. Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.

    PubMed Central

    Hornsby, P J; Hancock, J P; Vo, T P; Nason, L M; Ryan, R F; McAllister, J M

    1987-01-01

    Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17 alpha-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17 alpha-hydroxylase enzyme activity. The lower levels of 17 alpha-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells results from a general decline in response to several growth factors. However, the decline in 17 alpha-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17 alpha-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17 alpha-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17 alpha-hydroxylase. The loss of 17 alpha-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17 alpha-hydroxylase, because adrenocortical cell clones that had high levels of 17 alpha-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17 alpha-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17 alpha-hydroxylase

  5. The C1473G polymorphism in the Tryptophan hydroxylase-2 gene: involvement in ethanol-related behavior in mice.

    PubMed

    Bazovkina, Darya V; Lichman, Daria V; Kulikov, Alexander V

    2015-03-01

    Tryptophan hydroxylase-2 (Tph2) is the rate limiting enzyme of serotonin synthesis in the brain. The functional (C1473G) polymorphism in the mouse Tph2 gene affecting the enzymatic activity was suspected to be involved in behavioral actions of ethanol (EtOH). Congenic B6-1473C (C/C) and B6-1473G (G/G) lines bred from C57BL/6 mice were not different in EtOH-induced sleep time and hypothermia. B6-1473C mice displayed increased EtOH preference on the second and third days compared to that of the first day, but no differences in this parameter was found across genotypes. Both lines demonstrated the same responsiveness to hypothermic and hypnotic effect of acute EtOH treatment after repeated alcohol exposure. However, acute EtOH administration led to reduction of locomotor activity in B6-1473C, but not in B6-1473G animals and to increase of time spent in the center of open-field arena in B6-1473G, but not in B6-1473C mice. Thus, the present study indicates the involvement of C1473G polymorphism in mTph2 gene in the regulation of EtOH-induced effects on locomotor activity and anxiety-like behavior in mice.

  6. The C1473G polymorphism in the Tryptophan hydroxylase-2 gene: involvement in ethanol-related behavior in mice.

    PubMed

    Bazovkina, Darya V; Lichman, Daria V; Kulikov, Alexander V

    2015-03-01

    Tryptophan hydroxylase-2 (Tph2) is the rate limiting enzyme of serotonin synthesis in the brain. The functional (C1473G) polymorphism in the mouse Tph2 gene affecting the enzymatic activity was suspected to be involved in behavioral actions of ethanol (EtOH). Congenic B6-1473C (C/C) and B6-1473G (G/G) lines bred from C57BL/6 mice were not different in EtOH-induced sleep time and hypothermia. B6-1473C mice displayed increased EtOH preference on the second and third days compared to that of the first day, but no differences in this parameter was found across genotypes. Both lines demonstrated the same responsiveness to hypothermic and hypnotic effect of acute EtOH treatment after repeated alcohol exposure. However, acute EtOH administration led to reduction of locomotor activity in B6-1473C, but not in B6-1473G animals and to increase of time spent in the center of open-field arena in B6-1473G, but not in B6-1473C mice. Thus, the present study indicates the involvement of C1473G polymorphism in mTph2 gene in the regulation of EtOH-induced effects on locomotor activity and anxiety-like behavior in mice. PMID:25603476

  7. Polymorphism in the Tyrosine Hydroxylase (TH) Gene Is Associated with Activity-Impulsivity in German Shepherd Dogs

    PubMed Central

    Kubinyi, Enikő; Vas, Judit; Hejjas, Krisztina; Ronai, Zsolt; Brúder, Ildikó; Turcsán, Borbála; Sasvari-Szekely, Maria; Miklósi, Ádám

    2012-01-01

    We investigated the association between repeat polymorphism in intron 4 of the tyrosine hydroxylase (TH) gene and two personality traits, activity-impulsivity and inattention, in German Shepherd Dogs. The behaviour of 104 dogs was characterized by two instruments: (1) the previously validated Dog-Attention Deficit Hyperactivity Disorder Rating Scale (Dog-ADHD RS) filled in by the dog owners and (2) the newly developed Activity-impulsivity Behavioural Scale (AIBS) containing four subtests, scored by the experimenters. Internal consistency, inter-observer reliability, test-retest reliability and convergent validity were demonstrated for AIBS. Dogs possessing at least one short allele were proved to be more active-impulsive by both instruments, compared to dogs carrying two copies of the long allele (activity-impulsivity scale of Dog-ADHD RS: p = 0.007; AIBS: p = 0.023). The results have some potential to support human studies; however, further research should reveal the molecular function of the TH gene variants, and look for the effect in more breeds. PMID:22272320

  8. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

    PubMed

    Ortega-Molina, Ana; Boss, Isaac W; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A; Gascoyne, Randy D; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M; Wendel, Hans-Guido

    2015-10-01

    The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways. PMID:26366710

  9. Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

    PubMed

    Xiong, Shuo; Tian, Na; Long, Jinhua; Chen, Yuhong; Qin, Yu; Feng, Jinyu; Xiao, Wenjun; Liu, Shuoqian

    2016-08-01

    Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering. PMID:27070290

  10. Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin

    PubMed Central

    Castellarin, Simone D; Di Gaspero, Gabriele; Marconi, Raffaella; Nonis, Alberto; Peterlunger, Enrico; Paillard, Sophie; Adam-Blondon, Anne-Francoise; Testolin, Raffaele

    2006-01-01

    Background Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. Results Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. Conclusion We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between

  11. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  12. Genetics Home Reference: dopamine beta-hydroxylase deficiency

    MedlinePlus

    ... CONGENITAL Sources for This Page Cubells JF, Zabetian CP. Human genetics of plasma dopamine beta-hydroxylase activity: ... GeneReview: Dopamine Beta-Hydroxylase Deficiency Kim CH, Zabetian CP, Cubells JF, Cho S, Biaggioni I, Cohen BM, Robertson ...

  13. A large duplication in the gene for lysyl hydroxylase accounts for the type VI variant of Ehlers-Danlos syndrome in two siblings

    SciTech Connect

    Hautala, T.; Heikkinen, J.; Kivirikko, K.I.; Myllylae, R. )

    1993-02-01

    Ehlers-Danlos syndrome is a deterogeneous disorder characterized by joint hypermobility, skin hyperextensibility, fragility, and other sign of connective tissue involvement. In addition to these, the type VI variant of the disease has some special characteristics such as kyphoscoliosis and ocular abnormalities. The biochemical abnormality in most patients with this autosomal recessively inherited type IV variant is a deficiency in the activity of lysyl hydroxylase (EC 1.14,11.4), the enzyme catalyzing the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. The type VI variant of Ehlers-Danlos syndrome was first identified in two sisters with a reduced amount of lysyl hydroxylase activity in their skin fibroblasts (S.R. Pinnell, S.M. Krane, J.E. Kenzora, and M.J. Glimcher (1972) N. Engl. J. Med. 286; 1013-1020). Our recent molecular cloning of lysyl hydroxylase has now made it possible to study the mutations leading to the deficiency in lysyl dydroxylase activity in these cells. Our data indicate that the mRNA for lysyl hydroxylase produced in the affected cells is about 4 kb in size, whereas it is 3.2 kb in the control cells. The sequencing of the cDNA for lysyl hydroxylase from the affected cells revealed an apparently homozygous duplication rearrangement of nucleotides 1176 to 1955, corresponding to amino acids 326 to 585 in the normal sequence. From Southern blotting data, the duplicated area in the gene equals about 6-9 kb and corresponds to seven exons. 35 refs., 4 figs.

  14. Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains.

    PubMed

    Qin, Xiaoqiong; Zhang, Wenjun; Dubcovsky, Jorge; Tian, Li

    2012-12-01

    Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption.

  15. Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains.

    PubMed

    Qin, Xiaoqiong; Zhang, Wenjun; Dubcovsky, Jorge; Tian, Li

    2012-12-01

    Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption. PMID:23015203

  16. An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3'-hydroxylase gene.

    PubMed

    Momose, Masaki; Nakayama, Masayoshi; Itoh, Yoshio; Umemoto, Naoyuki; Toguri, Toshihiro; Ozeki, Yoshihiro

    2013-04-01

    The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3'H, the gene for flavonoid 3'-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.

  17. Studies on rat and human thymus to demonstrate immunoreactivity of calcitonin gene-related peptide, tyrosine hydroxylase and neuropeptide Y

    PubMed Central

    KRANZ, ANDREA; KENDALL, MARION D.; VON GAUDECKER, BRITA

    1997-01-01

    The peptidergic and noradrenergic innervation of rat and human thymus was investigated by immunohistochemistry at the light and electron microscopical level (avidin-biotin-complex, sucrose-phosphate-glyoxylic-acid, and immunogold techniques). The distribution of noradrenergic neural profiles, and positive immunoreactivity for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) is described in female rats during ageing, and in human children. In the neonatal rat thymus, the arteries and septa are well supplied by fine varicose nerves. In older animals (2 wk–1 y) the number of septa and blood vessels increase and consequently also the innervation. No nerves were found in the cortex. Apart from the innervation of the septal areas, immunoreactivity for CGRP and TH was present in thymic cells. Except for the young rats (neonatal–14 d), all rats showed CGRP positivity in subcapsular/perivascular epithelial cells (type 1 cells). All rat thymuses also contained a few TH positive cells in the medulla, which could only be confirmed as epithelial cells (type 6 cells) in children. Type 1 cells in the human thymus were not CGRP positive, but as in the rat, there were similar TH positive cells in the medulla. It was concluded that in addition to nerves containing CGRP, noradrenaline or dopamine, epithelial cells also contain these transmitters. They could therefore act on different cells (compared with neural targets) in a paracrine manner. PMID:9419001

  18. Lithium modulates tryptophan hydroxylase 2 gene expression and serotonin release in primary cultures of serotonergic raphe neurons.

    PubMed

    Scheuch, Kathrin; Höltje, Markus; Budde, Holger; Lautenschlager, Marion; Heinz, Andreas; Ahnert-Hilger, Gudrun; Priller, Josef

    2010-01-11

    Lithium salts are mood-stabilizing agents with acute antimanic properties and proven efficacy in the long-term prevention of manic and depressive episodes. Furthermore, lithium augmentation is a well-established strategy to treat depressed patients, which do not respond to antidepressants alone. There is evidence to suggest that these effects of lithium are due to a synergism with central serotonin (5-HT) neurotransmission. In this study, we investigated the effects of lithium chloride (LiCl, 1 mM) on 5-HT uptake and release in primary serotonergic neurons from rat raphe nuclei. Short-term (8 h) and long-term (14 days) treatment with LiCl resulted in a 20% and 23% increase in 5-HT release, but neither influenced 5-HT uptake across the plasma membrane nor vesicular 5-HT uptake. In lithium-treated raphe neurons, the inhibition of 5-HT uptake by fluoxetine was unchanged. Using real-time reverse transcriptase polymerase chain reaction and Western blotting, we examined the effect of lithium on tryptophan hydroxylase 2 (TPH2) expression, the rate-limiting enzyme in brain 5-HT biosynthesis. Short-term lithium treatment resulted in a 45% decrease in tph2 mRNA expression and a 31% reduction of TPH2 protein levels, which was completely compensated after long-term treatment. Our results suggest that lithium can modify tph2 gene expression and 5-HT release in raphe neurons, providing new insight into the serotonergic mechanisms of action of lithium.

  19. Terminal modification on mPEG-dendritic poly-(l)-lysine cationic diblock copolymer for efficient gene delivery.

    PubMed

    Sheng, Ruilong; Xia, Kejia; Chen, Jian; Xu, Yuhong; Cao, Amin

    2013-01-01

    The development of new non-viral gene vectors with the advantages of low cytotoxicity and high gene transfection efficiency is a recent trend in gene therapy. In this work, we developed a series of termini-modified mPEG-dendritic poly-(l)-lysine cationic diblock copolymers (mPEG5k-DPL4-CG) by coupling various cationic groups to the dendritic skeleton. Their molecular structures were characterized by (1)H NMR, and the buffering capacities were measured by acid titration. The plasmid DNA (pDNA) binding affinities of the mPEG5k-DPL4-CG copolymers were investigated by EB displacement and agarose gel retardation assay, and the average particle size and surface charge of the polyplexes were analyzed by dynamic light scattering. Cytotoxicity and in vitro gene transfection were evaluated in several cell lines in the presence and absence of serum by the luciferase expression assay. The results indicated that the low molecular weight polyethylenimine (PEI800) termini-modified copolymer, mPEG5k-DPL4-PEI800, possessed high pDNA binding affinity, low cytotoxicity, and high gene transfection capability which were maintained in the presence of serum (10% FBS). It is worth noting that the gene delivery efficiency of the dendritic poly-(l)-lysine gene vector was enhanced by termini modification of suitable cationic blocks. The low cytotoxicity and serum-resistance properties of mPEG5k-DPL4-PEI800 make it a potential long-circulating gene vector in gene therapy applications.

  20. Mechanism of Vitamin D Receptor Inhibition of Cholesterol 7α-Hydroxylase Gene Transcription in Human HepatocytesS⃞

    PubMed Central

    Han, Shuxin; Chiang, John Y. L.

    2009-01-01

    Lithocholic acid (LCA) is a potent endogenous vitamin D receptor (VDR) ligand. In cholestasis, LCA levels increase in the liver and intestine. The objective of this study is to test the hypothesis that VDR plays a role in inhibiting cholesterol 7α-hydroxylase (CYP7A1) gene expression and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the nucleus of the human hepatoma cell line HepG2 and human primary hepatocytes. 1α, 25-Dihydroxy-vitamin D3 or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA to VDR completely abrogated VDR inhibition of CYP7A1 mRNA expression in HepG2 cells. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/retinoid X receptor α in the human CYP7A1 promoter. Mammalian two-hybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligand-activated VDR specifically interacts with hepatocyte nuclear factor 4α (HNF4α) to block HNF4α interaction with coactivators or to compete with HNF4α for coactivators or to compete for binding to CYP7A1 chromatin, which results in the inhibition of CYP7A1 gene transcription. This study shows that VDR is expressed in human hepatocytes and may play a critical role in the inhibition of bile acid synthesis, thus protecting liver cells during cholestasis. PMID:19106115

  1. Advances in Tryptophan Hydroxylase-2 Gene Expression Regulation: New Insights into Serotonin-Stress Interaction and Clinical Implications

    PubMed Central

    Chen, Guo-Lin; Miller, Gregory M.

    2013-01-01

    Serotonin (5-HT) modulates the stress response by interacting with the hormonal hypothalamic-pituitary-adrenal (HPA) axis and neuronal sympathetic nervous system (SNS). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT biosynthesis, and the recent identification of a second, neuron-specific TPH isoform (TPH2) opened up a new area of research. While TPH2 genetic variance has been linked to numerous behavioral traits and disorders, findings on TPH2 gene expression have not only reinforced, but also provided new insights into, the long-recognized but not yet fully understood 5-HT-stress interaction. In this review, we summarize advances in TPH2 expression regulation and its relevance to the stress response and clinical implications. Particularly, based on findings on rhesus monkey TPH2 genetics and other relevant literature, we propose that: 1) upon activation of adrenal cortisol secretion, the cortisol surge induces TPH2 expression and de novo 5-HT synthesis; 2) the induced 5-HT in turn inhibits cortisol secretion by modulating the adrenal sensitivity to ACTH via the suprachiasmatic nuclei (SCN)-SNS-adrenal system, such that it contributes to the feedback inhibition of cortisol production; 3) basal TPH2 expression or 5-HT synthesis, as well as early-life experience, influence basal cortisol primarily via the hormonal HPA axis; and 4) 5′- and 3′-regulatory polymorphisms of TPH2 may differentially influence the stress response, presumably due to their differential roles in gene expression regulation. Our increasing knowledge of TPH2 expression regulation not only helps us better understand the 5-HT-stress interaction and the pathophysiology of neuropsychiatric disorders, but also provides new strategies for the treatment of stress-associated diseases. PMID:22241550

  2. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum).

    PubMed

    Fragkostefanakis, Sotirios; Sedeek, Khalid E M; Raad, Maya; Zaki, Marwa Samir; Kalaitzis, Panagiotis

    2014-07-01

    Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.

  3. Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

    PubMed

    Massimi, A; Malaponti, M; Federici, L; Vinciguerra, D; Manca Bitti, M L; Vottero, A; Ghizzoni, L; Maccarrone, M; Cappa, M; Bernardini, S; Porzio, O

    2014-06-01

    Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

  4. Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

    PubMed Central

    Yu, Hao; Li, Yangyang

    2015-01-01

    Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppAS and vppAL [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase. PMID:26407884

  5. Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways.

    PubMed

    Yu, Hao; Tang, Hongzhi; Li, Yangyang; Xu, Ping

    2015-12-01

    Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppAS and vppAL [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

  6. No association between schizophrenia and polymorphisms within the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT)

    SciTech Connect

    Daniels, J.; Williams, J.; Asherson, P.; McGuffin, P.; Owen, M.

    1995-02-27

    It has been suggested that the cytochrome P450 mono-oxygenase, debrisoquine 4-hydroxylase, is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells. It is also thought to be related to the dopamine transporter that acts to take released dopamine back up into presynaptic terminals. The present study used the association approach to test the hypothesis that mutations in the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT) confer susceptibility to schizophrenia. There were no differences in allele or genotype frequencies between patients and controls in the mutations causing the poor metaboliser phenotype in CYP2D6. In addition there was no association found between schizophrenia and a 48 bp repeat within the 3{prime} untranslated region of DAT. 18 refs., 2 tabs.

  7. Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3 and is required for efficient gene expression.

    PubMed

    Boa, Simon; Coert, Claudette; Patterton, Hugh-G

    2003-07-15

    Several homologues of the Drosophila Su(var)3-9 protein were recently reported to methylate lysine 9 of histone H3. Whereas this methylation signal served to recruit heterochromatin-associated proteins to transcriptionally silenced regions, histone H3 methylated at lysine 4 was associated with transcriptionally active areas of the genome. These findings suggested that the interplay between lysine 4 and 9 methylation is crucial in eukaryotic gene regulation. Here we provide evidence that Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3. In addition, we show that the absence of Set1p and lysine 4 methylation result in decreased transcription of approximately 80% of the genes in S. cerevisiae. Hierarchical clustering analysis of the set1(-) expression profile revealed a correspondence to that of a mad2(-) strain, suggesting that the transcriptional defect in the set1(-) strain may be due to changes in chromatin structure. These findings establish a central role for methylation of histone H3 lysine 4 in transcriptional regulation.

  8. Selection of Acinetobacter calcoaceticus mutants deficient in the p-hydroxybenzoate hydroxylase gene (pobA), a member of a supraoperonic cluster.

    PubMed Central

    Hartnett, G B; Averhoff, B; Ornston, L N

    1990-01-01

    p-Hydroxybenzoate hydroxylase, the product of the pobA gene, gives rise to protocatechuate, which is metabolized by enzymes encoded by the pca operon in Acinetobacter calcoaceticus. Mutations in pcaD prevented growth of A. calcoaceticus with succinate in the presence of p-hydroxybenzoate. Mutants selected on this medium contained the original mutation in pcaD and also carried spontaneous mutations in pobA. These independently expressed genes were cotransformed with a frequency of 15% and thus are components of a supraoperonic cluster. PMID:2211534

  9. Novel regulator MphX represses activation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR in Acinetobacter calcoaceticus.

    PubMed

    Yu, Haiying; Peng, Zixin; Zhan, Yuhua; Wang, Jin; Yan, Yongliang; Chen, Ming; Lu, Wei; Ping, Shuzhen; Zhang, Wei; Zhao, Zhonglin; Li, Shuying; Takeo, Masahiro; Lin, Min

    2011-03-24

    Acinetobacter calcoaceticus PHEA-2 utilizes phenol as its sole carbon and energy source and has a multi-component phenol hydroxylase-encoding gene operon (mphKLMNOP) for phenol degradation. Two additional genes, mphR and mphX, were found upstream and downstream of mphKLMNOP, respectively. The mphR gene encodes a XylR/DmpR-type regulator-like protein and is transcribed in the opposite direction to mphKLMNOP. The mphX gene is transcribed in the same direction as mphKLMNOP and encodes a protein with 293 amino acid residues showing weak identity with some unknown proteins encoded in the meta-cleavage pathway gene clusters for aromatic compound degradation. Disruption of mphR by homologous recombination resulted in the loss of phenol degradation while disruption of mphX caused significantly faster phenol degradation than in the wild type strain. Transcriptional assays for mphK, mphR, and mphX revealed that mphR activated mphKLMNOP transcription in the presence of phenol, but mphX partially repressed this activation. Gel mobility-shift assay demonstrated a direct interaction of MphR with the mphK promoter region. These results indicate the involvement of a novel repressor protein MphX in transcriptional regulation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR.

  10. Genetic Disruption of Both Tryptophan Hydroxylase Genes Dramatically Reduces Serotonin and Affects Behavior in Models Sensitive to Antidepressants

    PubMed Central

    Savelieva, Katerina V.; Rajan, Indrani; Yang, Qi; Cullinan, Emily; Lanthorn, Thomas H.

    2008-01-01

    The neurotransmitter serotonin (5-HT) plays an important role in both the peripheral and central nervous systems. The biosynthesis of serotonin is regulated by two rate-limiting enzymes, tryptophan hydroxylase-1 and -2 (TPH1 and TPH2). We used a gene-targeting approach to generate mice with selective and complete elimination of the two known TPH isoforms. This resulted in dramatically reduced central 5-HT levels in Tph2 knockout (TPH2KO) and Tph1/Tph2 double knockout (DKO) mice; and substantially reduced peripheral 5-HT levels in DKO, but not TPH2KO mice. Therefore, differential expression of the two isoforms of TPH was reflected in corresponding depletion of 5-HT content in the brain and periphery. Surprisingly, despite the prominent and evolutionarily ancient role that 5-HT plays in both vertebrate and invertebrate physiology, none of these mutations resulted in an overt phenotype. TPH2KO and DKO mice were viable and normal in appearance. Behavioral alterations in assays with predictive validity for antidepressants were among the very few phenotypes uncovered. These behavioral changes were subtle in the TPH2KO mice; they were enhanced in the DKO mice. Herein, we confirm findings from prior descriptions of TPH1 knockout mice and present the first reported phenotypic evaluations of Tph2 and Tph1/Tph2 knockout mice. The behavioral effects observed in the TPH2 KO and DKO mice strongly confirm the role of 5-HT and its synthetic enzymes in the etiology and treatment of affective disorders. PMID:18923670

  11. Overexpression of a tomato carotenoid ε-hydroxylase gene alleviates sensitivity to chilling stress in transgenic tobacco.

    PubMed

    Zhou, Bin; Deng, Yong-Sheng; Kong, Fan-Ying; Li, Bin; Meng, Qing-Wei

    2013-09-01

    Chilling is one of the most serious environmental stresses that disrupt the metabolic balance of cells and enhance the production of reactive oxygen species (ROS). Lutein plays important roles in dissipating excess excitation energy and eliminating ROS to maintain the normal physiological function of cells. A tomato carotenoid epsilon-ring hydroxylase gene (LeLUT1) was isolated, and the LeLUT1-GFP fusion protein was localized in the chloroplast of Arabidopsis mesophyll protoplast. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of LeLUT1 was the highest in the leaves and was down-regulated by various abiotic stresses in tomato. The transgenic tobacco plants overexpressing LeLUT1 had higher lutein content, which was decreased in cold condition. Under chilling stress, the non-photochemical quenching (NPQ) values were higher in the transgenic plants than in the wild type (WT) plants. Compared with the WT plants, the transgenic plants showed lower levels of hydrogen peroxide (H2O2), superoxide radical (O2(·-)), relative electrical conductivity, and malondialdehyde content (MDA), and relatively higher values of maximal photochemical efficiency of photosystem II (Fv/Fm), oxidizable P700 of PSI, and net photosynthetic rate (Pn). Therefore, the transgenic seedlings were less suppressed in growth and lost less cotyledon chlorophyll than the WT seedlings. These results suggested that the overexpression of LeLUT1 had a key function in alleviating photoinhibition and photooxidation, and decreased the sensitivity of photosynthesis to chilling stress. PMID:23796723

  12. Identification and characterization of cis-acting elements conferring insulin responsiveness on hamster cholesterol 7alpha-hydroxylase gene promoter.

    PubMed Central

    De Fabiani, E; Crestani, M; Marrapodi, M; Pinelli, A; Golfieri, V; Galli, G

    2000-01-01

    Bile acid biosynthesis occurs primarily through a pathway initiated by the 7alpha-hydroxylation of cholesterol, catalysed by cholesterol 7alpha-hydroxylase (encoded by CYP7A1). Insulin down-regulates CYP7A1 transcription. The aim of our study was to characterize the sequences of hamster CYP7A1 promoter, mediating the response to insulin. We therefore performed transient transfection assays with CYP7A1 promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster CYP7A1 promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster CYP7A1 insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster CYP7A1. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of CYP7A1; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster CYP7A1 proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin. PMID:10727413

  13. Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C.

    PubMed Central

    Vyas, S; Faucon Biguet, N; Mallet, J

    1990-01-01

    The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post-transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down-regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti-PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane-associated PKC increased within 15-30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti-PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down-regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two-fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig.2 Fig.3 Fig.6 PMID:1976513

  14. H28+C insertion in the CYP21 gene: a novel frameshift mutation in a Brazilian patient with the classical form of 21-hydroxylase deficiency.

    PubMed

    Lau, I F; Soardi, F C; Lemos-Marini, S H; Guerra Jr, G; Baptista, M T; De Mello, M P

    2001-12-01

    In the classical form of 21-hydroxylase deficiency, CYP21- affected genes either carry mutations present in the CYP21P pseudogene (microconversions) or bear a chimeric gene that replaces the active gene as a result of large conversion or deletion mutational events. Previous genotyping of 41 Brazilian patients revealed 64% microconversion, whereas deletions and large gene conversions accounted for up to 21% of the molecular defect. The present paper describes a new mutation disclosed by sequencing an entire gene in which no pseudogene-originated mutation had been found. The patient with the classical form of 21-hydroxylase deficiency is the daughter of a consanguineous marriage, and she is homozygous for a novel frameshift H28+C within exon 1. The mutation causes a stop codon at amino acid 78. Both parents are heterozygous for the mutation as confirmed by allele-specific oligonucleotide PCR. The H28+C is not present in the published CYP21P sequences and is likely to result in an enzyme with no activity.

  15. Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize

    PubMed Central

    Chang, Yujie; Shen, Erli; Wen, Liuying; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

    2015-01-01

    Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize. PMID:26580206

  16. Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.

    PubMed

    Mikula, Michal; Majewska, Aneta; Ledwon, Joanna Karolina; Dzwonek, Artur; Ostrowski, Jerzy

    2014-12-01

    Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.

  17. Ferulic acid 5-hydroxylase 1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis.

    PubMed

    Maruta, Takanori; Noshi, Masahiro; Nakamura, Maki; Matsuda, Shun; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2014-04-01

    Anthocyanins are important for preventing photoinhibition and photodamage. By comprehensive reverse genetic analysis of chloroplast-produced H2O2-responsive genes, we isolated here an anthocyanin-deficient mutant under photooxidative stress, which lacked ferulate 5-hydroxylase 1 (FAH1) involved in the phenylpropanoid pathway. Interestingly, the expression of anthocyanin biosynthesis-associated genes was also inhibited in this mutant. These findings suggest that FAH1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis. Furthermore, we found that estrogen-inducible silencing of thylakoid membrane-bound ascorbate peroxidase, which is a major H2O2-scavenging enzyme in chloroplasts, enhances the expression of FAH1 and anthocyanin biosynthesis-associated genes and accumulation of anthocyanin without any application of stress. Thus, it is likely that chloroplastic H2O2 activates FAH1 expression to induce anthocyanin accumulation for protecting cells from photooxidative stress.

  18. A bacteria deriving peptide modified dendrigraft poly-l-lysines (DGL) self-assembling nanoplatform for targeted gene delivery.

    PubMed

    Liu, Yang; He, Xi; Kuang, Yuyang; An, Sai; Wang, Chenyu; Guo, Yubo; Ma, Haojun; Lou, Jinning; Jiang, Chen

    2014-10-01

    Achieving effective gene therapy for glioma depends on gene delivery systems. The gene delivery system should be able to cross the blood-brain barrier (BBB) and further target glioma at its early stage. Active brain tumor targeted delivery can be achieved using the "Trojan horse" technology, which involves either endogenous ligands or extraneous substances that can recognize and bind to specific receptors in target sites. This method facilitates receptor-mediated endocytosis to cross the BBB and enter into glioma cells. Dendrigraft poly-l-lysines (DGLs), which are novel nonviral gene vectors, are conjugated to a peptide (sequence: EPRNEEK) derived from Streptococcus pneumonia, a pathogen causing meningitis. This process yields peptide-modified nanoparticles (NPs) after DNA loading. Cellular uptake and in vivo imaging results indicate that EPRNEEK peptide-modified NPs have a better brain tumor targeted effect compared with a pentapeptide derived from endogenous laminin after intravenous injection. The mechanism of this effect is further explored in the present study. Besides, EPRNEEK peptide-modified NPs also exhibited a prolonged median survival time. In conclusion, the EPRNEEK peptide-modified DGL NPs exhibit potential as a nonviral platform for efficient, noninvasive, and safe brain glioma dual-targeted gene delivery.

  19. High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine.

    PubMed

    Choi, S Y; Lee, S Y; Bock, R M

    1993-08-01

    A chemically synthesized gene, which encodes a 64 or 128 times-repeated tripeptide, aspartyl-phenylalanyl-lysine, has been cloned onto the yeast expression vector pAM82 containing the PHO5 promoter. The artificial gene (LAP gene) contains the untranslated leader sequence of the E. coli lipoprotein gene (lpp) with its transcription terminator sequence. When yeast AH22 cells transformed by recombinant plasmid containing repeated tripeptide gene were derepressed in low phosphate medium, the artificial polypeptides were synthesized to the amounts of about 30% of the total cell protein. SDS-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the artificial polypeptides synthesized in yeast have molecular weights ranging from about 30,000 and 60,000 and have immunoreactivity with the artificial polypeptides expressed in E. coli. The artificial popypeptides in whole cell extract were insoluble and seem to be synthesized as insoluble aggregates. Electron microscopy showed the presence of inclusion bodies in the cell. These polypeptides can be hydrolyzed to tripeptides with trypsin or chymotrypsin. These properties along with the high expression and easy separation may make the artificial polypeptides a potential raw material for the production of an artificial sweetener, Aspartame.

  20. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development

    PubMed Central

    Ortega-Molina, Ana; Boss, Isaac W.; Canela, Andres; Pan, Heng; Jiang, Yanwen; Zhao, Chunying; Jiang, Man; Hu, Deqing; Agirre, Xabier; Niesvizky, Itamar; Lee, Ji-Eun; Chen, Hua-Tang; Ennishi, Daisuke; Scott, David W.; Mottok, Anja; Hother, Christoffer; Liu, Shichong; Cao, Xing-Jun; Tam, Wayne; Shaknovich, Rita; Garcia, Benjamin A.; Gascoyne, Randy D.; Ge, Kai; Shilatifard, Ali; Elemento, Olivier; Nussenzweig, Andre; Melnick, Ari M.; Wendel, Hans-Guido

    2015-01-01

    The lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center (GC) involution, impedes B cell differentiation and class switch recombination (CSR). Integrative genomic analyses indicate that KMT2D affects H3K4 methylation and expression of a specific set of genes including those in the CD40, JAK-STAT, Toll-like receptor, and B cell receptor pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3, and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell activating pathways. PMID:26366710

  1. Isolated p.H62L Mutation in the CYP21A2 Gene in a Simple Virilizing 21-Hydroxylase Deficient Patient.

    PubMed

    Taboas, Melisa; Fernández, Cecilia; Belli, Susana; Buzzalino, Noemi; Alba, Liliana; Dain, Liliana

    2013-01-01

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%-95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation.

  2. Carnitine biosynthesis. Hydroxylation of N6-trimethyl-lysine to 3-hydroxy-N6-trimethyl-lysine.

    PubMed

    Sachan, D S; Hoppel, C L

    1980-05-15

    Rat kidney homogenates metabolize N6-trimethyl-lysine to N-trimethylammoniobutyrate, but not to carnitine. The first step in this conversion is the hydroxylation of trimethyl-lysine to form 3-hydroxy-N6-trimethyl-lysine. An assay system was developed in which hydroxylation of trimethyl-lysine is linear with respect to both time and homogenate protein concentration. The rate is 5 nmol of 3-hydroxy-N6-trimethyl-lysine formed/min per mg of homogenate protein. The cofactors required are ascorbate, alpha-oxoglutarate, FeSO4, and O2. Catalase and dithiothreitol give a 20% stimulation. Ca2+ produces a 2-fold increase in specific activity and cannot be replaced by Mg2+, Mn2+ or Zn2+. These last three bivalent cations lead to a decreased activity. Subcellular distribution studies demonstrate that trimethyl-lysine hydroxylase activity parallels the distribution profile of succinate dehydrogenase and citrate synthase. Thus trimethyl-lysine hydroxylase has a mitochondrial localization. Distribution of trimethyl-lysine hydroxylase activity between cortex and medulla of kidney if 67 and 33% respectively, similar to mitochondrial distribution.

  3. Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

    PubMed Central

    Liu, L; Shaw, P D

    1997-01-01

    The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis. PMID:8990304

  4. Dual function of histone H3 lysine 36 methyltransferase ASH1 in regulation of Hox gene expression.

    PubMed

    Tanaka, Yujiro; Kawahashi, Koji; Katagiri, Zen-Ichiro; Nakayama, Yasuhiro; Mahajan, Milind; Kioussis, Dimitris

    2011-01-01

    Hox genes play important roles in haematopoietic development in mammals. ASH1 is a member of the trithorax group (trxG) that is required for proper expression of Hox genes and is preferentially expressed in haematopoietic stem cells. We have recently reported that ASH1 methylates histone H3 at lysine 36 (K36) but its biological function has remained elusive. Here we show that ASH1 regulates Hox gene expression positively and negatively in a leukemic cell line K562 and is required for myelomonocytic differentiation of murine haematopoietic stem cells. ASH1 binds to endogenous Hox loci in K562 cells and its knockdown causes reduced expression of Hox genes. In addition, ASH1 and MLL1 induce more than 100-fold activation of Hox promoters in HeLa cells if expressed simultaneously but not individually. Notably, ASH1 harbouring a point mutation that kills methyltransferase activity is more efficient than wild type ASH1 in Hox gene activation, indicating that K36 methylation is not a prerequisite for Hox gene expression. Moreover, tethering wild type or catalytically inactive methyltransferase domain of ASH1 to a heterologous promoter causes downregulation or upregulation, respectively, of transcription, supporting a hypothesis that K36 methylation imparts repression. Knockdown of ASH1 in K562 cells in vitro causes increased expression of ε-globin gene and reduced expression of myelomonocytic markers GPIIb and GPIIIa, whereas knockdown of ASH1 in murine haematopoietic stem cells in vivo results in decreased number of macrophages and granulocytes, a phenotype similar to that induced by loss of mll1 function. Taken together, our data suggest that ASH1 and MLL1 synergize in activation of Hox genes and thereby regulate development of myelomonocytic lineages from haematopoietic stem cells.

  5. A leptin derived 30-amino-acid peptide modified pegylated poly-L-lysine dendrigraft for brain targeted gene delivery.

    PubMed

    Liu, Yang; Li, Jianfeng; Shao, Kun; Huang, Rongqin; Ye, Liya; Lou, Jinning; Jiang, Chen

    2010-07-01

    The blood-brain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specific ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormone-leptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-L-lysine (DGL) is used as non-viral gene vector in this study. DGL-PEG-Leptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGL-PEG-Leptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGL-PEG-Leptin30 provides a safe and noninvasive approach for the delivery of gene across the blood-brain barrier.

  6. Restoration of phytanic acid oxidation in Refsum disease fibroblasts from patients with mutations in the phytanoyl-CoA hydroxylase gene.

    PubMed

    Chahal, A; Khan, M; Pai, S G; Barbosa, E; Singh, I

    1998-06-01

    Refsum disease (RD) is biochemically characterized by the excessive accumulation of phytanic acid in tissues and body fluids due to deficiency of phytanoyl-CoA hydroxylase (PAHX). In this study, we screened three RD patients and identified a novel deletion (88 amino acids), and a missense mutation (Arg275Trp) in the previously reported PAHX cDNA (Jansen et al., 1997; Mihalik et al., 1997). Moreover, transfection of skin fibroblasts from two RD patients with wild-type PAHX gene restored the activity for alpha-oxidation of phytanic acid. Southern analysis on a somatic cell hybrid panel detected the PAHX gene on chromosome 10, corroborating radiation hybrid and homozygosity mapping data (Mihalik et al., 1997; Nadal et al., 1995).

  7. Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.

    PubMed

    Mahesh, Venkataramaiah; Million-Rousseau, Rachel; Ullmann, Pascaline; Chabrillange, Nathalie; Bustamante, José; Mondolot, Laurence; Morant, Marc; Noirot, Michel; Hamon, Serge; de Kochko, Alexandre; Werck-Reichhart, Danièle; Campa, Claudine

    2007-05-01

    Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

  8. Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

    PubMed Central

    Liang, Jie-Liang; JiangYang, Jing-Hong

    2015-01-01

    CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the −10 and −35 regions in Actinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria. PMID:26567302

  9. Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers.

    PubMed

    Jeong, Ji Hoon; Park, Tae Gwan

    2002-07-18

    Poly(lactic-co-glycolic acid) (PLGA)-grafted poly(L-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200-300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.

  10. Coarse-grained Molecular Simulation Studies of Complexation of Sulfobetaine-Lysine Copolymer and DNA for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Ghobadi, Ahmadreza F.; Jayaraman, Arthi

    2015-03-01

    Gene delivery involves successful transfection of therapeutic DNA by a vector into target cells and protein expression of that genetic material. Viral vectors are effective at gene delivery but elicit harmful immunogenic responses, thus motivating ongoing research on non-viral transfection agents. Cationic polymers are a promising class of non-viral vectors due to their low immugenic responses and low toxicity, and their ability to bind to the polyanionic DNA backbone to form a polycation-DNA complex (polyplex) that is then internalized in the target cell. While past studies have shown many polycations with differing DNA transfection efficacies, there is a need for general design guidelines that can relate the molecular features of the polycation to its DNA transfection efficiency. Using atomistic and coarse-grained molecular dynamics simulations we connect polycation design to polycation-DNA binding and experimentally observed transfection efficiency. Specifically in this presentation we will discuss our recent work looking into the effect of incorporating zwitterions into lysine based polycations on the resulting polyplex structure, shape, surface charge density and stability of DNA-polycation complexes.

  11. A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3',5'-hydroxylase gene.

    PubMed

    Fukui, Yuko; Tanaka, Yoshikazu; Kusumi, Takaaki; Iwashita, Takashi; Nomoto, Kyosuke

    2003-05-01

    Recently marketed genetically modified violet carnations cv. Moondust and Moonshadow (Dianthus caryophyllus) produce a delphinidin type anthocyanin that native carnations cannot produce and this was achieved by heterologous flavonoid 3',5'-hydroxylase gene expression. Since wild type carnations lack a flavonoid 3',5'-hydroxylase gene, they cannot produce delphinidin, and instead accumulate pelargonidin or cyanidin type anthocyanins, such as pelargonidin or cyanidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester. On the other hand, the anthocyanins in the transgenic flowers were revealed to be delphinidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester (main pigment), delphinidin 3,5-diglucoside-6"-malyl ester, and delphinidin 3,5-diglucoside-6",6"'- dimalyl ester. These are delphinidin derivatives analogous to the natural carnation anthocyanins. This observation indicates that carnation anthocyanin biosynthetic enzymes are versatile enough to modify delphinidin. Additionally, the petals contained flavonol and flavone glycosides. Three of them were identified by spectroscopic methods to be kaempferol 3-(6"'-rhamnosyl-2"'-glucosyl-glucoside), kaempferol 3-(6"'-rhamnosyl-2"'-(6-malyl-glucosyl)-glucoside), and apigenin 6-C-glucosyl-7-O-glucoside-6"'-malyl ester. Among these flavonoids, the apigenin derivative exhibited the strongest co-pigment effect. When two equivalents of the apigenin derivative were added to 1 mM of the main pigment (delphinidin 3,5-diglucoside-6"-O-4,6"'-O-1-cyclic-malyl diester) dissolved in pH 5.0 buffer solution, the lambda(max) shifted to a wavelength 28 nm longer. The vacuolar pH of the Moonshadow flower was estimated to be around 5.5 by measuring the pH of petal. We conclude that the following reasons account for the bluish hue of the transgenic carnation flowers: (1). accumulation of the delphinidin type anthocyanins as a result of flavonoid 3',5'-hydroxylase gene expression, (2). the presence of the flavone derivative strong co

  12. Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

    DOE PAGES

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; Rottmann, William H.; Kuhn, Sean A.; Foster, Cliff E.; Ziebell, Angela; Turner, Geoffrey B.; Decker, Stephen R.; Hinchee, Maud A. W.; et al

    2015-08-27

    In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

  13. Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

    PubMed Central

    Albiero, Elena; Ruggeri, Marco; Fortuna, Stefania; Finotto, Silvia; Bernardi, Martina; Madeo, Domenico; Rodeghiero, Francesco

    2012-01-01

    The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway. PMID:21828119

  14. Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis

    PubMed Central

    2013-01-01

    Background The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary. Results In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch. Conclusions The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm. PMID:23586588

  15. Cloning and expression in Escherichia coli of genes involved in the lysine pathway of Brevibacterium lactofermentum.

    PubMed Central

    Márquez, G; Sousa, J M; Sánchez, F

    1985-01-01

    The Brevibacterium lactofermentum genes which complement Escherichia coli lysA and asd-1 mutants were identified, respectively, as a 1.9-kilobase PstI-ClaI fragment and a 2.5-kilobase PstI fragment by cloning into pBR325. Southern blot transfers show hybridization to chromosomal fragments of identical size. The putative B. lactofermentum asd and lysA products are 44 and 48 kilodaltons, respectively. Images PMID:2864331

  16. Expression of flavonoid 3',5'-hydroxylase and acetolactate synthase genes in transgenic carnation: assessing the safety of a nonfood plant.

    PubMed

    Chandler, Stephen F; Senior, Michael; Nakamura, Noriko; Tsuda, Shinzo; Tanaka, Yoshikazu

    2013-12-01

    For 16 years, genetically modified flowers of carnation ( Dianthus caryophyllus ) have been sold to the floristry industry. The transgenic carnation carries a herbicide tolerance gene (a mutant gene encoding acetolactate synthase (ALS)) and has been modified to produce delphinidin-based anthocyanins in flowers, which conventionally bred carnation cannot produce. The modified flower color has been achieved by introduction of a gene encoding flavonoid 3',5'-hydroxylase (F3'5'H). Transgenic carnation flowers are produced in South America and are primarily distributed to North America, Europe, and Japan. Although a nonfood crop, the release of the genetically modified carnation varieties required an environmental risk impact assessment and an assessment of the potential for any increased risk of harm to human or animal health compared to conventionally bred carnation. The results of the health safety assessment and the experimental studies that accompanied them are described in this review. The conclusion from the assessments has been that the release of genetically modified carnation varieties which express F3'5'H and ALS genes and which accumulate delphinidin-based anthocyanins do not pose an increased risk of harm to human or animal health.

  17. Molecular cloning of the flavanone 3β-hydroxylase gene (FHT) from carnation (Dianthus caryophyllus) and analysis of stable and unstable FHT mutants.

    PubMed

    Dedio, J; Saedler, H; Forkmann, G

    1995-04-01

    Using a cDNA encoding the flavanone 3β-hydroxylase (FHT) from Dianthus caryophyllus (carnation) as a probe, we isolated the FHT gene from a genomic library. Sequence analysis revealed that the FHT gene consists of three exons and two introns. Two putative light-regulated elements were identified in the promoter region by sequence comparison. Southern blot analysis indicated that a single copy of the FHT gene is in the plant genome. Furthermore, a stable and an unstable FHT mutant of D. caryophyllus, both showing almost no FHT activity, were analyzed by Southern, Northern and Western blotting. It turned out that the FHT gene is present in both mutants, but no protein was detectable in the mutant flowers. FHT mRNA in amounts comparable to that found in the wildtype is present in flowers of the stable mutant, indicating a block in translation, but not in flowers of the unstable mutant, indicating a block in transcription. The translational block of the FHT mRNA of the stable mutant was demonstrated by in vitro translation of total flower mRNA followed by the specific measurement of FHT activity.

  18. Characterization of the Medium- and Long-Chain n-Alkanes Degrading Pseudomonas aeruginosa Strain SJTD-1 and Its Alkane Hydroxylase Genes

    PubMed Central

    Liu, Huan; Xu, Jing; Liang, Rubing; Liu, Jianhua

    2014-01-01

    A gram-negative aliphatic hydrocarbon-degrading bacterium SJTD-1 isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa by comparative analyses of the 16S rRNA sequence, phenotype, and physiological features. SJTD-1 could efficiently mineralize medium- and long-chain n-alkanes (C12-C30) as its sole carbon source within seven days, showing the most optimal growth on n-hexadecane, followed by n-octadecane, and n-eicosane. In 36 h, 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely; and 2 g/L n-hexadecane was degraded to undetectable levels within 72 h. Two putative alkane-degrading genes (gene 3623 and gene 4712) were characterized and our results indicated that their gene products were rate-limiting enzymes involved in the synergetic catabolism of C12–C16 alkanes. On the basis of bioinformatics and transcriptional analysis, two P450 monooxygenases, along with a putative AlmA-like oxygenase, were examined. Genetically defective mutants lacking the characteristic alkane hydroxylase failed to degrade n-octadecane, thereby suggesting a different catalytic mechanism for the microbial transformation of alkanes with chain lengths over C18. PMID:25165808

  19. Characterization of the medium- and long-chain n-alkanes degrading Pseudomonas aeruginosa strain SJTD-1 and its alkane hydroxylase genes.

    PubMed

    Liu, Huan; Xu, Jing; Liang, Rubing; Liu, Jianhua

    2014-01-01

    A gram-negative aliphatic hydrocarbon-degrading bacterium SJTD-1 isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa by comparative analyses of the 16S rRNA sequence, phenotype, and physiological features. SJTD-1 could efficiently mineralize medium- and long-chain n-alkanes (C12-C30) as its sole carbon source within seven days, showing the most optimal growth on n-hexadecane, followed by n-octadecane, and n-eicosane. In 36 h, 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely; and 2 g/L n-hexadecane was degraded to undetectable levels within 72 h. Two putative alkane-degrading genes (gene 3623 and gene 4712) were characterized and our results indicated that their gene products were rate-limiting enzymes involved in the synergetic catabolism of C12-C16 alkanes. On the basis of bioinformatics and transcriptional analysis, two P450 monooxygenases, along with a putative AlmA-like oxygenase, were examined. Genetically defective mutants lacking the characteristic alkane hydroxylase failed to degrade n-octadecane, thereby suggesting a different catalytic mechanism for the microbial transformation of alkanes with chain lengths over C18. PMID:25165808

  20. Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

    PubMed Central

    Schneider, P M; Carroll, M C; Alper, C A; Rittner, C; Whitehead, A S; Yunis, E J; Colten, H R

    1986-01-01

    Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease. Images PMID:3018042

  1. Alu-alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ethlers-Danlos syndrome

    SciTech Connect

    Pousi, B.; Hautala, T.; Heikkinen, J.; Pajunen, L.; Kivirikko, K.I.; Myllylae, R.

    1994-11-01

    The type VI variant of the Ethlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hyptonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS. 38 refs., 6 figs.

  2. Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco.

    PubMed

    Song, Xinyu; Diao, Jinjin; Ji, Jing; Wang, Gang; Guan, Chunfeng; Jin, Chao; Wang, Yurong

    2016-01-01

    Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L. chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101 bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42 kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol in vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells in vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L. chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H

  3. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus.

  4. CYP17A1 gene mutations and hypertension variations found in 46, XY females with combined 17α-hydroxylase/17, 20-lyase deficiency.

    PubMed

    Wang, Yue-Ping; Zhao, Yun-Jing; Zhou, Guang-Yu; He, Bing

    2014-06-01

    The aim of this study was to analyze the structural consequences of the mutations in CYP17A1 gene and their relationship with the variations of clinical manifestations in three patients who presented with complete or partial combined 17α-hydroxylase/17,20-lyase deficiency (17OHD). DNA sequences of the coding exons and intron/exon boundaries of the CYP17A1 gene were analyzed for mutations. In silico analysis with computational three-dimensional model of human P450c17 and multiple alignments analysis were performed to evaluate the spatial conformational changes by missense mutations. Five mutations p.S117fs (c.351_352delCT), p.H373L (c.1184 A>T), p.Y329fs (c.985_987delTACinsAA), p.A82D (c.245 C>A) and p.L209P (c.626 T>C) were identified in three patients, respectively. The novel mutation p.S117fs (c.351_352delCT) has not been reported previously. In silico analysis explained the conformational changes by the described mutations, which resulted in different severe 17OHD. Our studies also suggest that molecular data accompanying with in silico analysis of the CYP17A1 gene are much helpful for the diagnosis, management and genetic counseling of 17OHD.

  5. Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

    PubMed Central

    Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

    2015-01-01

    SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

  6. The mutation spectrum of the phenylalanine hydroxylase (PAH) gene and associated haplotypes reveal ethnic heterogeneity in the Taiwanese population.

    PubMed

    Liang, Ying; Huang, Miao-Zeng; Cheng, Cheng-Yi; Chao, Hung-Kun; Fwu, Victor Tramjay; Chiang, Szu-Hui; Hsiao, Kwang-Jen; Niu, Dau-Ming; Su, Tsung-Sheng

    2014-03-01

    Phenylalanine hydroxylase (PAH) deficiency is responsible for most cases of phenylketonuria (PKU). In this study of the PAH mutation spectrum in the Taiwanese population, 139 alleles were identified including 34 different mutations. The V190G, Q267R and F392I mutations are first reported in this study. The most common mutations, R241C, R408Q and Ex6-96A>G, account for 23.2%, 12.0% and 9.2%, of the mutant alleles, respectively. Haplotype analysis shows that R241C and Ex6-96A>G are exclusively associated with haplotype 4.3 to suggest founder effects. On the other hand, R408Q is found on two distinct haplotypes suggesting recurrent mutations. The spectrum of PAH mutations in Taiwan shows various links to those of other Asian regions, yet remarkable differences exist. Notably, R408Q, E286K and -4173_-407del, accounting for 21% of all mutant alleles in Taiwan, are very rare or are undetected among PKU cohorts of other Asian regions to suggest local founder effects. Moreover, the low homozygosity value of 0.092 hints at a high degree of ethnic heterogeneity within the Taiwanese population. Our study of PAH mutation spectrum and the associated haplotypes is useful for subsequent study on the origin and migration pattern via Taiwan, an island at the historical crossroad of migration of ancient populations.

  7. A new allele of flower color gene W1 encoding flavonoid 3'5'-hydroxylase is responsible for light purple flowers in wild soybean Glycine soja

    PubMed Central

    2010-01-01

    Background Glycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat. Results B09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants. Conclusions B09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be

  8. 5′-Untranslated Region of the Tryptophan Hydroxylase-2 Gene Harbors an Asymmetric Bidirectional Promoter but not Internal Ribosome Entry Site in vitro

    PubMed Central

    Chen, Guo-Lin; Miller, Gregory M.

    2009-01-01

    Tryptophan hydroxylase-2 (TPH2) catalyzes the synthesis of neuronal serotonin, a major neurotransmitter involved in many brain functions and psychiatric disorders. We have previously revealed a critical role of the human TPH2 (hTPH2) 5′-UTR in gene expression regulation. This study aimed to further characterize mechanism(s) by which the hTPH2 5′-UTR regulates gene expression. An internal ribosome entry site (IRES) activity in hTPH2 5′-UTR was suggested by the conventional bicistronic reporter assay; however, further stringent experiments, including in vitro translation, quantitative real-time PCR, Northern blot, ribonuclease protection assay, and monocistronic reporter assay, demonstrated that the hTPH2 5′-UTR harbors a bidirectional promoter, but not IRES, within its downstream segment (61~141). The antisense promoter is much stronger than the sense promoter, but the strength of both promoters are cell-line dependent, with the highest and lowest activities being observed in HEK-293T and SK-N-MC cells, respectively. In accordance with our previous findings, the upstream segment (1~60) of hTPH2 5′-UTR suppresses the neighboring promoter of both direction, independent of the cell line and its location in the 5′- or 3′-flanking regions of the gene. In summary, this study demonstrates that no IRES but an asymmetric bidirectional promoter is present in the downstream segment of hTPH2 5′-UTR, and this promoter is susceptible to a gene silencing effect caused by the upstream segment (1~60) of hTPH2 5′-UTR. Our findings point to the potential involvement of antisense transcription and non-coding RNA in the regulation of TPH2 gene expression. PMID:19344641

  9. Diurnal variation in cholesterol 7α-hydroxylase activity is determined by the -203A>C polymorphism of the CYP7A1 gene

    PubMed Central

    Vlachová, Miluše; Blahová, Tereza; Lánská, Věra; Leníček, Martin; Piťha, Jan; Vítek, Libor; Kovář, Jan

    2016-01-01

    Aim To determine whether the promoter polymorphism -203A>C of cholesterol-7α-hydroxylase encoding gene (CYP7A1) affects diurnal variation in CYP7A1 enzyme activity. Methods The study included 16 healthy male volunteers – 8 homozygous for -203A and 8 homozygous for the -203C allele of CYP7A1. Three 15-hour examinations (from 7am to 10pm) were carried out for each of the participants: after one-day treatment with cholestyramine; after one-day treatment with chenodeoxycholic acid (CDCA); and a control examination without any treatment. The plasma concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, was determined in all the experiments at 90-min intervals. Results CYP7A1 activity was up-regulated after treatment with cholestyramine and suppressed after treatment with CDCA. There were no differences between -203A and -203C allele carriers in the response of enzyme activity to both drugs. In the control experiment, -203A allele carriers displayed diurnal variation in enzyme activity, whereas CYP7A1 activity did not change in -203C allele carriers. These results were confirmed by modeling the dynamics of C4 using polynomial regression. Conclusion The promoter polymorphism of the CYP7A1 gene has a pronounced impact on diurnal variation in CYP7A1 activity. PMID:27106353

  10. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms. PMID:26300047

  11. MORF-RELATED GENE702, a Reader Protein of Trimethylated Histone H3 Lysine 4 and Histone H3 Lysine 36, Is Involved in Brassinosteroid-Regulated Growth and Flowering Time Control in Rice.

    PubMed

    Jin, Jing; Shi, Jinlei; Liu, Bing; Liu, Yanchao; Huang, Ying; Yu, Yu; Dong, Aiwu

    2015-08-01

    The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes and is involved in controlling flowering time in rice (Oryza sativa). Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MORF-RELATED GENE702 (MRG702) binds to trimethylated H3K4 and H3K36 (H3K4me3 and H3K36me3) in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase gene SET DOMAIN GROUP725 (SDG725), the MRG702 knockdown mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed that MRG702 knockdown resulted in the down-regulation of BR-related genes, including DWARF11, BRASSINOSTEROD INSENSITIVE1, and BRASSINOSTEROID UPREGULATED1, and several flowering genes, including Early heading date1 (Ehd1), Ehd2, Ehd3, OsMADS50, Heading date 3a, and RICE FLOWERING LOCUS T1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of trimethylated H3K36, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information. PMID:25855537

  12. MORF-RELATED GENE702, a Reader Protein of Trimethylated Histone H3 Lysine 4 and Histone H3 Lysine 36, Is Involved in Brassinosteroid-Regulated Growth and Flowering Time Control in Rice.

    PubMed

    Jin, Jing; Shi, Jinlei; Liu, Bing; Liu, Yanchao; Huang, Ying; Yu, Yu; Dong, Aiwu

    2015-08-01

    The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes and is involved in controlling flowering time in rice (Oryza sativa). Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MORF-RELATED GENE702 (MRG702) binds to trimethylated H3K4 and H3K36 (H3K4me3 and H3K36me3) in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase gene SET DOMAIN GROUP725 (SDG725), the MRG702 knockdown mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed that MRG702 knockdown resulted in the down-regulation of BR-related genes, including DWARF11, BRASSINOSTEROD INSENSITIVE1, and BRASSINOSTEROID UPREGULATED1, and several flowering genes, including Early heading date1 (Ehd1), Ehd2, Ehd3, OsMADS50, Heading date 3a, and RICE FLOWERING LOCUS T1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of trimethylated H3K36, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information.

  13. MORF-RELATED GENE702, a Reader Protein of Trimethylated Histone H3 Lysine 4 and Histone H3 Lysine 36, Is Involved in Brassinosteroid-Regulated Growth and Flowering Time Control in Rice1[OPEN

    PubMed Central

    Jin, Jing; Shi, Jinlei; Liu, Bing; Liu, Yanchao; Huang, Ying; Yu, Yu; Dong, Aiwu

    2015-01-01

    The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes and is involved in controlling flowering time in rice (Oryza sativa). Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MORF-RELATED GENE702 (MRG702) binds to trimethylated H3K4 and H3K36 (H3K4me3 and H3K36me3) in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase gene SET DOMAIN GROUP725 (SDG725), the MRG702 knockdown mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed that MRG702 knockdown resulted in the down-regulation of BR-related genes, including DWARF11, BRASSINOSTEROD INSENSITIVE1, and BRASSINOSTEROID UPREGULATED1, and several flowering genes, including Early heading date1 (Ehd1), Ehd2, Ehd3, OsMADS50, Heading date 3a, and RICE FLOWERING LOCUS T1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of trimethylated H3K36, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information. PMID:25855537

  14. [Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea].

    PubMed

    Qiang, Wei; Hou, Yan-ling; Li, Xiao; Xia, Ke; Liao, Zhi-hua

    2015-10-01

    Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.

  15. The effects of child maltreatment on early signs of antisocial behavior: genetic moderation by tryptophan hydroxylase, serotonin transporter, and monoamine oxidase A genes.

    PubMed

    Cicchetti, Dante; Rogosch, Fred A; Thibodeau, Eric L

    2012-08-01

    Gene-environment interaction effects in predicting antisocial behavior in late childhood were investigated among maltreated and nonmaltreated low-income children (N = 627, M age = 11.27). Variants in three genes were examined: tryptophan hydroxylase 1 (TPH1), serotonin transporter linked polymorphic region (5-HTTLPR), and monoamine oxidase A (MAOA) upstream variable number tandem repeat. In addition to child maltreatment status, we considered the impact of maltreatment subtypes, developmental timing of maltreatment, and chronicity. Indicators of antisocial behavior were obtained from self-, peer, and adult counselor reports. In a series of analyses of covariance, child maltreatment and its parameters demonstrated strong main effects on early antisocial behavior as assessed by all report forms. Genetic effects operated primarily in the context of gene-environment interactions, moderating the impact of child maltreatment on outcomes. Across the three genes, among nonmaltreated children no differences in antisocial behavior were found based on genetic variation. In contrast, among maltreated children specific polymorphisms of TPH1, 5-HTTLPR, and MAOA were each related to heightened self-report of antisocial behavior; the interaction of 5-HTTLPR and developmental timing of maltreatment also indicated more severe antisocial outcomes for children with early onset and recurrent maltreatment based on genotype. TPH1 and 5-HTTLPR interacted with maltreatment subtype to predict peer reports of antisocial behavior; genetic variation contributed to larger differences in antisocial behavior among abused children. The TPH1 and 5-HTTLPR polymorphisms also moderated the effects of maltreatment subtype on adult reports of antisocial behavior; again, the genetic effects were strongest for children who were abused. In addition, TPH1 moderated the effect of developmental timing of maltreatment and chronicity on adult reports of antisocial behavior. The findings elucidate how genetic

  16. [Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea].

    PubMed

    Qiang, Wei; Hou, Yan-ling; Li, Xiao; Xia, Ke; Liao, Zhi-hua

    2015-10-01

    Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering. PMID:26837185

  17. Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene β-cyclase and β-carotene hydroxylase gene's cDNA

    PubMed Central

    Bhore, Subhash Janardhan; Amelia, Kassim; Wang, Edina; Priyadharsini, Sindhuja; Shah, Farida Habib

    2013-01-01

    The identification of genes and understanding of genes' expression and regulation in common bean (Phaseolus vulgaris L.) is necessary in order to strategize its improvement using genetic engineering techniques. Generation of expressed sequence tags (ESTs) is useful in rapid isolation, identification and characterization of the genes. To study the gene expression in P. vulgaris pods tissue, ESTs generation work was initiated. Early stage and late stage bean-pod-tissues cDNA libraries were constructed using CloneMiner cDNA library construction kit. In total, 5972 EST clones were isolated using random method of gene isolation. While processing ESTs, we found lycopene β-cyclase (PvLCY-β) and β-carotene hydroxylase (PvCHY-β) gene's cDNA. In carotenoid biosynthesis pathway, PvLCY-β catalyzes the production of carotene; and PvCHY-β is known to function as a catalyst in the production of lutein and zeaxanthin. To understand more about PvLCY-β and PvCHY-β, both strands of both cDNA clones were sequenced using M13 forward and reverse primers. Nucleotide and deduced protein sequences were analyzed and annotated using online bioinformatics tools. Results showed that PvLCY-β and PvCHY-β cDNAs are 1639 and 1107 bp in length, respectively. Analysis results showed that PvLCY-β and PvCHY-β gene's cDNA contains an open reading frame (ORF) that encodes for 502 and 305 amino acid residues, respectively. The deduced protein sequence analysis results also showed the presence of conserved domains needed for PvLCY-β and PvCHY-β functions. The phylogenetic analysis of both PvLCY-β and PvCHY-β proteins showed it's closeness with the LCY-β and CHY-β proteins from Glycine max, respectively. The nucleotide sequence of PvLCY-β and PvCHY-β gene's cDNA and it's annotation is reported in this paper. PMID:23519320

  18. Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions.

    PubMed

    Li, Xiangjun; Li, Chaoyuan; Li, Xiaoxia; Cui, Peihe; Li, Qifeng; Guo, Qiaoyan; Han, Hongbo; Liu, Shujun; Sun, Guangdong

    2016-01-01

    Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN. PMID:27652271

  19. Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions

    PubMed Central

    Li, Chaoyuan; Li, Xiaoxia; Cui, Peihe; Li, Qifeng; Han, Hongbo; Liu, Shujun

    2016-01-01

    Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

  20. Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions

    PubMed Central

    Li, Chaoyuan; Li, Xiaoxia; Cui, Peihe; Li, Qifeng; Han, Hongbo; Liu, Shujun

    2016-01-01

    Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN. PMID:27652271

  1. Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

    PubMed

    Trubetskoy, V S; Torchilin, V P; Kennel, S J; Huang, L

    1992-01-01

    A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.

  2. Proline with or without hydroxyproline influences collagen concentration and regulates prolyl 4-hydroxylase α (I) gene expression in juvenile turbo ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Zhang, Kaikai; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Liufu, Zhiguo; Zhang, Yanjiao; Peng, Mo; Ai, Qinghui

    2015-06-01

    This study was conducted to investigate the effect of dietary proline (Pro), and Pro and hydroxyproline (Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I) (P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro (Pro-0.75) or 0.75% Pro and 0.75% Hyp (Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot (P > 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased (P <0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets (P <0.05). The expression of P4H a(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control (P <0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75 (P <0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.

  3. The dopamine beta-hydroxylase gene polymorphism rs1611114 is associated with schizophrenia in the Chinese Zhuang but not Chinese Han population.

    PubMed

    Long, Jianxiong; Huang, Guifeng; Liang, Baoyun; Ling, Weijun; Guo, Xiaojing; Jiang, Juan; Su, Li

    2016-10-01

    Schizophrenia (SCZ) is a devastating neurodevelopmental disorder. However, the mechanism underlying this highly heritable disorder remains unclear. The dopamine beta-hydroxylase (DBH) gene encodes a key metabolic enzyme of dopamine. Consequently, DBH is considered a candidate gene for SCZ. However, previous studies on its association with SCZ susceptibility have shown conflicting results. Here, we examined association between the rs1611114 polymorphism of DBH and SCZ susceptibility and related clinical symptoms. A total of 691 SCZ patients and 698 age- and gender-matched healthy controls were examined. mRNA expression levels of DBH were measured by quantitative real-time polymerase chain reaction, and the rs1611114 polymorphism was genotyped using the Sequenom MassARRAY platform. Also, the Positive and Negative Syndrome Scale (PANSS) was used to assess SCZ clinical symptoms. Our results show lower DBH mRNA expression levels in SCZ patients than healthy controls (Zhuang: p = 0.000; Han: p = 0.037). Interestingly, the rs1611114 polymorphism was significantly associated with SCZ susceptibility (overdominant model: p = 0.010) in only the Chinese Zhuang population. Furthermore, the rs1611114 polymorphism was associated with PANSS total score (allele T/C: p = 0.015) and general psychopathology score (allele T/C: p = 0.027) in Chinese Zhuang SCZ patients. These results suggest that the DBH gene may play an important role in the occurrence of SCZ. Also, rs1611114 may be associated with SCZ susceptibility and related clinical symptoms in the Chinese Zhuang but not Han Chinese population. Further studies with larger samples of different ethnicities are needed to confirm the role of DBH in SCZ. PMID:27236774

  4. Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis.

    PubMed

    Giannetto, Alessia; Maisano, Maria; Cappello, Tiziana; Oliva, Sabrina; Parrino, Vincenzo; Natalotto, Antonino; De Marco, Giuseppe; Barberi, Chiara; Romeo, Orazio; Mauceri, Angela; Fasulo, Salvatore

    2015-12-01

    Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5'-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

  5. Novel mutation of the CYP17 gene in two unrelated patients with combined 17alpha-hydroxylase/17,20-lyase deficiency: demonstration of absent enzyme activity by expressing the mutant CYP17 gene and by three-dimensional modeling.

    PubMed

    Patocs, Attila; Liko, István; Varga, Ibolya; Gergics, Peter; Boros, Andras; Futo, Laszlo; Kun, Imre; Bertalan, Rita; Toth, Szilvia; Pazmany, Tamas; Toth, Miklós; Szücs, Nikolette; Horanyi, Janos; Glaz, Edit; Racz, Karoly

    2005-11-01

    The CYP17 gene, located on chromosome 10q24-q25, encodes the cytochrome P450c17 enzyme. Mutations of this gene cause the 17alpha-hydroxylase/17,20-lyase deficiency, which is a rare, autosomal recessive form of congenital adrenal hyperplasia. Approximately 50 different mutations of the CYP17 gene have been described, of which some mutations have been identified in certain ethnic groups. In this study, we present the clinical history, hormonal findings and mutational analysis of two patients from unrelated families, who were evaluated for hypertension, hypokalemia and sexual infantilism. In the first patient, who was a 37-year-old female, additional studies showed a large myelolipoma in the left adrenal gland, and a smaller tumor in the right adrenal gland. In the second patient, who was a 31-year-old phenotypic female, clinical work-up revealed a 46,XY kariotype, absence of ovaries and presence of testes located in the inner opening of both inguinal canals. Analysis of the CYP17 gene by polymerase chain reaction amplification and direct sequencing demonstrated a novel homozygous mutation of codon 440 from CGC (Arg) to TGC (Cys) in both patients. The effect of this novel mutation on 17alpha-hydroxylase/17,20-lyase activity was assessed by in vitro studies on the mutant and wild-type P450c17 generated by site-directed mutagenesis and transfected in nonsteroidogenic COS-1 cells. These studies showed that the mutant P450c17 protein was produced in transfected COS-1 cells, but it had negligible 17alpha-hydroxylase and 17,20-lyase activities. In addition, three-dimensional computerized modeling of the heme-binding site of the P450c17 enzyme indicated that replacement of Arg by Cys at amino acid position 440 predicts a loss of the catalytic activity of the enzyme, as the mutant enzyme containing Cys440 fails to form a hydrogen bond with the propionate group of heme, which renders the mutant enzyme unable to stabilize the proper position of heme. Based on these findings we

  6. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene

    SciTech Connect

    Wilson, R.C.; Wei, J.Q.; Cheng, K.C.

    1995-05-01

    Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radio active probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient`s other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent`s phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity. 26 refs., 3 figs., 2 tabs.

  7. Ligand specificity of MobR, a transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s

    SciTech Connect

    Yoshida, Mariko; Hiromoto, Takeshi; Hosokawa, Keiichi; Yamaguchi, Hiroshi; Fujiwara, Shinsuke

    2007-10-19

    MobR from Comamonas testosteroni KH122-3s is a member of the MarR family of transcriptional regulators and functions as a repressor for the mobA gene that encodes a 3-hydroxybenzoate 4-hydroxylase. 3-Hydroxybenzoate binds to MobR as a ligand, resulting in an efficient induction of mobA. Various 3-hydroxybenzoate analogues were examined for their inducibilities using the mobA::lacZ transcriptional fusion system. {beta}-Galactosidase was induced by the addition of 2,3-dihydroxybenzoate or 3,5-dihydroxybenzoate besides 3-hydroxybenzoate, suggesting that the hydroxyl group at position 3 is critical in addition to the carboxyl group on the aromatic ring. A gel mobility-shift assay also showed that MobR was released from the target DNA in the presence of these compounds. Circular dichroism studies demonstrated that MobR adopted two conformational states corresponding to the 3-hydroxybenzoate-bound and unbound forms. Other ligands also induced the structural change as well; however, the tertiary structures of converted forms were different from those by 3-hydroxybenzoate.

  8. Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Hwang, L R; Cha, S; Jong, J E; Jang, J H; Seo, T

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV. PMID:25283865

  9. Poly(2-hydroxyethyl methacrylate)-b-poly(L-Lysine) cationic hybrid materials for non-viral gene delivery in NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Johnson, Renjith P; Uthaman, Saji; John, Johnson V; Heo, Min Seon; Park, In Kyu; Suh, Hongsuk; Kim, Il

    2014-09-01

    In order to develop efficient and nontoxic gene delivery vectors, a series of biocompatible block copolymers, poly[(2-hydroxyethyl methacrylate)40 -block-(L-lysine)n ] (n = 40, 80, 120, 150), are prepared by combining an atom transfer radical polymerization of 2-hydroxyethyl methacrylate with a ring-opening polymerization of N(ϵ) -(carbobenzoxy)-L-lysine N-carboxyanhydride. The block copolymers are successfully condensed with plasmid DNA (pDNA) into nanosized (<200 nm) polyplexes. As a representative sample, p(HEMA)40 -b-p(lys)150 is utilized to confirm the effective cellular and nuclear uptake of pDNA. The polymer/pDNA polyplexes exhibit very low cytotoxicity and enhanced transfection activity by being easily taken up into mouse embryonic fibroblast cell line (NIH 3T3). Thus, the chimeric block copolymers provide a means for developing versatile nonviral gene vectors harboring the ideal requirements of low cytotoxicity, good stability, and high transfection efficiency for gene therapy. PMID:24862905

  10. Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Hwang, L R; Cha, S; Jong, J E; Jang, J H; Seo, T

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV.

  11. Role of hMOF-dependent histone H4 lysine 16 acetylation in the maintenance of TMS1/ASC gene activity1

    PubMed Central

    Kapoor-Vazirani, Priya; Kagey, Jacob D.; Powell, Doris R.; Vertino, Paula M.

    2008-01-01

    Epigenetic silencing of tumor suppressor genes in human cancers is associated with aberrant methylation of promoter region CpG islands and local alterations in histone modifications. However, the mechanisms that drive these events remain unclear. Here, we establish an important role for histone H4 lysine 16 acetylation (H4K16Ac) and the histone acetyltransferase hMOF in the regulation of TMS1/ASC, a proapoptotic gene that undergoes epigenetic silencing in human cancers. In the unmethylated and active state, the TMS1 CpG island is spanned by positioned nucleosomes and marked by histone H3K4 methylation. H4K16Ac was uniquely localized to two sharp peaks that flanked the unmethylated CpG island and corresponded to strongly positioned nucleosomes. Aberrant methylation and silencing of TMS1 was accompanied by loss of the H4K16Ac peaks, loss of nucleosome positioning, hypomethylation of H3K4 and hypermethylation of H3K9. In addition, a single peak of histone H4 lysine 20 trimethylation was observed near the transcription start site. Downregulation of hMOF or another component of the MSL complex resulted in a gene-specific decrease in H4K16Ac, loss of nucleosome positioning and silencing of TMS1. Gene silencing induced by H4K16 deacetylation occurred independently of changes in histone methylation and DNA methylation and was reversed upon hMOF re-expression. These results indicate that the selective marking of nucleosomes flanking the CpG island by hMOF is required to maintain TMS1 gene activity, and suggest that the loss of H4K16Ac, mobilization of nucleosomes and transcriptional downregulation may be important events in the epigenetic silencing of certain tumor suppressor genes in cancer. PMID:18701507

  12. Effect of mouse chromosome 13 terminal fragment on liability to catalepsy and expression of tryptophane hydroxylase-2, serotonin transporter, and 5-HT1A receptor genes in the brain.

    PubMed

    Kulikov, A V; Naumenko, V S; Bazovkina, D V; Dee, V Yu; Osipova, D V; Popova, N K

    2009-05-01

    Congenic mice obtained by genome fragments transfer from one strain to another are a potent tool for studies of the molecular mechanisms of behavioral mutations. The 59-70 cM fragment of chromosome 13 containing the locus determining predisposition to freezing reaction (catalepsy) and the gene encoding 5-HT(1A) receptor were transferred from cataleptic CBA/Lac mice into the genome of catalepsy-resistant AKR/J mice. The impact of this fragment for the severity of catalepsy and expression of genes encoding tryptophane hydroxylase-2, serotonin transporter, and 5-HT(1A) receptor was studied. Half of mice of the resultant congenic AKR.CBA-D13Mit76 strain exhibited pronounced catalepsy, similarly to donor CBA animals. The expression of 5-HT(1A) receptor gene in the midbrain of AKR animals was significantly higher than in CBA. The level of 5-HT(1A) receptor mRNA in AKR.CBA-D13Mit76 animals was significantly higher than in the donor strain. Mice of parental AKR and CBA strains did not differ from each other and from AKR.CBA-D13Mit76 animals by the levels of tryptophane hydroxylase-2 and serotonin transporter genes mRNA. These data prove the location of catalepsy regulating gene in the distal fragment of chromosome 13. The recipient strain genome enhanced the expression of 5-HT(1A) receptor gene in the brain without modulating the expression of catalepsy gene.

  13. Is there a link between Depressive Disorders and Tryptophan Hydroxylase 1 (TPH1) Gene Polymorphism? - Study from a Distressed Area, Kashmir (India)

    PubMed Central

    Tarfarosh, Shah Faisal Ahmad; Dar, Mohammad Maqbool; Hussain, Arshad; Shoib, Sheikh; Shah, Tabindah; Shah, Sahil; Manzoor, Mushbiq

    2016-01-01

    Background The progress that man has made in all domains of life, during all these years of reign over the earth, is utterly remarkable. However, it always came at a price. Each epoch of progress has seen human beings inflicted with trauma and cynical consequences. During the last two decades, Kashmiri (Indian) people have experienced continuous violence, a reign of terror, and political turmoil. Each of these disastrous events has contributed to the increase in psychiatric disorders in this part of the world, especially major depressive disorders. We can observe that besides the environmental influences, gene polymorphism also plays a crucial role in the development of depressive disorders. The role of Tryptophan Hydroxylase 1 (TPH1) gene is implicated in various psychiatric disorders, including depression. However, no study has investigated TPH1 A779C gene polymorphism in depressive disorders in a distressed society like Kashmir (India). Aims To study TPH1 A779C single nucleotide polymorphism in depressive disorders in Kashmiri (Indian) population. Materials and Methods Two hundred and forty patients diagnosed with depressive disorder, and 160 unrelated healthy volunteers (control), were studied in a case-control study design. Polymorphism was determined using polymerase chain reaction (PCR) and agarose gel electrophoresis, after digestion with HAP II enzyme. Genotypes and allele frequencies were compared using Chi-square tests, Fisher’s exact test, odds ratio, 95% confidence interval (C.I.) and a p-value of <0.05 was considered to be statistically significant. Results The mean age ± standard deviation (SD) of depression and control group was 32.02±10.99 and 31.75±9.93, respectively (p= 0.512). It was found that the patients from depression group had AA genotype (51.7%) in comparison to control group (17.5%) and these results were statistically significant (p≤0.0001). Calculation of allelic frequency revealed a stronger association of A allele with

  14. Is there a link between Depressive Disorders and Tryptophan Hydroxylase 1 (TPH1) Gene Polymorphism? - Study from a Distressed Area, Kashmir (India)

    PubMed Central

    Tarfarosh, Shah Faisal Ahmad; Dar, Mohammad Maqbool; Hussain, Arshad; Shoib, Sheikh; Shah, Tabindah; Shah, Sahil; Manzoor, Mushbiq

    2016-01-01

    Background The progress that man has made in all domains of life, during all these years of reign over the earth, is utterly remarkable. However, it always came at a price. Each epoch of progress has seen human beings inflicted with trauma and cynical consequences. During the last two decades, Kashmiri (Indian) people have experienced continuous violence, a reign of terror, and political turmoil. Each of these disastrous events has contributed to the increase in psychiatric disorders in this part of the world, especially major depressive disorders. We can observe that besides the environmental influences, gene polymorphism also plays a crucial role in the development of depressive disorders. The role of Tryptophan Hydroxylase 1 (TPH1) gene is implicated in various psychiatric disorders, including depression. However, no study has investigated TPH1 A779C gene polymorphism in depressive disorders in a distressed society like Kashmir (India). Aims To study TPH1 A779C single nucleotide polymorphism in depressive disorders in Kashmiri (Indian) population. Materials and Methods Two hundred and forty patients diagnosed with depressive disorder, and 160 unrelated healthy volunteers (control), were studied in a case-control study design. Polymorphism was determined using polymerase chain reaction (PCR) and agarose gel electrophoresis, after digestion with HAP II enzyme. Genotypes and allele frequencies were compared using Chi-square tests, Fisher’s exact test, odds ratio, 95% confidence interval (C.I.) and a p-value of <0.05 was considered to be statistically significant. Results The mean age ± standard deviation (SD) of depression and control group was 32.02±10.99 and 31.75±9.93, respectively (p= 0.512). It was found that the patients from depression group had AA genotype (51.7%) in comparison to control group (17.5%) and these results were statistically significant (p≤0.0001). Calculation of allelic frequency revealed a stronger association of A allele with

  15. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27.

    PubMed

    Aronson, B E; Rabello Aronson, S; Berkhout, R P; Chavoushi, S F; He, A; Pu, W T; Verzi, M P; Krasinski, S D

    2014-11-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal ('jejunal') identity while repressing a distal intestinal ('ileal') identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal versus ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- versus up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wild-type control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27.

  16. IFN-γ Induces Histone 3 Lysine 27 Trimethylation in a Small Subset of Promoters to Stably Silence Gene Expression in Human Macrophages.

    PubMed

    Qiao, Yu; Kang, Kyuho; Giannopoulou, Eugenia; Fang, Celeste; Ivashkiv, Lionel B

    2016-09-20

    The mechanisms by which IFN-γ activates expression of interferon-stimulated genes that have inflammatory and host defense functions are well understood. In contrast, little is known about how IFN-γ represses gene expression. By using transcriptomic and epigenomic analysis, we found that stable repression of a small group of genes by IFN-γ is associated with recruitment of the histone methyltransferase EZH2 and deposition of the negative mark histone 3 lysine 27 trimethylation (H3K27me3) at their promoters. Repressed genes included MERTK, PPARG, and RANK, which have anti-inflammatory functions and promote osteoclast differentiation. Gene repression and H3K27me3 persisted after IFN-γ signaling was terminated, and these silenced genes were no longer responsive to glucocorticoids, IL-4, and M-CSF. These results identify cytokine-induced H3K27 trimethylation as a mechanism that stabilizes gene silencing in macrophages. IFN-γ-induced macrophage activation is thus reinforced by a chromatin-based mechanism that blocks anti-inflammatory and opposing pathways. PMID:27653678

  17. Identification of a brain specific protein that associates with a refsum disease gene product, phytanoyl-CoA alpha-hydroxylase.

    PubMed

    Lee, Z H; Kim, H; Ahn, K Y; Seo, K H; Kim, J K; Bae, C S; Kim, K K

    2000-02-22

    Refsum disease is an autosomal recessive neurologic disorder of the lipid metabolism. Major diagnostic clinical findings include retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, increased cerebrospinal fluid protein without pleocytosis, nerve deafness, and cardiac involvement. We have identified a novel protein (PAHX-AP #1) associated with phytanoyl-CoA alpha-hydroxylase (PAHX), a Refsum disease gene product, using the yeast-based two-hybrid assay. The middle portion (amino acids 83-264) of PAHX was used as a bait and a mouse brain cDNA library was searched. The ability of PAHX-AP #1 to interact with PAHX was confirmed using immunoprecipitation and Western blot studies in NIH3T3 cells which stably expressed both PAHX and PAHX-AP #1. Northern and Western blot analyses demonstrated a unique pattern of developmental PAHX-AP #1 expression which was targeted to the adult brain, but ubiquitous expressions of PAHX were observed in all examined tissues. In situ hybridization analyses of the brain showed specific localization of PAHX-AP #1 to the supragranular layer in the cerebral cortex, dentate gyrus, hippocampus, Purkinje cell layer, deep cerebellar nucleus, trigeminal nucleus, abducent nucleus, facial nucleus, cochlear and vestibular nucleus, ganglion cell and nuclear layer of the retina. These data indicate that localization of PAHX-AP #1 in the brain is correlated with central neurologic symptoms of Refsum disease such as retinitis pigmentosa, cerebellar ataxia, nerve deafness and suggest that PAHX-AP #1 may be involved in the development of the central neurologic deficits of Refsum disease.

  18. [Influence of chronic alcohol treatment on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes in the mouse brain: Role of the C1473G polymorphism in the gene encoding tryptophan hydroxylase 2].

    PubMed

    Bazovkina, D V; Tsybko, A S; Filimonova, E A; Ilchibaeva, T V; Naumenko, V S

    2016-01-01

    Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment.

  19. [Influence of chronic alcohol treatment on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes in the mouse brain: Role of the C1473G polymorphism in the gene encoding tryptophan hydroxylase 2].

    PubMed

    Bazovkina, D V; Tsybko, A S; Filimonova, E A; Ilchibaeva, T V; Naumenko, V S

    2016-01-01

    Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment. PMID:27239851

  20. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

    PubMed

    Gatto, Gregory J; Boyne, Michael T; Kelleher, Neil L; Walsh, Christopher T

    2006-03-22

    Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner. PMID:16536560

  1. The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    PubMed Central

    Zsindely, Nóra; Pankotai, Tibor; Újfaludi, Zsuzsanna; Lakatos, Dániel; Komonyi, Orbán; Bodai, László; Tora, László; Boros, Imre M.

    2009-01-01

    In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently. PMID:19740772

  2. Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1

    PubMed Central

    Wang, Meizhen; Liu, Chen; Li, Shixue; Zhu, Dengyun; Zhao, Qian; Yu, Jingjuan

    2013-01-01

    Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9–10 and 19–11, ZmMYC1 in line 19–11 and ZmSDR in line 19–11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt

  3. The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

    PubMed Central

    Taylor, D S; Dahl, H H; Mercer, J F; Green, A K; Fisher, M J

    1989-01-01

    The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression. Images Fig. 1. Fig. 3. PMID:2532505

  4. Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

    PubMed Central

    Alwani, Saniya; Kaur, Randeep; Michel, Deborah; Chitanda, Jackson M; Verrall, Ronald E; Karunakaran, Chithra; Badea, Ildiko

    2016-01-01

    Purpose Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. Methods lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry. Results Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization

  5. The genetics of aflatoxin B1 metabolism. Association of the induction of aflatoxin B1-4-hydroxylase with the transcriptional activation of cytochrome P3-450 gene.

    PubMed

    Koser, P L; Faletto, M B; Maccubbin, A E; Gurtoo, H L

    1988-09-01

    The association between murine cytochrome P3-450 and hepatic aflatoxin B1-4-hydroxylase, a cytochrome P-450-dependent enzyme which converts aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1), was examined by (a) purification of the cytochrome P-450 which preferentially metabolizes AFB1 to AFM1; (b) isolation of the specific cDNA clone; and (c) correlating induction of transcriptional activation of the specific message with the enzyme activity in the hepatic microsomes. Isolation of cytochromes P-450 from C57BL/6 mice, an Ah-responsive strain, pretreated with a 150 mg/kg dose of beta-naphthoflavone resulted in the partial purification of the cytochrome P-450 with preference for the metabolism of AFB1 to AFM1. Antibodies raised against this cytochrome P-450 were used to enrich hepatic mRNA for cDNA cloning. A cDNA library screened with a rat cytochrome P-450c gene probe yielded only two types of cDNA clones that contained inserts corresponding to cytochrome P1-450 and cytochrome P3-450. Specific restriction fragments of near full-length P1-450 cDNA and full-length P3-450 cDNA, hybridizing only with their respective messages, were isolated and used to assess transcriptional activation of these messages in liver and extrahepatic tissues from C57BL/6 mice treated with 3-methylcholanthrene, beta-naphthoflavone, indolylacetonitrile, and Aroclor-1254. Dose-dependent induction of the two messenger RNAs, when compared with the induction of specific enzyme activities, demonstrated the association of cytochrome P1-450 with aryl hydrocarbon hydroxylase activity and the association of cytochrome P3-450 with AFB1-4-hydroxylase activity. This supports our earlier hypothesis that AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase, although regulated by the Ah locus, are the products of two separate genes (Gurtoo, H.L., Dahms, R.P., Kanter, P., and Vaught, J.B. (1978) J. Biol. Chem. 253, 3952-3961). PMID:3137229

  6. Down-regulation of flavonoid 3'-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence.

    PubMed

    Nagamatsu, Atsushi; Masuta, Chikara; Matsuura, Hideyuki; Kitamura, Keisuke; Abe, Jun; Kanazawa, Akira

    2009-01-01

    Changes in flavonoid content are often manifested as altered pigmentation in plant tissues. Two loci have been identified as controlling pigmentation in soybean pubescence. Of these, the T locus appears to encode flavonoid 3'-hydroxylase (F3'H) protein: the T and t alleles are associated with tawny and gray colors, respectively, in pubescence. We previously down-regulated F3'H gene expression by virus-induced gene silencing (VIGS) in soybean. Despite this successful VIGS, the tawny pubescence pigmentation proved to be unchanged in greenhouse-grown plants. We hypothesized that the reduced mRNA level of the F3'H gene resulting from VIGS remained high enough to induce pigmentation. To verify this hypothesis, in the present study, we performed F3'H VIGS on plants grown under controlled conditions, in which the steady-state mRNA level of the F3'H gene was reduced to approximately 5% of that of greenhouse-grown plants. This VIGS treatment resulted in the loss of tawny pigmentation in pubescence, suggesting that the sf3'h1 gene is involved in the control of pigmentation in pubescence. We detected a marked decrease in target mRNA, an accumulation of short interfering RNAs (siRNAs), and a decrease in quercetin content relative to kaempferol in leaf tissues, indicating that sequence-specific mRNA degradation of the F3'H gene was induced. These results suggest that leaf tissues have a threshold mRNA level of the F3'H gene, which is associated with the occurrence of tawny pigmentation in pubescence. The estimated threshold mRNA level for pigmentation in pubescence was approximately 3% of the steady-state mRNA level of the F3'H gene in greenhouse-grown plants.

  7. Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch.

    PubMed

    Shimada, Masaya; Mochizuki, Kazuki; Goda, Toshinao

    2013-03-01

    Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.

  8. Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds.

    PubMed

    Ozga, Jocelyn A; Yu, Jody; Reinecke, Dennis M

    2003-03-01

    To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (-2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6-10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

  9. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

    PubMed

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  10. A star-shaped porphyrin-arginine functionalized poly(L-lysine) copolymer for photo-enhanced drug and gene co-delivery.

    PubMed

    Ma, Dong; Lin, Qian-Ming; Zhang, Li-Ming; Liang, Yuan-Yuan; Xue, Wei

    2014-05-01

    The co-delivery of drug and gene has become the primary strategy in cancer and other disease therapy. To co-deliver hydrophobic drug and functional gene efficiently into tumor cells, a star-shaped copolymer (PP-PLLD-Arg) with a photochemical internalization effect consisting of a porphyrin (PP) core and arginine-functionalized poly(L-lysine) dendron (PLLD-Arg) arms has been designed, and used to co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid for nasopharyngeal cancer therapy. It was found that PP-PLLD-Arg/MMP-9 nanocomplex showed the photo-enhanced gene transfection efficiency in vitro, and could mediate a significant reduce of MMP-9 protein expression in HNE-1 cells. For co-delivery analysis, the obtained PP-PLLD-Arg/DOC/MMP-9 complexes could induce a more significant apoptosis than DOC or MMP-9 used only, and decreased invasive capacity of HNE-1 cells. Moreover, the star-shaped copolymer exhibited better blood compatibility and lower cytotoxicity compared to PEI-25k in the hemolysis and MTT assays, and also showed a good biocompatibility in vivo. Therefore, PP-PLLD-Arg with suited irradiation is a promising non-toxic and photo-inducible effective drug and gene delivery strategy, which should be encouraged in tumor therapy.

  11. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27

    PubMed Central

    Aronson, B. E.; Aronson, S. Rabello; Berkhout, R. P.; Chavoushi, S. F.; He, A.; Pu, W. T.; Verzi, M. P.; Krasinski, S. D.

    2015-01-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal (‘jejunal’) identity while repressing a distal intestinal (‘ileal’) identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal vs. ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- vs. up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wildtype control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27. PMID:24878542

  12. Epistatic and gene wide effects in YWHA and aromatic amino hydroxylase genes across ADHD and other common neuropsychiatric disorders: Association with YWHAE

    PubMed Central

    Jacobsen, Kaya K.; Kleppe, Rune; Johansson, Stefan; Zayats, Tetyana

    2015-01-01

    Monoamines critically modulate neurophysiological functions affected in several neuropsychiatric disorders. We therefore examined genes encoding key enzymes of catecholamine and serotonin biosynthesis (tyrosine and tryptophan hydroxylases—TH and TPH1/2) as well as their regulatory 14‐3‐3 proteins (encoded by YWHA‐genes). Previous studies have focused mainly on the individual genes, but no analysis spanning this regulatory network has been reported. We explored interactions between these genes in Norwegian patients with adult attention deficit hyperactivity disorder (aADHD), followed by gene‐complex association tests in four major neuropsychiatric conditions; childhood ADHD (cADHD), bipolar disorder, schizophrenia, and major depressive disorder. For interaction analyses, we evaluated 55 SNPs across these genes in a sample of 583 aADHD patients and 637 controls. For the gene‐complex tests, we utilized the data from large‐scale studies of The Psychiatric Genomics Consortium (PGC). The four major neuropsychiatric disorders were examined for association with each of the genes individually as well as in three complexes as follows: (1) TPH1 and YWHA‐genes; (2) TH, TPH2 and YWHA‐genes; and (3) all genes together. The results show suggestive epistasis between YWHAE and two other 14‐3‐3‐genes ‐ YWHAZ, YWHAQ ‐ in aADHD (nominal P‐value of 0.0005 and 0.0008, respectively). In PGC data, association between YWHAE and schizophrenia was noted (P = 1.00E‐05), whereas the combination of TPH1 and YWHA‐genes revealed signs of association in cADHD, schizophrenia, and bipolar disorder. In conclusion, polymorphisms in the YWHA‐genes and their targets may exert a cumulative effect in ADHD and related neuropsychiatric conditions, warranting the need for further investigation of these gene‐complexes. © 2015 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc. PMID:26172220

  13. Histone H3 methylation at lysine 4 on the SLC2A5 gene in intestinal Caco-2 cells is involved in SLC2A5 expression.

    PubMed

    Inamochi, Yuko; Mochizuki, Kazuki; Osaki, Ayumi; Ishii, Takeshi; Nakayama, Tsutomu; Goda, Toshinao

    2010-01-29

    Histone H3 methylation at lysine 4 (K4) is associated with euchromatic regions and is thought to be important for the transcriptional activation of genes during differentiation. In this study, we found that di- and tri-methylation of histone H3 at K4 and acetylation of histones H3 and H4 from the promoter/enhancer to the transcribed region close to the transcription initiation site of the solute carrier family 2, member 5 (SLC2A5) gene, and its expression, were induced by differentiation of intestine-like Caco-2 cells. These effects were accompanied by contact inhibition of cell growth of these cells. Furthermore, these modifications were induced by co-treatment with a synthetic glucocorticoid hormone dexamethasone and a p44/42 mitogen-activated protein kinase inhibitor PD89059. Our results suggest that methylation of histone H3 at K4 and acetylation of histones H3 and H4 are involved in SLC2A5 gene induction associated with intestinal differentiation of Caco-2 cells.

  14. Role of polymeric endosomolytic agents in gene transfection: a comparative study of poly(L-lysine) grafted with monomeric L-histidine analogue and poly(L-histidine).

    PubMed

    Hwang, Hee Sook; Hu, Jun; Na, Kun; Bae, You Han

    2014-10-13

    Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(L-lysine) (PLL), a stacking form of poly(L-histidine) (PLL-g-PHis) and a mono-L-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups from PHis also provided higher gene expression than mHis due to its ability to escape the endosome. mHis or PHis grafting reduced the cytotoxicity of PLL and changed the rate of cellular uptake by changing the quantity of free ε-amines available for gene condensation. The subcellular localization of PLL-g-PHis/pDNA measured by YOYO1-pDNA intensity was highest inside the nucleus, while the lysotracker, which stains the acidic compartments was lowest among these polymers. Thus, the polymeric histidine arrangement demonstrate the ability to escape the endosome and trigger rapid release of polyplexes into the cytosol, resulting in a greater amount of pDNA available for translocation to the nucleus and enhanced gene expression.

  15. Prolyl 4-hydroxylase

    PubMed Central

    Gorres, Kelly L.; Raines, Ronald T.

    2010-01-01

    Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on–off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein–protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor α, which plays a key role in sensing molecular oxygen, and could act on inhibitory κB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification. PMID:20199358

  16. Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

    NASA Astrophysics Data System (ADS)

    Hashimoto, Tomoko; Kawazu, Takeshi; Nagasaki, Takeshi; Murakami, Akira; Yamaoka, Tetsuji

    2012-02-01

    Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

  17. Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

    PubMed Central

    Brasacchio, Daniella; Okabe, Jun; Tikellis, Christos; Balcerczyk, Aneta; George, Prince; Baker, Emma K.; Calkin, Anna C.; Brownlee, Michael; Cooper, Mark E.; El-Osta, Assam

    2009-01-01

    OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes. PMID:19208907

  18. Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library

    PubMed Central

    Bhore, Subhash J; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

    2010-01-01

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. Abbreviations ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster PMID:21364789

  19. Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.

    PubMed

    Mersman, Douglas P; Du, Hai-Ning; Fingerman, Ian M; South, Paul F; Briggs, Scott D

    2012-01-20

    Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss of H3K4 methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between the C terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishes H3K4 methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counterparts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals. PMID:22147691

  20. Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content.

    PubMed

    Winter, Andreas; Krämer, Wolfgang; Werner, Fabian A O; Kollers, Sonja; Kata, Srinivas; Durstewitz, Gregor; Buitkamp, Johannes; Womack, James E; Thaller, Georg; Fries, Ruedi

    2002-07-01

    DGAT1 encodes diacylglycerol O-acyltransferase (EC ), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein-Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof.

  1. Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content

    PubMed Central

    Winter, Andreas; Krämer, Wolfgang; Werner, Fabian A. O.; Kollers, Sonja; Kata, Srinivas; Durstewitz, Gregor; Buitkamp, Johannes; Womack, James E.; Thaller, Georg; Fries, Ruedi

    2002-01-01

    DGAT1 encodes diacylglycerol O-acyltransferase (EC 2.3.1.20), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein–Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof. PMID:12077321

  2. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element*S⃞

    PubMed Central

    Garst, Andrew D.; Héroux, Annie; Rambo, Robert P.; Batey, Robert T.

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  3. Crystal structure of the lysine riboswitch regulatory mRNA element.

    PubMed

    Garst, Andrew D; Héroux, Annie; Rambo, Robert P; Batey, Robert T

    2008-08-15

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  4. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

    SciTech Connect

    Garst, A.; Heroux, A; Rambo, R; Batey, R

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8{angstrom} resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

  5. The relationship between lysine 4 on histone H3 methylation levels of alcohol tolerance genes and changes of ethanol tolerance in Saccharomyces cerevisiae

    PubMed Central

    Wang, Hang; Ji, Binfeng; Ren, Hongzhen; Meng, Chun

    2014-01-01

    We evaluated whether epigenetic changes contributed to improve ethanol tolerance in mutant populations of Saccharomyces cerevisiae (S. cerevisiae). Two ethanol-tolerant variants of S. cerevisiae were used to evaluate the genetic stability in the process of stress-free passage cultures. We found that acquired ethanol tolerance was lost and transcription level of some genes (HSP104, PRO1, TPS1, and SOD1) closely related to ethanol tolerance decreased significantly after the 10th passage in ethanol-free medium. Tri-methylation of lysine 4 on histone H3 (H3K4) enhanced at the promoter of HSP104, PRO1, TPS1 and SOD1 in ethanol-tolerant variants of S. cerevisiae was also diminished after tenth passage in stress-free cultures. The ethanol tolerance was reacquired when exogenous SOD1 transferred in some tolerance-lost strains. This showed that H3K4 methylation is involved in phenotypic variation with regard to ethanol tolerance with respect to classic breeding methods used in yeast. PMID:24779776

  6. Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8.

    PubMed

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

    2014-09-01

    The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the K(m) and V(max) of recombinant phenol hydroxylase were 0.21 mmol·L(-1) and 0.077 μmol·L(-1)·min(-1), respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

  7. Functional analysis of abscisic acid 8'-hydroxylase.

    PubMed

    Endo, Akira; Kimura, Mitsuhiro; Kawakami, Naoto; Nambara, Eiji

    2011-01-01

    Abscisic acid (ABA) plays an important role in the control of seed dormancy and germination. Identification of hormone metabolism genes from a particular plant species of interest is an essential step in hormone research. The function of these gene products is validated by biochemical analysis using heterologous expression systems, such as E. coli and yeast. ABA 8'-hydroxylase is a subfamily of P450 monooxygenases and is encoded by CYP707A genes. CYP707A catalyzes the committed step in the major ABA catabolic pathway. In this chapter, we describe the methods for RNA extraction from seeds, cloning the CYP707A cDNAs, protein expression in yeast, and biochemical analysis of their gene products.

  8. Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.

    PubMed

    Zehnder, D; Bland, R; Williams, M C; McNinch, R W; Howie, A J; Stewart, P M; Hewison, M

    2001-02-01

    The mitochondrial enzyme 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D(3), in the kidney. However, enzyme activity assays indicate that 1 alpha-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1 alpha-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1 alpha-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1 alpha-hydroxylase in both normal and diseased tissues. Specific staining for 1 alpha-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Further studies using psoriatic skin highlighted overexpression of 1 alpha-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1alpha-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin from these patients 1 alpha-hydroxylase expression was observed in cells positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1 alpha-hydroxylase in several extrarenal tissues, such as skin, placenta, and lymph nodes. The function of this enzyme at novel extrarenal sites, such as adrenal medulla, brain, pancreas, and colon, remains to be determined. However, the discrete patterns of staining in these tissues emphasizes a possible role for 1 alpha-hydroxylase

  9. C1473G polymorphism in mouse tph2 gene is linked to tryptophan hydroxylase-2 activity in the brain, intermale aggression, and depressive-like behavior in the forced swim test.

    PubMed

    Osipova, Daria V; Kulikov, Alexander V; Popova, Nina K

    2009-04-01

    Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme of brain serotonin synthesis. The C1473G polymorphism in the mouse tryptophan hydroxylase-2 gene affects the enzyme's activity. In the present study, we investigated the linkage between the C1473G polymorphism, enzyme activity in the brain, and behavior in the forced swim, intermale aggression, and open field tests using mice of the C57BL/6 (C/C) and CC57BR/Mv (G/G) strains and the B6-1473C (C/C) and B6-1473G (G/G) lines created by three successive backcrossings on C57BL/6. Mice of the CC57BR/Mv strain had decreased brain enzyme activity, aggression intensity, and immobility in the forced swim test, but increased locomotor activity and time spent in the central part of the open field arena compared with animals of the C57BL/6 strain. Mice of the B6-1473G line homozygous for the 1473G allele had lower TPH2 activity in the brain, aggression intensity, and immobility time in the forced swim test compared with animals of the B6-1473C line homozygous for the 1473C allele. No differences were found between the B6-1473G and B6-1473C mice in locomotor activity and time spent in the central part of the arena in the open field test. Thus, the C1473G polymorphism is involved in the determination of TPH2 activity and is linked to aggression intensity and forced-swim immobility in mice. At the same time, the polymorphism does not affect locomotion and anxiety-related behavior in the open field test. The B6-1473C and B6-1473G mice represent a valuable experimental model for investigating molecular mechanisms of serotonin-related behavior.

  10. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans.

    PubMed

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-03-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l(-1) when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24-30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis.

  11. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans

    PubMed Central

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-01-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24–30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis. PMID:24423427

  12. Aryl hydrocarbon hydroxylase in cultured lymphocytes of twins.

    PubMed Central

    Paigen, B; Ward, E; Steenland, K; Houten, L; Gurtoo, H L; Minowada, J

    1978-01-01

    Measurement of aryl hydrocarbon hydroxylase (AHH) in cultured lymphocytes of 18 monozygotic and 30 dizygotic twin pairs showed that basal and induced AHH activity and AHH inducibility are heritable traits. The data are consistent with AHH inducibility being determined by a single or a very few polymorphic genes. PMID:569973

  13. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  14. Dopaminergic inhibition by G9a/Glp complex on tyrosine hydroxylase in nerve injury-induced hypersensitivity

    PubMed Central

    Wang, Nan; Shen, Xiaofeng; Bao, Senzhu; Feng, Shan-Wu; Wang, Wei; Liu, Yusheng; Wang, Yiquan; Wang, Xian; Guo, Xirong; Shen, Rong; Wu, Haibo; Lei, Liming; Wang, Fuzhou

    2016-01-01

    The neural balance between facilitation and inhibition determines the final tendency of central sensitization. Nerve injury-induced hypersensitivity was considered as the results from the enhanced ascending facilitation and the diminished descending inhibition. The role of dopaminergic transmission in the descending inhibition has been well documented, but its underlying molecular mechanisms are unclear. Previous studies demonstrated that the lysine dimethyltransferase G9a/G9a-like protein (Glp) complex plays a critical role in cocaine-induced central plasticity, and given cocaine’s role in the nerve system is relied on its function on dopamine system, we herein proposed that the reduced inhibition of dopaminergic transmission was from the downregulation of tyrosine hydroxylase expression by G9a/Glp complex through methylating its gene Th. After approval by the Animal Care and Use Committee, C57BL/6 mice were used for pain behavior using von Frey after spared nerve injury, and Th CpG islands methylation was measured using bisulfite sequencing at different nerve areas. The inhibitor of G9a/Glp, BIX 01294, was administered intraventricularly daily with bolus injection. The protein levels of G9a, Glp, and tyrosine hydroxylase were measured with immunoblotting. Dopamine levels were detected using high-performance liquid chromatography. The expression of G9a but not Glp was upregulated in ventral tegmental area at post-injury day 4 till day 49 (the last day of the behavioral test). Correspondingly, the Th CpG methylation is increased, but the tyrosine hydroxylase expression was downregulated and the dopamine level was decreased. After the intracerebroventriclar injection of BIX 01294 since the post-injury days 7 and 14 for consecutive three days, three weeks, and six weeks, the expression of tyrosine hydroxylase was upregulated with a significant decrease in Th methylation and increase in dopamine level. Moreover, the pain after G9a/Glp inhibitor was attenuated

  15. Dopaminergic inhibition by G9a/Glp complex on tyrosine hydroxylase in nerve injury-induced hypersensitivity.

    PubMed

    Wang, Nan; Shen, Xiaofeng; Bao, Senzhu; Feng, Shan-Wu; Wang, Wei; Liu, Yusheng; Wang, Yiquan; Wang, Xian; Guo, Xirong; Shen, Rong; Wu, Haibo; Lei, Liming; Xu, Shiqin; Wang, Fuzhou

    2016-01-01

    The neural balance between facilitation and inhibition determines the final tendency of central sensitization. Nerve injury-induced hypersensitivity was considered as the results from the enhanced ascending facilitation and the diminished descending inhibition. The role of dopaminergic transmission in the descending inhibition has been well documented, but its underlying molecular mechanisms are unclear. Previous studies demonstrated that the lysine dimethyltransferase G9a/G9a-like protein (Glp) complex plays a critical role in cocaine-induced central plasticity, and given cocaine's role in the nerve system is relied on its function on dopamine system, we herein proposed that the reduced inhibition of dopaminergic transmission was from the downregulation of tyrosine hydroxylase expression by G9a/Glp complex through methylating its gene Th After approval by the Animal Care and Use Committee, C57BL/6 mice were used for pain behavior using von Frey after spared nerve injury, and Th CpG islands methylation was measured using bisulfite sequencing at different nerve areas. The inhibitor of G9a/Glp, BIX 01294, was administered intraventricularly daily with bolus injection. The protein levels of G9a, Glp, and tyrosine hydroxylase were measured with immunoblotting. Dopamine levels were detected using high-performance liquid chromatography. The expression of G9a but not Glp was upregulated in ventral tegmental area at post-injury day 4 till day 49 (the last day of the behavioral test). Correspondingly, the Th CpG methylation is increased, but the tyrosine hydroxylase expression was downregulated and the dopamine level was decreased. After the intracerebroventriclar injection of BIX 01294 since the post-injury days 7 and 14 for consecutive three days, three weeks, and six weeks, the expression of tyrosine hydroxylase was upregulated with a significant decrease in Th methylation and increase in dopamine level. Moreover, the pain after G9a/Glp inhibitor was attenuated

  16. Lysine supplementation of commercial fishmeal-free diet in hybrid striped bass Morone chrysops x M. saxatilis affects expression of growth related genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our recent results in hybrid striped bass (HSB) concluded that ideal protein theory accurately predicts first-limiting amino acids in commercial diet formulations if accurate amino acid availability data are used and that appropriate levels of supplemental lysine are needed in order to improve fish ...

  17. Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus.

    PubMed Central

    DiMarco, A A; Averhoff, B; Ornston, L N

    1993-01-01

    We have identified pobR, a gene encoding a transcriptional activator that regulates expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase (PobA) in Acinetobacter calcoaceticus ADP1. Inducible expression of cloned pobA in Escherichia coli depended upon the presence of a functional pobR gene, and mutations within pobR prevented pobA expression in A. calcoaceticus. A pobA-lacZ operon fusion was used to demonstrate that pobA expression in A. calcoaceticus is enhanced up to 400-fold by the inducer p-hydroxybenzoate. Inducer concentrations as low as 10(-7) M were sufficient to elicit partial induction. Some structurally related analogs of p-hydroxybenzoate, unable to cause induction by themselves, were effective anti-inducers. The nucleotide sequence of pobR was determined, and the activator gene was shown to be transcribed divergently from pobA; the genes are separated by 134 DNA base pairs. The deduced amino acid sequence yielded a polypeptide of M(r) = 30,764. Analysis of this sequence revealed at the NH2 terminus a stretch of residues with high potential for forming a helix-turn-helix structure that could serve as a DNA-binding domain. A conservative amino acid substitution (Arg-61-->His-61) in this region inactivated PobR. The primary structure of PobR appears to be evolutionarily distinct from the four major families of NH2-terminal helix-turn-helix containing bacterial regulatory proteins that have been identified thus far. PMID:8331077

  18. Induction by fructose force-feeding of histone H3 and H4 acetylation at their lysine residues around the Slc2a5 gene and its expression in mice.

    PubMed

    Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2013-01-01

    It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.

  19. 21-Hydroxylase gene mutant allele CYP21A2*15 strongly linked to the resistant HLA haplotype B*14:02-DRB1*01:02 in chronic Chagas disease.

    PubMed

    del Puerto, Florencia; Kikuchi, Mihoko; Nishizawa, Juan Eiki; Roca, Yelin; Avila, Cinthia; Gianella, Alberto; Lora, Javier; Gutierrez Velarde, Freddy Udalrico; Hirayama, Kenji

    2013-06-01

    We previously reported protective haplotype HLA-B*14:02-DRB1*01:02 against chronic Chagas disease in Bolivia. The V281L mutant allele of the 21-Hydroxylase gene, CYP21A2*15, is reported to be located in the Class III region of the Human leukocyte antigen region and linked to the haplotype HLA-B*14:02-DRB1*01:02. The mutant allele might play a primary role in the pathogenesis of chronic Chagas disease in the associated HLA region. We analyzed the frequency of this allele in the same subjects for the previous one. The statistical analysis showed a significant association of the CYP21A2*15 with resistance to severe chronic Chagas disease (OR=0.207273; Pv=0.0041). However, there is no significant tendency of the mutant gene contribution to the resistance after the elimination of the HLA-B*14:02-DRB1*01:02 linked mutants (OR=0.38; Pv=0.1533). Although the frequency of the CYP21A2*15 was small, we found no primary contribution of this mutation to the protection against chronic Chagas disease.

  20. Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

    SciTech Connect

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; Rottmann, William H.; Kuhn, Sean A.; Foster, Cliff E.; Ziebell, Angela; Turner, Geoffrey B.; Decker, Stephen R.; Hinchee, Maud A. W.; Davis, Mark F.

    2015-08-27

    In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

  1. Role of several histone lysine methyltransferases in tumor development

    PubMed Central

    LI, JIFU; ZHU, SHUNQIN; KE, XIAO-XUE; CUI, HONGJUAN

    2016-01-01

    The field of cancer epigenetics has been evolving rapidly in recent decades. Epigenetic mechanisms include DNA methylation, histone modifications and microRNAs. Histone modifications are important markers of function and chromatin state. Aberrant histone methylation frequently occurs in tumor development and progression. Multiple studies have identified that histone lysine methyltransferases regulate gene transcription through the methylation of histone, which affects cell proliferation and differentiation, cell migration and invasion, and other biological characteristics. Histones have variant lysine sites for different levels of methylation, catalyzed by different lysine methyltransferases, which have numerous effects on human cancers. The present review focused on the most recent advances, described the key function sites of histone lysine methyltransferases, integrated significant quantities of data to introduce several compelling histone lysine methyltransferases in various types of human cancers, summarized their role in tumor development and discussed their potential mechanisms of action. PMID:26998265

  2. Substrate-Induced Transcriptional Activation of the MoCel7C Cellulase Gene Is Associated with Methylation of Histone H3 at Lysine 4 in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Vu, Ba Van; Pham, Kieu Thi Minh

    2013-01-01

    The mechanisms involved in substrate-dependent regulation of a Magnaporthe oryzae gene encoding a cellulase which we designate MoCel7C (MGG_14954) were investigated. The levels of MoCel7C transcript were dramatically increased more than 1,000-fold, 16 to 24 h after transfer to a medium containing 2% carboxymethylcellulose (CMC), while levels were very low or undetectable in conventional rich medium. Green fluorescent protein reporter assays showed that the MoCel7C promoter was activated by cello-oligosaccharides larger than a pentamer. CMC-induced activation of the MoCel7C promoter was suppressed by glucose and cellobiose. Chromatin immunoprecipitation assays revealed that histone H3 methylation on lysine 4 (H3K4) at the MoCel7C locus was associated with activation of the gene by CMC. Consistently, CMC-induced MoCel7C gene activation was drastically diminished in a knockout (KO) mutant of the MoSET1 gene, which encodes a histone lysine methyltransferase that catalyzes H3K4 methylation in M. oryzae. Interestingly, however, MoCel7C transcript levels under noninducing conditions were significantly increased in the MoSET1 KO mutant, suggesting that MoSET1 directly or indirectly plays a role in both activation and suppression of the MoCel7C gene in response to environmental signals. In addition, gene expression and silencing vectors using the MoCel7C promoter were constructed. PMID:23995923

  3. Biodegradable tri-block copolymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a non-viral vector to enhance gene transfection.

    PubMed

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-02-23

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH(2)-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.

  4. l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

    PubMed

    Takeno, Seiki; Hori, Kazumasa; Ohtani, Sachiko; Mimura, Akinori; Mitsuhashi, Satoshi; Ikeda, Masato

    2016-09-01

    We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the

  5. l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

    PubMed

    Takeno, Seiki; Hori, Kazumasa; Ohtani, Sachiko; Mimura, Akinori; Mitsuhashi, Satoshi; Ikeda, Masato

    2016-09-01

    We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the

  6. LIGNIFICATION IN TRANSGENICS DEFICIENT IN P-COUMARATE 3-HYDROXYLASE (C3H) AND THE ASSOCIATED HYDROXYCINNAMOYL TRANSFERASE (HCT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied in angiosperms. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), and its associated hydroxycinnamyl transferase (HCT),...

  7. Mimivirus Collagen Is Modified by Bifunctional Lysyl Hydroxylase and Glycosyltransferase Enzyme*

    PubMed Central

    Luther, Kelvin B.; Hülsmeier, Andreas J.; Schegg, Belinda; Deuber, Stefan A.; Raoult, Didier; Hennet, Thierry

    2011-01-01

    Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology. PMID:22045808

  8. [Steroid 21-hydroxylase deficiencies and female infertility: pathophysiology and management].

    PubMed

    Robin, G; Decanter, C; Baffet, H; Catteau-Jonard, S; Dewailly, D

    2014-06-01

    Steroid 21-hydroxylase deficiency is the most common adrenal genetic disease and is also named congenital adrenal hyperplasia. Depending on the severity of CYP21A2 gene mutations, there are severe or "classical" forms and moderate or "nonclassical" forms of 21-hydroxylase deficiency. The enzyme deficiency causes a disruption of adrenal steroidogenesis, which induces hyperandrogenism and elevated plasma levels of progesterone and 17-hydroxyprogesterone, the two substrates of 21-hydroxylase. These endocrine abnormalities will disrupt gonadal axis, endometrial growth and maturation and finally secretion of cervical mucus. All these phenomena contribute to a female hypofertility. Infertility is more severe in classical forms. When to become pregnant, treatment with hydrocortisone or dexamethasone can limit the production of adrenal androgens and progesterone and improves spontaneous pregnancy rates while minimizing the risk of miscarriage, which is usually relatively high in this disease. When planning pregnancy in patients with a 21-hydroxylase deficiency, genotyping the partner is required to screen for heterozygozity (1/50) and to assess the risk of transmission of a classical form in the progeny.

  9. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  10. IDDM2 and the polymorphism of the human tyrosine hydroxylase (hTH) gene in African Americans with type-1 diabetes.

    PubMed Central

    Berka, Noureddine; Nunlee-Bland, Gail; Erabhaoui, Elhajja; Belmamoun, Maher; Dunston, Georgia M.

    2004-01-01

    In this study, we investigate the polymorphic microsatellite repeat (TCAT)n, in the insulin gene region that has been associated with susceptibility to type-1 diabetes in some Caucasian populations. The microsatellite repeat polymorphism begins at base pair 1,170 in intron 1 of the hTH gene, which is located on the short arm of chromosome 11. This study is the first to investigate the association of this microsatellite repeat polymorphism in African-American type-1 diabetes patients and controls. The predicted amplified sequence was 254 bp. We found five alleles among African Americans in the Washington, DC area. The alleles were labeled K5 (244 bp), K4 (248 bp), K3 (252 bp), K2 (256 bp), and K1 (260 bp), and heterozygosity was greater than 0.75. The most frequent allele of the hTH microsatellite repeats was K5 (248 bp) with a frequency 0.62 in controls and 0.66 in type-1 diabetes patients, which did not differ significantly. Although the largest allele was more frequent in controls, the difference was not statistically significant. The five alleles of the hTH microsatellite generated 15 different genotypes. The most frequent genotype in controls and patients was K5/K4, whose frequencies were 0.19 and 0.17, respectively. No significant differences in genotype frequencies were found between type-1 diabetes patients and controls. This data shifts the focus from hTH to the VNTR at the insulin gene for IDDM2, the second major candidate gene for type-1 diabetes. PMID:15303408

  11. Localization of Romano-Ward long QT syndrome gene, LQTI, to the interval between tyrosine hydroxylase (TH) and D11S1349

    SciTech Connect

    Russell, M.W. |; Hulse, J.E.; Campbell, R.M.

    1995-08-01

    The Romano-Ward long-QT syndrome (RWLQTS) is an autosomal dominant disorder that is characterized by heritable prolongation of the QT interval, syncope, and sudden death. Identification of the gene responsible for this syndrome may aid the diagnosis, management, and treatment of patients with this disease. Furthermore, it may lead to improved understanding of and therapy for other sympathetic-dependent ventricular arrhythmias. 20 refs., 1 fig., 1 tab.

  12. Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro model

    PubMed Central

    Giangreco, Angeline A.; Dambal, Shweta; Wagner, Dennis; Van der Kwast, Theodorus; Vieth, Reinhold; Prins, Gail S.; Nonn, Larisa

    2014-01-01

    Previous work on vitamin D in the prostate has focused on the prostatic epithelium, from which prostate cancer arises. Prostatic epithelial cells are surrounded by stroma, which has well-established regulatory control over epithelial proliferation, differentiation, and the inflammatory response. Here we examined the regulation of vitamin D-related genes and inflammatory genes by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) in laser-capture microdissected prostate tissue from a vitamin D3 clinical trial and in an in vitro model that facilitates stromal–epithelial crosstalk. Analysis of the trial tissues showed that VDR was present in both cell types, whereas expression of the hydroxylases was the highest in the epithelium. Examination of gene expression by prostatic (1,25(OH)2D) concentrations showed that VDR was significantly lower in prostate tissues with the highest concentration of 1,25(OH)2D, and down-regulation of VDR by 1,25(OH) 2D was confirmed in the primary cell cultures. Analysis of inflammatory genes in the patient tissues revealed that IL-6 expression was the highest in the prostate stroma while PTGS2 (COX2) levels were lowest in the prostate cancer tissues from men in the highest tertile of prostatic 1,25(OH)2D. In vitro, TNF-α, IL-6 and IL-8 were suppressed by 1,25 (OH)2D in the primary epithelial cells, whereas TNF-α and PTGS2 were suppressed by 1,25(OH) 2D in the stromal cells. Importantly, the ability of 1,25(OH)2D to alter pro-inflammatory-induced changes in epithelial cell growth were dependent on the presence of the stromal cells. In summary, whereas both stromal and epithelial cells of the prostate express VDR and can presumably respond to 1,25(OH)2D, which appears to be primarily produced by the prostatic epithelium. Further, while the prostate epithelium was more responsive to the anti-inflammatory activity of 1,25 (OH)2D than stromal cells, stroma–epithelial crosstalk enhanced the phenotypic effects of 1,25(OH)2D and the inflammatory

  13. A Novel Interaction between Tryptophan Hydroxylase 2 (TPH2) Gene Polymorphism (rs4570625) and BDNF Val66Met Predicts a High-Risk Emotional Phenotype in Healthy Subjects

    PubMed Central

    Latsko, Maeson S.; Gilman, T. Lee; Matt, Lindsey M.; Nylocks, K. Maria; Coifman, Karin G.; Jasnow, Aaron M.

    2016-01-01

    Poor inhibitory processing of negative emotional content is central to many psychiatric disorders, including depression and anxiety. Moreover, increasing evidence suggests that core aspects of emotion-inhibitory processing are largely inherited and as such may represent a key intermediate or risk-related phenotype for common affective diseases (e.g., unipolar depressive, anxiety disorders). The current study employed a candidate-gene approach in order to most effectively examine this complex behavioral phenotype. We examined the novel interaction between BDNF (Val66Met) and TPH2 (rs4570625) polymorphisms and their influence on behavioral inhibition of negative emotion in two independent investigations of healthy adults. BDNF Met carriers consistently report greater symptoms of affective disease and display corresponding behavioral rigidity, while TPH2 T carriers display poor inhibitory processing. These genotypes are traditionally perceived as ‘risk’ genotypes when compared to their respective major Val and G homozygous genotypes, but evidence is mixed. Recent studies in humans and mutant mouse models suggest biological epistasis between BDNF and genes involved in serotonin regulation. Moreover, polymorphisms in the TPH2 gene may have greater influence on serotonergic function than other more commonly studied polymorphisms (e.g., 5-HTTLPR). We observed consistent evidence across two different emotion-inhibition paradigms, one with high internal validity (Study 1, n = 119) and one with high ecological validity (Study 2, n = 115) that the combination of Val/Val and G/G genotypes was clearly associated with impaired inhibition of negative emotional content. This was followed by individuals carrying the BDNF—Met allele (including Met/Val and Met/Met) when combined with the TPH2—T allele (including T/G and T/T combinations). The consistency of these results across tasks and studies suggests that these two groups may be particularly vulnerable to the most common

  14. Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System

    PubMed Central

    Tada, Naomi; Ochiai, Kazuhiko; Chong, Yong Hwa; Kato, Yuiko; Mitsui, Hiroko; Gin, Azusa; Oda, Hitomi; Azakami, Daigo; Tamura, Kyoichi; Sako, Toshinori; Inagaki, Takeshi; Sakamoto, Atsushi; Tsutsui, Toshihiko; Bonkobara, Makoto; Tsuchida, Shuichi; Ikemoto, Shigenori

    2016-01-01

    Cat’s AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system. PMID:27755584

  15. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

    PubMed

    Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

    2016-02-01

    Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor.

  16. The Presence of Clitoromegaly in the Nonclassical Form of 21-Hydroxylase Deficiency Could Be Partially Modulated by the CAG Polymorphic Tract of the Androgen Receptor Gene

    PubMed Central

    Garcia Gomes, Larissa; Bugano Diniz Gomes, Diogo; Marcondes, José Antônio Miguel; Madureira, Guiomar; de Mendonca, Berenice Bilharinho; Bachega, Tânia A. Sartori Sanchez

    2016-01-01

    Background In the nonclassical form (NC), good correlation has been observed between genotypes and 17OH-progesterone (17-OHP) levels. However, this correlation was not identified with regard to the severity of hyperandrogenic manifestations, which could depend on interindividual variability in peripheral androgen sensitivity. Androgen action is modulated by the polymorphic CAG tract (nCAG) of the androgen receptor (AR) gene and by polymorphisms in 5α-reductase type 2 (SRD5A2) enzyme, both of which are involved in the severity of hyperandrogenic disorders. Objectives To analyze whether nCAG-AR and SRD5A2 polymorphisms influence the severity of the nonclassical phenotype. Patients NC patients (n = 114) diagnosed by stimulated-17OHP ≥10 ng/mL were divided into groups according to the beginning of hyperandrogenic manifestations (pediatric and adolescent/adult) and CYP21A2 genotypes (C/C: homozygosis for mild mutations; A/C: compound heterozygosis for severe/mild mutations). Methods CYP21A2 mutations were screened by allelic-specific PCR, MLPA and/or sequencing. HpaII-digested and HpaII-undigested DNA samples underwent GeneScan analysis to study nCAG, and the SRD5A2 polymorphisms were screened by RLFP. Results Mean nCAG did not differ among pediatric, adolescent/adult and asymptomatic subjects. In the C/C genotype, we observed a significantly lower frequency of longer CAG alleles in pediatric patients than in adolescent/adults (p = 0.01). In patients carrying the A/C genotype, the frequencies of shorter and longer CAG alleles did not differ between pediatric patients and adolescent/adults (p>0.05). Patients with clitoromegaly had significantly lower weighted CAG biallelic mean than those without it: 19.1±2.7 and 21.6±2.5, respectively (p = 0.007), independent of the CYP21A2 genotype's severity. The SRD5A2 polymorphisms were not associated with the variability of hyperandrogenic NC phenotypes. Conclusions In this series, we observed a modulatory effect of the CAG

  17. Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection

    PubMed Central

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-01-01

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo. PMID:21541064

  18. Differential vitamin D 24-hydroxylase/CYP24A1 gene promoter methylation in endothelium from benign and malignant human prostate

    PubMed Central

    Karpf, Adam R; Omilian, Angela R; Bshara, Wiam; Tian, Lili; Tangrea, Michael A; Morrison, Carl D; Johnson, Candace S

    2011-01-01

    Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment. PMID:21725204

  19. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8'-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition.

    PubMed

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-03-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8'-hydroxyase gene which was highly expressed during seed development (TaABA8'OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8'OH1 on the D genome (TaABA8'OH1-D) was identified in Japanese cultivars including 'Tamaizumi'. However, a single mutation in TaABA8'OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8'OH1 on the A genome (TaABA8'OH1-A), TM1833, was identified from gamma-ray irradiation lines of 'Tamaizumi'. TM1833 (a double mutant in TaABA8'OH1-A and TaABA8'OH1-D) showed lower TaABA8'OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in 'Tamaizumi' (a single mutant in TaABA8'OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8'OH1 may be effective in germination inhibition in field-grown wheat.

  20. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8′-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition

    PubMed Central

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-01-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8′-hydroxyase gene which was highly expressed during seed development (TaABA8′OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8′OH1 on the D genome (TaABA8′OH1-D) was identified in Japanese cultivars including ‘Tamaizumi’. However, a single mutation in TaABA8′OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8′OH1 on the A genome (TaABA8′OH1-A), TM1833, was identified from gamma-ray irradiation lines of ‘Tamaizumi’. TM1833 (a double mutant in TaABA8′OH1-A and TaABA8′OH1-D) showed lower TaABA8′OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in ‘Tamaizumi’ (a single mutant in TaABA8′OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8′OH1 may be effective in germination inhibition in field-grown wheat. PMID:23641187

  1. Bovine NK-lysin: Copy number variation and functional diversification.

    PubMed

    Chen, Junfeng; Huddleston, John; Buckley, Reuben M; Malig, Maika; Lawhon, Sara D; Skow, Loren C; Lee, Mi Ok; Eichler, Evan E; Andersson, Leif; Womack, James E

    2015-12-29

    NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer's patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants. PMID:26668394

  2. Bovine NK-lysin: Copy number variation and functional diversification

    PubMed Central

    Chen, Junfeng; Huddleston, John; Buckley, Reuben M.; Malig, Maika; Lawhon, Sara D.; Skow, Loren C.; Lee, Mi Ok; Eichler, Evan E.; Andersson, Leif; Womack, James E.

    2015-01-01

    NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30–35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer’s patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants. PMID:26668394

  3. Insights into the regulatory landscape of the lysine riboswitch

    PubMed Central

    Garst, Andrew D.; Porter, Ely B.; Batey, Robert T.

    2012-01-01

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, the relative contributions of the polar functional groups to binding affinity and the regulatory response have been determined. Our results reveal that the lysine riboswitch has >1,000-fold specificity for lysine over other amino acids. To achieve this specificity, the aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main chain functional groups. At low NTP concentrations, we observe good agreement between the half-maximal regulatory activity (T50) and the affinity of the receptor for lysine (KD) as well many of its analogs. However, above 400 µM [NTP] the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that under physiologically relevant conditions riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  4. Insights into the regulatory landscape of the lysine riboswitch.

    PubMed

    Garst, Andrew D; Porter, Ely B; Batey, Robert T

    2012-10-12

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  5. Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

    PubMed

    Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

    2016-05-01

    Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

  6. Feed intake and brain neuropeptide Y (NPY) and cholecystokinin (CCK) gene expression in juvenile cobia fed plant-based protein diets with different lysine to arginine ratios.

    PubMed

    Nguyen, Minh Van; Jordal, Ann-Elise Olderbakk; Espe, Marit; Buttle, Louise; Lai, Hung Van; Rønnestad, Ivar

    2013-07-01

    Cobia (Rachycentron canadum, Actinopterygii, Perciformes;10.5±0.1g) were fed to satiation with three plant-based protein test diets with different lysine (L) to arginine (A) ratios (LL/A, 0.8; BL/A, 1.1; and HL/A, 1.8), using a commercial diet as control for six weeks. The test diets contained 730 g kg(-1) plant ingredients with 505-529 g protein, 90.2-93.9 g lipid kg(-1) dry matter; control diet contained 550 g protein and 95 g lipid kg(-1) dry matter. Periprandial expression of brain NPY and CCK (npy and cck) was measured twice (weeks 1 and 6). At week one, npy levels were higher in pre-feeding than postfeeding cobia for all diets, except LL/A. At week six, npy levels in pre-feeding were higher than in postfeeding cobia for all diets. cck in pre-feeding cobia did not differ from that in postfeeding for all diets, at either time point. Cobia fed LL/A had lower feed intake (FI) than cobia fed BL/A and control diet, but no clear correlations between dietary L/A ratio and FI, growth and expression of npy and cck were detected. The data suggest that NPY serves as an orexigenic factor, but further studies are necessary to describe links between dietary L/A and regulation of appetite and FI in cobia.

  7. Lysine nutrition in swine and the related monogastric animals: muscle protein biosynthesis and beyond.

    PubMed

    Liao, Shengfa F; Wang, Taiji; Regmi, Naresh

    2015-01-01

    Improving feed efficiency of pigs with dietary application of amino acids (AAs) is becoming increasingly important because this practice can not only secure the plasma AA supply for muscle growth but also protect the environment from nitrogen discharge with feces and urine. Lysine, the first limiting AA in typical swine diets, is a substrate for generating body proteins, peptides, and non-peptide molecules, while excess lysine is catabolized as an energy source. From a regulatory standpoint, lysine is at the top level in controlling AA metabolism, and lysine can also affect the metabolism of other nutrients. The effect of lysine on hormone production and activities is reflected by the change of plasma concentrations of insulin and insulin-like growth factor 1. Lysine residues in peptides are important sites for protein post-translational modification involved in epigenetic regulation of gene expression. An inborn error of a cationic AA transporter in humans can lead to a lysinuric protein intolerance condition. Dietary deficiency of lysine will impair animal immunity and elevate animal susceptibility to infectious diseases. Because lysine deficiency has negative impact on animal health and growth performance and it appears that dietary lysine is non-toxic even at a high dose of supplementation, nutritional emphasis should be put on lysine supplementation to avoid its deficiency rather than toxicity. Improvement of muscle growth of monogastric animals such as pigs via dietary lysine supply may be due to a greater increase in protein synthesis rather than a decrease in protein degradation. Nevertheless, the underlying metabolic and molecular mechanisms regarding lysine effect on muscle protein accretion merits further clarification. Future research undertaken to fully elucidate the metabolic and regulatory mechanisms of lysine nutrition could provide a sound scientific foundation necessary for developing novel nutritional strategies to enhance the muscle growth and

  8. Understanding the relationship between DNA methylation and histone lysine methylation☆

    PubMed Central

    Rose, Nathan R.; Klose, Robert J.

    2014-01-01

    DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24560929

  9. Effect of L-lysine on expression of selected genes, serum concentration of amino acids, muscle growth and performance of growing pigs.

    PubMed

    Morales, A; García, H; Arce, N; Cota, M; Zijlstra, R T; Araiza, B A; Cervantes, M

    2015-08-01

    Lysine (Lys) is the first limiting amino acid (AA) in most feed formulations for pigs and most abundant, along with leucine, in muscle proteins. An experiment was conducted with 17 pigs (17.7 ± 0.05 kg initial BW) to identify a role of dietary Lys in the control of protein synthesis in pigs. Fourteen pigs were randomly assigned to one of the two wheat-based dietary treatments: Lys-deficient, 3.0 g/kg (DEF) and Lys-adequate, 10.8 g/kg (ADE). Samples from jejunum mucosa, liver, Longissumus and Semitendinosus muscles, and blood were collected. The other three pigs were sacrificed at the beginning of the trial to measure basal carcass composition. Weight gain, gain:feed ratio, Lys intake and loin eye area were greater in ADE than in DEF pigs (p < 0.01). Muscle-related carcass characteristics were better, and myosin heavy chain IIb expression (MyHC IIb) in Semitendinosus was higher in ADE than in DEF pigs. Expression of AA transporters CAT-1 was lower (p < 0.05), serum Lys was higher and serum Val was lower in pigs fed the ADE diet. The higher muscularity, MyHC IIb expression in Semitendinosus muscle and Lys serum of pigs fed the ADE diet suggest that Lys increases growth rate not only by functioning as protein construction unit but also as potential control of the protein synthesis process.

  10. Genetics Home Reference: 21-hydroxylase deficiency

    MedlinePlus

    ... deficiency is an inherited disorder that affects the adrenal glands . The adrenal glands are located on top of the kidneys and ... body. In people with 21-hydroxylase deficiency , the adrenal glands produce excess androgens, which are male sex hormones. ...

  11. Phenylalanine hydroxylase: function, structure, and regulation.

    PubMed

    Flydal, Marte I; Martinez, Aurora

    2013-04-01

    Mammalian phenylalanine hydroxylase (PAH) catalyzes the rate-limiting step in the phenylalanine catabolism, consuming about 75% of the phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of phenylalanine in mammalian organisms.

  12. The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

    PubMed Central

    Murray-Stewart, Tracy; Woster, Patrick M.; Casero, Robert A.

    2013-01-01

    Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl- H3K4 histone marks that are associated with active gene expression. As most posttranslational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N5-[3,3-(diphenyl)propyl]-N1-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the reexpression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the ecadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated

  13. Distribution of histone3 lysine4 trimethylation at T3-responsive loci in the heart during reversible changes in gene expression

    PubMed Central

    Pandya, Kumar; Kohro, Takahide; Mimura, Imari; Kobayashi, Mika; Wada, Youichiro; Kodama, Tatsuhiko; Smithies, Oliver

    2013-01-01

    Expression in the adult heart of a number of cardiac genes, including the two genes comprising the cardiac Myosin heavy chain locus (Myh), is controlled by thyroid hormone (T3) levels, but there is minimal information concerning the epigenetic status of the genes when their expressions change. We fed mice normal chow or a Propyl thio uracil (PTU, an inhibitor of T3 production)-diet for 6 weeks, or the PTU diet for 6 weeks followed by normal chow for a further two weeks. Heart ventricles from these groups were then used for ChIP-seq analyses with an antibody to H3K4me3, a well documented epigenetic marker of gene activation. The resulting data show that, at the Myh7 locus, H3K4me3 modifications are induced primarily at 5’ transcribed region in parallel with increased expression of beta myosin heavy chain (MHC). At the Myh6 locus, decreases in H3K4me3 modifications occurred at the promoter and 5’ transcribed region. Extensive H3K4me3 modifications also occurred at the intergenic region between the two Myh genes which extended into the 3’ transcribed region of Myh7. The PTU-induced changes in H3K4me3 levels are, for the most part, reversible but are not invariably complete. We found full restoration of Myh6 gene expression upon PTU withdrawal, however the H3K4me3 pattern was only partially restored at Myh6, suggesting that full re-expression of Myh6 does not require that the H3K4me3 modifications return fully to the untreated conditions. Together, our data show that the H3K4me3 modification is an epigenetic marker closely associated with changes in Myh gene expression. PMID:22783727

  14. Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

    PubMed Central

    Liu, Yuwei; Xie, Shaojun; Yu, Jingjuan

    2016-01-01

    Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed. PMID:26829553

  15. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii.

    PubMed

    Li, Xiaolong; Hu, Xin; Wan, Yujing; Xie, Guizhen; Li, Xiangzhi; Chen, Di; Cheng, Zhongyi; Yi, Xingling; Liang, Shaohui; Tan, Feng

    2014-12-01

    Lysine succinylation is a new posttranslational modification identified in histone proteins of Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa. However, very little is known about their scope and cellular distribution. Here, using LC-MS/MS to identify parasite peptides enriched by immunopurification with succinyl lysine antibody, we produced the first lysine succinylome in this parasite. Overall, a total of 425 lysine succinylation sites that occurred on 147 succinylated proteins were identified in extracellular Toxoplasma tachyzoites, which is a proliferative stage that results in acute toxoplasmosis. With the bioinformatics analysis, it is shown that these succinylated proteins are evolutionarily conserved and involved in a wide variety of cellular functions such as metabolism and epigenetic gene regulation and exhibit diverse subcellular localizations. Moreover, we defined five types of definitively conserved succinylation site motifs, and the results imply that lysine residue of a polypeptide with lysine on the +3 position and without lysine at the -1 to +2 position is a preferred substrate of lysine succinyltransferase. In conclusion, our findings suggest that lysine succinylation in Toxoplasma involves a diverse array of cellular functions, although the succinylation occurs at a low level.

  16. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.

    PubMed

    Seo, Ho Seong; Sullam, Paul M

    2011-09-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin(102-198)). This region has no sequence homology to other known fibrinogen binding proteins. Lysin(102-198) bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin(102-198) also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin(102-198) also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis. PMID:21690235

  17. Recombinant bacteriophage lysins as antibacterials

    PubMed Central

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential. PMID:21327123

  18. Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.

    PubMed

    Khan, Mohammed A; Wu, Victoria M; Ghosh, Shreya; Uskoković, Vuk

    2016-06-01

    Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(l-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100×. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with

  19. Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2.

    PubMed

    Gjaltema, Rutger A F; van der Stoel, Miesje M; Boersema, Miriam; Bank, Ruud A

    2016-06-28

    Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer. PMID:27298363

  20. Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2.

    PubMed

    Gjaltema, Rutger A F; van der Stoel, Miesje M; Boersema, Miriam; Bank, Ruud A

    2016-06-28

    Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

  1. Phenylalanine hydroxylase deficiency: diagnosis and management guideline.

    PubMed

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A

    2014-02-01

    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  2. Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency.

    PubMed

    Korner, Germaine; Noain, Daniela; Ying, Ming; Hole, Magnus; Flydal, Marte I; Scherer, Tanja; Allegri, Gabriella; Rassi, Anahita; Fingerhut, Ralph; Becu-Villalobos, Damasia; Pillai, Samyuktha; Wueest, Stephan; Konrad, Daniel; Lauber-Biason, Anna; Baumann, Christian R; Bindoff, Laurence A; Martinez, Aurora; Thöny, Beat

    2015-10-01

    Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

  3. Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). A full-length C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog...

  4. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    PubMed

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons. PMID:25904808

  5. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    PubMed

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons.

  6. Phosphatidylinositol-4-phosphate 5-Kinase 1α Modulates Ribosomal RNA Gene Silencing through Its Interaction with Histone H3 Lysine 9 Trimethylation and Heterochromatin Protein HP1-α*

    PubMed Central

    Chakrabarti, Rajarshi; Sanyal, Sulagna; Ghosh, Amit; Bhar, Kaushik; Das, Chandrima; Siddhanta, Anirban

    2015-01-01

    Phosphoinositide signaling has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, cellular motility, vesicle trafficking, and gene transcription. Studies have also shown that nuclear phosphoinositide(s) regulates processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We have shown previously that the nuclear form of phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K), the enzyme responsible for phosphatidylinositol 4,5-bisphosphate synthesis, is modified by small ubiquitin-like modifier (SUMO)-1. In this study, we have shown that due to the site-specific Lys to Ala mutations of PIP5K at Lys-244 and Lys-490, it is unable to localize in the nucleus and nucleolus, respectively. Furthermore, by using chromatin immunoprecipitation assays, we have observed that PIP5K associates with the chromatin silencing complex constituted of H3K9me3 and heterochromatin protein 1α at multiple ribosomal DNA (rDNA) loci. These interactions followed a definite cyclical pattern of occupancy (mostly G1) and release from the rDNA loci (G1/S) throughout the cell cycle. Moreover, the immunoprecipitation results clearly demonstrate that PIP5K SUMOylated at Lys-490 interacts with components of the chromatin silencing machinery, H3K9me3 and heterochromatin protein 1α. However, PIP5K does not interact with the gene activation signature protein H3K4me3. This study, for the first time, demonstrates that PIP5K, an enzyme actively associated with lipid modification pathway, has additional roles in rDNA silencing. PMID:26157143

  7. Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.

    PubMed

    Bertea, Cinzia; Schalk, Michel; Mau, Christopher J D; Karp, Frank; Wildung, Mark R; Croteau, Rodney

    2003-12-01

    Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.

  8. Bacteriophage lysin mediates the binding of streptococcus mitis to human platelets through interaction with fibrinogen.

    PubMed

    Seo, Ho Seong; Xiong, Yan Q; Mitchell, Jennifer; Seepersaud, Ravin; Bayer, Arnold S; Sullam, Paul M

    2010-01-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aalpha and Bbeta chains of fibrinogen, but not the gamma subunit. Binding of lysin to the Bbeta chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83+/-3.1% reduction (mean +/- SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis. PMID:20714354

  9. Bacteriophage lysin mediates the binding of streptococcus mitis to human platelets through interaction with fibrinogen.

    PubMed

    Seo, Ho Seong; Xiong, Yan Q; Mitchell, Jennifer; Seepersaud, Ravin; Bayer, Arnold S; Sullam, Paul M

    2010-08-12

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aalpha and Bbeta chains of fibrinogen, but not the gamma subunit. Binding of lysin to the Bbeta chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83+/-3.1% reduction (mean +/- SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis.

  10. Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

    PubMed Central

    Seo, Ho Seong; Xiong, Yan Q.; Mitchell, Jennifer; Seepersaud, Ravin; Bayer, Arnold S.; Sullam, Paul M.

    2010-01-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis. PMID:20714354

  11. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  12. Characterization of two carnation petal prolyl 4 hydroxylases.

    PubMed

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis

    2010-10-01

    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

  13. Investigation of Lysine-Functionalized Dendrimers as Dichlorvos Detoxification Agents.

    PubMed

    Durán-Lara, Esteban F; Marple, Jennifer L; Giesen, Joseph A; Fang, Yunlan; Jordan, Jacobs H; Godbey, W Terrence; Marican, Adolfo; Santos, Leonardo S; Grayson, Scott M

    2015-11-01

    Lysine-containing polymers have seen broad application due to their amines' inherent ability to bind to a range of biologically relevant molecules. The synthesis of multiple generations of polyester dendrimers bearing lysine groups on their periphery is described in this report. Their hydrolytic stabilities with respect to pH and time, their toxicity to a range of cell lines, and their possible application as nano-detoxification agents of organophosphate compounds are all investigated. These zeroth-, first-, and second-generation water-soluble dendrimers have been designed to bear exactly 4, 8, and 16 lysine groups, respectively, on their dendritic periphery. Such monodisperse bioactive polymers show potential for a range of applications including drug delivery, gene delivery, heavy metal binding, and the sequestration of organic toxins. These monodisperse bioactive dendrimers were synthesized using an aliphatic ester dendritic core (prepared from pentaerythritol) and protected amino acid moieties. This library of lysine-conjugated dendrimers showed the ability to efficiently capture the pesticide dichlorvos, confirming the potential of dendrimer-based antidotes to maintain acetylcholinesterase activity in response to poisoning events. PMID:26460283

  14. CPLM: a database of protein lysine modifications

    PubMed Central

    Liu, Zexian; Wang, Yongbo; Gao, Tianshun; Pan, Zhicheng; Cheng, Han; Yang, Qing; Cheng, Zhongyi; Guo, Anyuan; Ren, Jian; Xue, Yu

    2014-01-01

    We reported an integrated database of Compendium of Protein Lysine Modifications (CPLM; http://cplm.biocuckoo.org) for protein lysine modifications (PLMs), which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. The CPLM database was updated from our previously developed database of Compendium of Protein Lysine Acetylation (CPLA), which contained 7151 lysine acetylation sites in 3311 proteins. Here, we manually collected experimentally identified substrates and sites for 12 types of PLMs, including acetylation, ubiquitination, sumoylation, methylation, butyrylation, crotonylation, glycation, malonylation, phosphoglycerylation, propionylation, succinylation and pupylation. In total, the CPLM database contained 203 972 modification events on 189 919 modified lysines in 45 748 proteins for 122 species. With the dataset, we totally identified 76 types of co-occurrences of various PLMs on the same lysine residues, and the most abundant PLM crosstalk is between acetylation and ubiquitination. Up to 53.5% of acetylation and 33.1% of ubiquitination events co-occur at 10 746 lysine sites. Thus, the various PLM crosstalks suggested that a considerable proportion of lysines were competitively and dynamically regulated in a complicated manner. Taken together, the CPLM database can serve as a useful resource for further research of PLMs. PMID:24214993

  15. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  16. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

    PubMed

    Yang, Qing-Qing; Zhang, Chang-Quan; Chan, Man-Ling; Zhao, Dong-Sheng; Chen, Jin-Zhu; Wang, Qing; Li, Qian-Feng; Yu, Heng-Xiu; Gu, Ming-Hong; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2016-07-01

    Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice. PMID:27252467

  17. Recent advances in biochemical and molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    PubMed Central

    Kim, Gu-Hwan; Yoo, Han-Wook

    2016-01-01

    The term congenital adrenal hyperplasia (CAH) covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency. PMID:27104172

  18. Recent advances in biochemical and molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    PubMed

    Choi, Jin-Ho; Kim, Gu-Hwan; Yoo, Han-Wook

    2016-03-01

    The term congenital adrenal hyperplasia (CAH) covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency.

  19. Segregation for endosperm lysine in F2, F 3 and F 4 progeny from a cross of in vitro-selected and unselected cultivar of rice.

    PubMed

    Schaeffer, G W; Sharpe, F T; Dudley, J T

    1989-02-01

    Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F2 populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R(2) 0.52 and 0.56). The F2 segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F3 and F4 data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility. PMID:24232525

  20. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma.

    PubMed

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S; Watanabe, Takeshi; Kim, Min P; Blackmon, Shanda H; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I; Minelli, Rosalba; Scott, Kenneth L; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R; Creighton, Chad J; Martinez, Elisabeth D; Zal, Tomasz; Grande-Allen, K Jane; Yamauchi, Mitsuo; Kurie, Jonathan M

    2015-03-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  1. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma.

    PubMed

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S; Watanabe, Takeshi; Kim, Min P; Blackmon, Shanda H; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I; Minelli, Rosalba; Scott, Kenneth L; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R; Creighton, Chad J; Martinez, Elisabeth D; Zal, Tomasz; Grande-Allen, K Jane; Yamauchi, Mitsuo; Kurie, Jonathan M

    2015-03-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

  2. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  3. Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

    PubMed

    Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

    2014-12-01

    We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells.

  4. Cytochrome P450c17 (steroid 17. cap alpha. -hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues

    SciTech Connect

    Chung, B.; Picado-Leonard, J.; Haniu, M.; Bienkowski, M.; Hall, P.F.; Shively, J.E.; Miller, W.L.

    1987-01-01

    P450c17 is the single enzyme mediating both 17..cap alpha..-hydroxylase (steroid 17..cap alpha..-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. The authors sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac 17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17.

  5. Identification and characterization of lysine-methylated sites on histones and non-histone proteins.

    PubMed

    Lee, Tzong-Yi; Chang, Cheng-Wei; Lu, Cheng-Tzung; Cheng, Tzu-Hsiu; Chang, Tzu-Hao

    2014-06-01

    Protein methylation is a kind of post-translational modification (PTM), and typically takes place on lysine and arginine amino acid residues. Protein methylation is involved in many important biological processes, and most recent studies focused on lysine methylation of histones due to its critical roles in regulating transcriptional repression and activation. Histones possess highly conserved sequences and are homologous in most species. However, there is much less sequence conservation among non-histone proteins. Therefore, mechanisms for identifying lysine-methylated sites may greatly differ between histones and non-histone proteins. Nevertheless, this point of view was not considered in previous studies. Here we constructed two support vector machine (SVM) models by using lysine-methylated data from histones and non-histone proteins for predictions of lysine-methylated sites. Numerous features, such as the amino acid composition (AAC) and accessible surface area (ASA), were used in the SVM models, and the predictive performance was evaluated using five-fold cross-validations. For histones, the predictive sensitivity was 85.62% and specificity was 80.32%. For non-histone proteins, the predictive sensitivity was 69.1% and specificity was 88.72%. Results showed that our model significantly improved the predictive accuracy of histones compared to previous approaches. In addition, features of the flanking region of lysine-methylated sites on histones and non-histone proteins were also characterized and are discussed. A gene ontology functional analysis of lysine-methylated proteins and correlations of lysine-methylated sites with other PTMs in histones were also analyzed in detail. Finally, a web server, MethyK, was constructed to identify lysine-methylated sites. MethK now is available at http://csb.cse.yzu.edu.tw/MethK/.

  6. Class I Lysine Deacetylases Facilitate Glucocorticoid-induced Transcription*

    PubMed Central

    Kadiyala, Vineela; Patrick, Nina M.; Mathieu, Wana; Jaime-Frias, Rosa; Pookhao, Naruekamol; An, Lingling; Smith, Catharine L.

    2013-01-01

    Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi exposure has a significant impact on GR signaling and thus has ramifications for the clinical use of these drugs. PMID:23946490

  7. LambdaSa1 and LambdaSa2 Prophage Lysins of Streptococcus agalactiae▿

    PubMed Central

    Pritchard, David G.; Dong, Shengli; Kirk, Marion C.; Cartee, Robert T.; Baker, John R.

    2007-01-01

    Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited γ-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed β-d-N-acetylglucosaminidase activity. PMID:17905888

  8. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain

    PubMed Central

    Lund, Erik G.; Guileyardo, Joseph M.; Russell, David W.

    1999-01-01

    The turnover of cholesterol in the brain is thought to occur via conversion of excess cholesterol into 24S-hydroxycholesterol, an oxysterol that is readily secreted from the central nervous system into the plasma. To gain molecular insight into this pathway of cholesterol metabolism, we used expression cloning to isolate cDNAs that encode murine and human cholesterol 24-hydroxylases. DNA sequence analysis indicates that both proteins are localized to the endoplasmic reticulum, share 95% identity, and represent a new cytochrome P450 subfamily (CYP46). When transfected into cultured cells, the cDNAs produce an enzymatic activity that converts cholesterol into 24S-hydroxycholesterol, and to a lesser extent, 25-hydroxycholesterol. The cholesterol 24-hydroxylase gene contains 15 exons and is located on human chromosome 14q32.1. Cholesterol 24-hydroxylase is expressed predominantly in the brain as judged by RNA and protein blotting. In situ mRNA hybridization and immunohistochemistry localize the expression of this P450 to neurons in multiple subregions of the brain. The concentrations of 24S-hydroxycholesterol in serum are low in newborn mice, reach a peak between postnatal days 12 and 15, and thereafter decline to baseline levels. In contrast, cholesterol 24-hydroxylase protein is first detected in the brain of mice at birth and continues to accumulate with age. We conclude that the cloned cDNAs encode cholesterol 24-hydroxylases that synthesize oxysterols in neurons of the brain and that secretion of 24S-hydroxycholesterol from this tissue in the mouse is developmentally regulated. PMID:10377398

  9. Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus, and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG), or low (LG) plasma...

  10. TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling.

    PubMed

    Remst, Dennis F G; Blaney Davidson, Esmeralda N; Vitters, Elly L; Bank, Ruud A; van den Berg, Wim B; van der Kraan, Peter M

    2014-01-01

    Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

  11. High carrier frequency of 21-hydroxylase deficiency in Cyprus.

    PubMed

    Phedonos, A A P; Shammas, C; Skordis, N; Kyriakides, T C; Neocleous, V; Phylactou, L A

    2013-12-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is a common autosomal recessive disorder caused by mutations in the CYP21A2 gene. The carrier frequency of CYP21A2 mutations has been estimated to be 1:25 to 1:10 on the basis of newborn screening. The main objective of this study was to determine the carrier frequency in the Cypriot population of mutations in the CYP21A2 gene. Three hundred unrelated subjects (150 males and 150 females) from the general population of Cyprus were screened for mutations in the CYP21A2 gene and its promoter. The CYP21A2 genotype analysis identified six different mutants and revealed a carrier frequency of 9.83% with the mild p.Val281Leu being the most frequent (4.3%), followed by p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) and p.Met283Val (0.17%). The notable high CYP21A2 carrier frequency of the Cypriot population is one of the highest reported so far by genotype analysis. Knowledge of the mutational spectrum of CYP21A2 will enable to optimize mutation detection strategy for genetic diagnosis of 21-OHD not only in Cyprus, but also the greater Mediterranean region.

  12. Characterization of mutations at the mouse phenylalanine hydroxylase locus

    SciTech Connect

    McDonald, J.D.; Charlton, C.K.

    1997-02-01

    Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAH{sup ENU1}, the phenotype is mild. The Pah{sup enu1} mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAH{sup ENU2} the phenotype is severe. The Pah{sup enu2} mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAH{sup ENU2}, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site. 26 refs., 2 figs., 1 tab.

  13. Identification of the molecular basis for the functional difference between flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase.

    PubMed

    Seitz, Christian; Ameres, Stefanie; Forkmann, Gert

    2007-07-24

    Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3'- or the 3'- and 5'-position, respectively. Sequence identity between F3'H and F3'5'H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3'5'H from an F3'H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3'H and F3'5'H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5'-hydroxylation activity to recombinant Gerbera hybrida F3'H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3'5'H into an F3'H with low remaining 5'-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3'- to 3',5'-hydroxylation activity.

  14. Linkages in thermal copolymers of lysine

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Suzuki, F.

    1975-01-01

    The thermal copolymerization of lysine with other alpha-amino acids was studied. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins.

  15. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity.

    PubMed

    Boniface, Audrey; Bouhss, Ahmed; Mengin-Lecreulx, Dominique; Blanot, Didier

    2006-06-01

    The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.

  16. Heterosis and components of genetic variation for protein and lysine content in some grain sorghums.

    PubMed

    Rana, B S; Murty, B R

    1975-06-01

    Twelve varieties representing six geographical regions and nine taxonomic groups from a World Collection ofSorghum were used in a diallel and line X tester analysis of the nature of genetic variation for protein and lysine content. In a majority of crosses, heterosis was negative for protein, and positive for lysine.Analysis of combining ability indicated that both additive and non-additive variation were important for protein, while the non-additive component was predominant for lysine. Heterosis inlow Xlow andmedium Xlow crosses also indicated the presence of substantial non-allelic gene interactions for both the characters.Associations between protein, lysine and yield in parents were significantly different from those in the hybrids indicating considerable scope for genetic manipulation. Negative correlations between protein and lysine (% protein) and between protein and grain yield were very low. The results indicated that a high level of protein and moderately high lysine may be incorporated in high yielding varieties of sorghum, by using Caudatum kaura, Roxburghii shallu and durra from Nigeria and Sudan in the hybridisation programme. PMID:24419465

  17. Methylation of translation-associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases.

    PubMed

    Couttas, Timothy A; Raftery, Mark J; Padula, Matthew P; Herbert, Ben R; Wilkins, Marc R

    2012-04-01

    This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-α, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-α is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1. PMID:22522802

  18. SPOTing Acetyl-Lysine Dependent Interactions.

    PubMed

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-08-17

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  19. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation. PMID:27600229

  20. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  1. Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio)

    PubMed Central

    Chun, Rene F.; Blatter, Elizabeth; Elliott, Stephanie; Fitz-Gibbon, Sorel; Rieger, Sandra; Sagasti, Alvaro; Adams, John S.; Hewison, Martin

    2015-01-01

    Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (1,25D) is a pivotal step in vitamin D physiology, catalyzed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). To establish new models for assessing the physiological importance of the 1α-hydroxylase-25D-axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5-150 nM) or active 1,25D (0.1-10 nM) induced dose responsive expression (15-95 fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 cDNA was then generated using RACE and RT-PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC-8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than 2-fold induction of CYP24A1 mRNA expression and a 25-fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. PMID:25290078

  2. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  3. Effect of some agents on the activity of cell-free progesterone 11 alpha-hydroxylase and 11 beta-hydroxylase from Aspergillus niger 12Y.

    PubMed

    Abdel-Fattah, A F; Badawi, M A

    1978-01-01

    The effect of some agents on the activity of cell-free progesterone 11 alpha-hydroxylase and 11 beta-hydroxylase from Aspergillus niger 12Y was studied. Calcium chloride, sodium chloride, magnesium sulphate, copper sulphate, and EDTA inhibited 11 alpha-hydroxylase and 11 beta-hydroxylase, while mercuric chloride inhibited only 11 alpha-hydroxylase. Inhibition of both the enzymes was also brought about by iodine, p-chloromercuribenzoate, iodoacetic acid, maleic acid, and cystine as well as potassium ferricyanide for 11 alpha-hydroxylase. Reduced glutathione and cysteine-HCl brought about activation of 11 alpha-hydroxylase and 11 beta-hydroxylase. The probability of the presence of reactive sulfhydryl groups in the active sites of both enzymes was discussed. Urea inhibited both fungal progesterone hydroxylases, probably due to enzyme protein denaturation. PMID:107681

  4. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase.

    PubMed

    Abromaitis, Stephanie; Hefty, P Scott; Stephens, Richard S

    2009-03-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases (AroAA-Hs). AroAA-Hs hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine, and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-Hs are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylate host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  5. Lysine requirement of growing male Pekin ducks.

    PubMed

    Bons, A; Timmler, R; Jeroch, H

    2002-12-01

    1. One growth experiment and one balance test were conducted to study the response to increasing levels of dietary lysine supplementation in male Pekin ducks with special reference to the growth periods from 1 to 3 weeks and 4 to 7 weeks of age. 2. Two different low-lysine diets were used as basal diets in both periods. The basal lysine levels were 7.6 g/kg (d 1 to 21) and 6.2 g/kg (d 22 to 49) and the ranges in lysine concentration were 7.6 to 12.6 g/kg (d 1 to 21) and 6.2 to 11.2 g/kg (d 22 to 49). 3. Growth performance, feed conversion efficiency and meat yield increased (P < 0.05) with increasing lysine concentration (requirement defined as 95% of the asymptote). 4. It is concluded that the dietary lysine concentration should be 0.93 g/MJ nitrogen corrected apparent metabolisable energy (AMEN) (11.7 g/kg) for the starter period (until d 21) and 0.75 g/MJ AMEN (10.0 g/kg) for the grower period (from d 22 onwards).

  6. Structural studies of dopamine. beta. -hydroxylase

    SciTech Connect

    Papadopoulos, N.J.

    1985-01-01

    Dopamine ..beta..-hydroxylase catalyzes the conversion of dopamine to norepinephrine, a ..beta..-hydroxylation reaction, utilizing ascorbic acid as reducing agent and molecular oxygen as cosubstrate. Modifications of the previously published purification procedure for D..beta..H have produced findings which show that (1) enzyme is inactivated by ascorbate autooxidation during the isolation procedure, (2) active as well as inactive D..beta..H co-purify throughout the entire purification procedure and (3) beef liver catalase totally protects against this time dependent inactivation. The stoichiometry of copper binding to the active sites of D..beta..H has been investigated using /sup 19/F-NMR and radioactive binding experiments. The data unequivocally show that homogeneous D..beta..H (isolated in the presence of catalase) specifically binds up to approx.8 copper atoms per enzyme tetramer. Distance determinations done using NMR relaxation rate theory show that anion activators of the catalytic reaction are bound at a fairly far distance from the Cu/sup 2 +/ centers. Spin-echo electron paramagnetic resonance spectroscopy indicates that at least one, possibly two, histidines are bound as equatorial ligands to each Cu/sup 2 +/ ion. The combined data indicate that highly purified dopamine ..beta..-hydroxylase contains a 2 copper atom active site, composed of magnetically non-interacting metal centers. Active site components are distant from the Cu/sup 2 +/ centers, suggesting a possible movement of active site residues or components after reduction of enzyme bound copper in order to achieve the insertion of 1 atom of oxygen into the benzylic C-H bond of dopamine.

  7. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene. PMID:25504221

  8. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma. PMID:27535933

  9. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

  10. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics

    PubMed Central

    Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.

    2013-01-01

    The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression. PMID:23059474

  11. Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

    SciTech Connect

    Witchel, S.S.; Lee, P.A.; Trucco, M.

    1996-01-02

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population. 29 refs., 3 figs., 2 tabs.

  12. Identification of flavonoid 3'-hydroxylase in the yellow flower of Delphinium zalil.

    PubMed

    Miyahara, Taira; Hamada, Arisa; Okamoto, Mitsutoshi; Hirose, Yukio; Sakaguchi, Kimitoshi; Hatano, Shoji; Ozeki, Yoshihiro

    2016-09-01

    The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors. PMID:27478933

  13. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases*

    PubMed Central

    Chan, Mun Chiang; Ilott, Nicholas E.; Schödel, Johannes; Sims, David; Tumber, Anthony; Lippl, Kerstin; Mole, David R.; Pugh, Christopher W.; Ratcliffe, Peter J.; Ponting, Chris P.; Schofield, Christopher J.

    2016-01-01

    The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable. PMID:27502280

  14. N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

    PubMed Central

    Jiang, Tao; Zhou, Xinfeng; Taghizadeh, Koli; Dong, Min; Dedon, Peter C.

    2007-01-01

    The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N6-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3′-formylphosphate residues arising from 5′-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N6-amino groups of lysine side chains. A liquid chromatography (LC)–tandem mass spectrometry (MS) method was developed to quantify the resulting N6-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04–0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose–response relationship for the formation of N6-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N6-formylation of the linker histone H1. The N6-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N6-acetylation recognized as an important determinant of gene expression in mammalian cells. The N6-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress. PMID:17190813

  15. Simultaneous analysis of Nε-(carboxymethyl)lysine, reducing sugars, and lysine during the dairy thermal process.

    PubMed

    Xu, Xian-Bing; Ma, Fei; Yu, Shu-Juan; Guan, Yong-Guang

    2013-09-01

    A new analytical method allowing the simultaneous quantification of Nε-(carboxymethyl)lysine (CML), lysine, and reducing sugars (glucose, lactose, and galactose) is described. It is based on high performance anion-exchange chromatography with pulsed amperometric electrochemical detection. This method demonstrated a low limit of quantification (0.385 to 0.866 mg/L), excellent linear correlation (R(2)>0.997), and desired calibration range (3.125 to 25 mg/L). In addition, lactose-lysine solutions containing sulfite (4 to 400 mmol/L) were heated at 110°C for 2h. The results showed that sulfite inhibited the formation of CML and promoted the consumption of reducing sugars and lysine in the Maillard reaction model. The method proved to be useful for simultaneous analysis of CML, lysine, and reducing sugars (glucose, galactose, and lactose) in the Maillard reaction system. Moreover, sulfite was an effective inhibitor of CML formation.

  16. Expression of the CYP4F3 gene. tissue-specific splicing and alternative promoters generate high and low K(m) forms of leukotriene B(4) omega-hydroxylase.

    PubMed

    Christmas, P; Ursino, S R; Fox, J W; Soberman, R J

    1999-07-23

    Cytochrome P450 4F3 (CYP4F3) catalyzes the inactivation of leukotriene B(4) by omega-oxidation in human neutrophils. To understand the regulation of CYP4F3 expression, we analyzed the CYP4F3 gene and cloned a novel isoform (CYP4F3B) that is expressed in fetal and adult liver, but not in neutrophils. The CYP4F3 gene contains 14 exons and 13 introns. The cDNAs for CYP4F3A (the neutrophil isoform) and CYP4F3B have identical coding regions, except that they contain exons 4 and 3, respectively. Both exons code for amino acids 66-114 but share only 27% identity. When expressed in COS-7 cells, the K(m) of CYP4F3B was determined to be 26-fold higher than the K(m) of CYP4F3A using leukotriene B(4) as a substrate. 5'-Rapid amplification of cDNA end studies reveal that the CYP4F3A and CYP4F3B transcripts have 5'-termini derived from different parts of the gene and are initiated from distinct transcription start sites located 519 and 71 base pairs (bp), respectively, from the ATG initiation codon. A consensus TATA box is located 27 bp upstream of the CYP4F3B transcription start site, and a TATA box-like sequence is located 23 bp upstream of the CYP4F3A transcription start site. The data indicate that the tissue-specific expression of functionally distinct CYP4F3 isoforms is regulated by alternative promoter usage and mutually exclusive exon splicing.

  17. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGES

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  18. Prolonged incubation time in sheep with prion protein containing lysine at position 171

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheep scrapie susceptibility or resistance is a function of genotype with polymorphisms at codon 171 in the sheep prion gene playing a major role. Glutamine (Q) at 171 contributes to scrapie susceptibility while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 1...

  19. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

    PubMed

    Calvo, Ana C; Pey, Angel L; Ying, Ming; Loer, Curtis M; Martinez, Aurora

    2008-08-01

    In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; K(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable V(max)], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution. PMID:18460651

  20. Tryptophan hydroxylase 2 in seasonal affective disorder: underestimated perspectives?

    PubMed

    Kulikov, Alexander V; Popova, Nina K

    2015-01-01

    Seasonal affective disorder (SAD) is characterized by recurrent depression occurring generally in fall/winter. Numerous pieces of evidence indicate the association of SAD with decreased brain neurotransmitter serotonin (5-HT) system functioning. Tryptophan hydroxylase 2 (TPH2) is the key and rate-limiting enzyme in 5-HT synthesis in the brain. This paper concentrates on the relationship between TPH2 activity and mood disturbances, the association between human TPH2 gene expression and the risk of affective disorder, application of tryptophan to SAD treatment and the animal models of SAD. The main conclusions of this review are as follows: (i) the brain 5-HT deficiency contributes to the mechanism underlying SAD, (ii) TPH2 is involved in the regulation of some kinds of genetically defined affective disorders and (iii) the activation of 5-HT synthesis with exogenous l-tryptophan alone or in combination with light therapy could be effective in SAD treatment. The synergic effect of these combined treatments will have several advantages compared to light or tryptophan therapy alone. First, it is effective in the treatment of patients resistant to light therapy. Secondly, l-tryptophan treatment prolongs the antidepressant effect of light therapy.

  1. Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia.

    PubMed

    Abeykoon, Amila H; Chao, Chien-Chung; Wang, Guanghui; Gucek, Marjan; Yang, David C H; Ching, Wei-Mei

    2012-12-01

    Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

  2. Biochemical Characterization of an FAD-Dependent Monooxygenase, the Ornithine Hydroxylase from Pseudomonas aeruginosa, Suggests a Novel Reaction Mechanism†

    PubMed Central

    Meneely, Kathleen M.; Lamb, Audrey L.

    2008-01-01

    Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both FAD and NADPH for activity, and was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics using an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a non-substrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA in relation to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (para-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady state experiments indicate that PvdA:FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to para-hydroxybenzoate hydroxylase (PHBH from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. PMID:17900176

  3. Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families.

    PubMed Central

    Wang, J T; Lin, C J; Burridge, S M; Fu, G K; Labuda, M; Portale, A A; Miller, W L

    1998-01-01

    Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency." PMID:9837822

  4. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  5. The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

    PubMed

    Do, Eunsoo; Park, Minji; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-09-01

    The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis. PMID:27353379

  6. Novel intronic CYP21A2 mutation in a Japanese patient with classic salt-wasting steroid 21-hydroxylase deficiency.

    PubMed

    Katsumata, Noriyuki; Shinagawa, Takashi; Horikawa, Reiko; Fujikura, Kaori

    2010-11-01

    Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder caused by the defective CYP21A2 gene that leads to various degrees of impaired secretion of both cortisol and aldosterone. In the present study, we analyzed the CYP21A2 gene in a Japanese male patient with 21-OHD and functionally characterized the mutant CYP21A2 gene. The patient presented with hypoglycemia and a salt-losing crisis during the neonatal period, and was diagnosed as having the salt-wasting form of 21-OHD based on the clinical and laboratory findings. Analysis of the CYP21A2 gene revealed that the patient is homozygous for a novel C to A conversion at -9 position of intron 9 (IVS9-9C>A) and that his parents are heterozygous for the IVS9-9C>A mutation. Transient expression of the IVS9-9C>A mutant CYP21A2 gene in COS-1 cells demonstrated that the mutation creates an aberrant splice acceptor site at -7 position of intron 9 and totally inactivates the authentic splice acceptor site of intron 9, which results in complete deficiency of 21-hydroxylase activity and loss of immunoreactive 21-hydroxylase protein. Clinical presentations of the patient as the severe salt-wasting form of 21-OHD are in good agreement with these results of the expression study. In conclusion, the patient is a homozygote for the novel intronic IVS9-9C>A mutation, which affects messenger RNA splicing and totally inactivates 21-hydroxylase to give rise to clinically manifest classic salt-wasting 21-OHD.

  7. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  8. Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm

    PubMed Central

    Liu, Chen; Li, Shixue; Yue, Jing; Xiao, Wenhan; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

    2015-01-01

    Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality. PMID:26703573

  9. Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata).

    PubMed

    Azizi, Sheida; Nematollahi, Mohammad Ali; Mojazi Amiri, Bagher; Vélez, Emilio J; Lutfi, Esmail; Navarro, Isabel; Capilla, Encarnación; Gutiérrez, Joaquim

    2016-01-01

    Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of

  10. Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata)

    PubMed Central

    Azizi, Sheida; Nematollahi, Mohammad Ali; Mojazi Amiri, Bagher; Vélez, Emilio J.; Lutfi, Esmail; Navarro, Isabel; Capilla, Encarnación; Gutiérrez, Joaquim

    2016-01-01

    Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of

  11. Tryptophan Hydroxylase-2: An Emerging Therapeutic Target for Stress Disorders

    PubMed Central

    Chen, Guo-Lin; Miller, Gregory M.

    2013-01-01

    Serotonin (5-HT) has been long recognized to modulate the stress response, and dysfunction of 5-HT has been implicated in numerous stress disorders. Accordingly, the 5-HT system has been targeted for the treatment of stress disorders. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT synthesis, and the recent identification of a second, neuron-specific TPH isoform (TPH2) opened up a new area of research. With a decade of extensive investigation, it is now recognized that: 1) TPH2 exhibits a highly flexible gene expression that is modulated by an increasing number of internal and external environmental factors including the biological clock, stressors, endogenous hormones, and antidepressant therapies; and 2) genetically determined TPH2 activity is linked to a growing body of stress-related neuronal correlates and behavioral traits. These findings reveal an active role of TPH2 in the stress response and provide new insights into the long recognized but not yet fully understood 5-HT-stress interaction. As a major modulator of 5-HT neurotransmission and the stress response, TPH2 is of both pathophysiological and pharmacological significance, and is emerging as a new therapeutic target for the treatment of stress disorders. Given that numerous antidepressant therapies influence TPH2 gene expression, TPH2 is already inadvertently targeted for the treatment of stress disorders. With increased understanding of the regulation of TPH2 activity we can now purposely utilize TPH2 as a target to develop new or optimize current therapies, which are expected to greatly improve the prevention and treatment of a wide variety of stress disorders. PMID:23435356

  12. Co- and Post-Treatment with Lysine Protects Primary Fish Enterocytes against Cu-Induced Oxidative Damage.

    PubMed

    Li, Xue-Yin; Liu, Yang; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-01-01

    The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1-19.6 g kg-1 diet) for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0-300 mg/L in media) prior to (pre-treatment), along with (co-treatment) or following (post-treatment) with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD), glutathioneperoxidase (GPx), glutathione-S-transferase (GST) and reductase (GR)), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) OD values, along with alkaline phosphatase (ALP) and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b) followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT) and GST activities (P<0.01 and P<0.001). GR activity and gene

  13. Co- and Post-Treatment with Lysine Protects Primary Fish Enterocytes against Cu-Induced Oxidative Damage

    PubMed Central

    Li, Xue-Yin; Liu, Yang; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-01-01

    The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1–19.6 g kg-1 diet) for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0–300 mg/L in media) prior to (pre-treatment), along with (co-treatment) or following (post-treatment) with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD), glutathioneperoxidase (GPx), glutathione-S-transferase (GST) and reductase (GR)), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) OD values, along with alkaline phosphatase (ALP) and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b) followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT) and GST activities (P<0.01 and P<0.001). GR activity and gene

  14. Bioavailability of free lysine and protein-bound lysine from casein and fishmeal in juvenile turbot (Psetta maxima).

    PubMed

    Kroeckel, Saskia; Dietz, Carsten; Schulz, Carsten; Susenbeth, Andreas

    2015-03-14

    In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (k(Lys)) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25-4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and L-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (P< 0·001). CP, crude lipid and crude ash contents in the whole body were affected by the dietary treatments. The linear regression slope between lysine retention and lysine intake (k(Lys)) was similar between all the dietary lysine sources. The k(Lys) values for the diets supplemented with Cas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7-23·4 mg/kg(0·8) per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

  15. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  16. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  17. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  18. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  19. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  20. Radioactive Lysine in Protein Metabolism Studies

    DOE R&D Accomplishments Database

    Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

    1950-01-09

    Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

  1. A new dopamine-β-hydroxylase inhibitor

    PubMed Central

    Andén, N. -E.; Fuxe, K.

    1971-01-01

    1. The dopamine-β-hydroxylase inhibitor bis(4-methyl-1-homopiperazinyl-thiocarbonyl) disulphide (FLA-63; 25 mg/kg i.p.) caused within 4 h a 65% loss of noradrenaline throughout the intact rat spinal cord and also cranial to a transection of the cut spinal cord. Caudal to the lesion, there was only an insignificant depletion of 17% indicating the importance of nerve impulses for the disappearance of noradrenaline. 2. Dopamine accumulated in the spinal cord after treatment with FLA-63 although the amounts were not sufficient to replace the missing noradrenaline. Even after treatment with L-3,4-dihydroxyphenylalanine (L-DOPA), the catecholamine store was incompletely replenished by dopamine. 3. After a large depletion of the noradrenaline stores, induced by repeated doses of FLA-63 or by reserpine plus FLA-63, the L-DOPA-induced increase in flexor reflex activity of the hind limbs of spinal rats was inhibited much more than after pretreatment with α-methyl-tyrosine or reserpine. FLA-63 blocked the formation of noradrenaline but not of dopamine from L-DOPA. 4. The increase in flexor reflex activity induced by the noradrenaline receptor stimulating agent clonidine was not changed by FLA-63, indicating that the noradrenaline receptor sensitivity was not influenced. 5. After depletion of the noradrenaline stores, the small formation of noradrenaline from L-DOPA may be of greater functional significance for the noradrenaline receptor stimulation than the greater formation of dopamine, but the dopamine formed also has a slight action. With intact noradrenaline stores, displacement of endogenous noradrenaline by newly formed dopamine contributes, at least after monoamine oxidase inhibition, to the increase in the flexor reflex activity caused by L-DOPA. PMID:4339882

  2. Ribosomes slide on lysine-encoding homopolymeric A stretches.

    PubMed

    Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

    2015-01-01

    Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

  3. Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency

    SciTech Connect

    Fardella, C.E.; Hum, D.W.; Miller, W.L.; Homoki, J.

    1994-07-01

    Genetic disorders in the gene encoding P450c17 cause 17{alpha}-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralorcorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1 cells showed that the mutant P450c17 protein was produced, but it lacked both 17{alpha}-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17{alpha}-hydroxylase deficiency, indicating that mutations in this gene are due to random events. 36 refs., 3 figs., 2 tabs.

  4. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  5. Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

    PubMed Central

    Menabò, Soara; Polat, Seher; Baldazzi, Lilia; Kulle, Alexandra E; Holterhus, Paul-Martin; Grötzinger, Joachim; Fanelli, Flaminia; Balsamo, Antonio; Riepe, Felix G

    2014-01-01

    Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. PMID:24022297

  6. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

    PubMed Central

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  7. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

    PubMed

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  8. Bioavailability of lysine in Maillard browned protein as determined by plasma lysine response in rainbow trout (Salmo gairdneri).

    PubMed

    Plakas, S M; Lee, T C; Wolke, R E

    1988-01-01

    The bioavailability of lysine in Maillard browned protein was investigated by plasma lysine response in rainbow trout (Salmo gairdneri). The concentrations of free lysine in the plasma were measured after feeding control and browned protein diets supplemented with graded levels of lysine. Bioavailability of lysine was estimated based on the amounts of supplemental lysine in the diets that resulted in rapid increases in plasma lysine. An approximately 80% loss in bioavailable lysine content was determined by this method in a fish protein isolate subjected to the Maillard browning reaction under mild conditions (40 d incubation at 37 degrees C). The nutritional damage to lysine determined by plasma lysine response was similar to that estimated in vitro by enzymatic hydrolysis and fluorodinitrobenzene reagent, but was underestimated by acid hydrolysis and trinitrobenzene sulfonic acid reagent. Rainbow trout are similar to other animals in their inability to utilize the deoxyketosyl (Amadori) compound of lysine formed in early Maillard reaction, and in their plasma response to dietary levels of essential amino acids. PMID:3121813

  9. Molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: an update of new CYP21A2 mutations.

    PubMed

    Concolino, Paola; Mello, Enrica; Zuppi, Cecilia; Capoluongo, Ettore

    2010-08-01

    Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia, an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Approximately 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. A list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http:www.imm.Ki.se/CYPalleles/cyp21.htm). Unfortunately, the last update of this database was in 2006. However, over the last 4 years many other novel CYP21A2 mutations have been reported in PubMed. The aim of this review is to provide a focus on the molecular and genetic aspects of the diagnosis of 21-hydroxylase deficiency. In addition, an updated list of the last new CYP21A2 mutations is included.

  10. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga

    PubMed Central

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Gándara, Lautaro; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-01-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses. PMID:27223464

  11. Expression of M6 and M7 lysin in Mytilus edulis is not restricted to sperm, but occurs also in oocytes and somatic tissue of males and females.

    PubMed

    Heß, Anne-Katrin; Bartel, Manuela; Roth, Karina; Messerschmidt, Katrin; Heilmann, Katja; Kenchington, Ellen; Micheel, Burkhard; Stuckas, Heiko

    2012-08-01

    Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females. PMID:22674895

  12. Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency.

    PubMed

    Vrzalová, Zuzana; Hrubá, Zuzana; St'ahlová Hrabincová, Eva; Pouchlá, Slavka; Votava, Felix; Kolousková, Stanislava; Fajkusová, Lenka

    2010-10-01

    Congenital adrenal hyperplasia (CAH) is comprised of a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in most of the severe cases, also the biosynthesis of aldosterone. Approximately 90-95% of all the CAH cases are due to mutations in the steroid 21-hydroxylase gene (CYP21A2). In this study, the molecular genetic analysis of CYP21A2 was performed in 267 Czech probands suspected of 21-hydroxylase deficiency (21OHD). 21OHD was confirmed in 241 probands (2 mutations were detected). In 26 probands, a mutation was found only in 1 CYP21A2 allele. A set of 30 different mutant alleles was determined. We describe i) mutated CYP21A2 alleles carrying novel point mutations (p.Thr168Asn, p.Ser169X and p.Pro386Arg), ii) mutated CYP21A2 alleles carrying the novel chimeric gene designated as CH-7, which was detected in 21.4% of the mutant alleles, iii) an unusual genotype with a combination of the CYP21A2 duplication, 2 point mutations and the CYP21A2 large-scale gene conversion on the second allele, and (iv) a detailed analysis of the chimeric CYP21A1P/CYP21A2 genes. In conclusion, our genotyping approach allowed for the accurate identification of the CYP21A2 gene mutations in 21OHD patients and their families and provided some useful information on diagnosis and genetic counselling.

  13. Methane monooxygenase hydroxylase and B component interactions.

    PubMed

    Zhang, Jingyan; Wallar, Bradley J; Popescu, Codrina V; Renner, Daniel B; Thomas, David D; Lipscomb, John D

    2006-03-01

    The interaction of the soluble methane monooxygenase regulatory component (MMOB) and the active site-bearing hydroxylase component (MMOH) is investigated using spin and fluorescent probes. MMOB from Methylosinus trichosporium OB3b is devoid of cysteine. Consequently, site-directed mutagenesis was used to incorporate single cysteine residues, allowing specific placement of the probe molecules. Sixteen MMOB Cys mutants were prepared and labeled with the EPR spin probe 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (MSL). Spectral evaluation of probe mobility and accessibility to the hydrophilic spin-relaxing agent NiEDDA showed that both properties decrease dramatically for a subset of the spin labels as the complex with MMOH forms, thereby defining the likely interaction surface on MMOB. This surface contains MMOB residue T111 thought to play a role in substrate access into the MMOH active site. The surface also contains several hydrophilic residues and is ringed by charged residues. The surface of MMOB opposite the proposed binding surface is highly charged, consistent with solvent exposure. Probes of both of the disordered N- and C-terminal regions remain highly mobile and exposed to solvent in the MMOH complex. Spin-labeling studies show that residue A62 of MMOB is located in a position where it can be used to monitor MMOH-MMOB complex formation without perturbing the process. Accordingly, steady-state kinetic assays show that it can be changed to Cys (A62C) and labeled with the fluorescent probes 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) or 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) without loss of the ability of MMOB to promote turnover. The BADAN fluorescence is partially quenched and red shifted as the complex with MMOH forms, allowing affinity measurements. It is shown that the high affinity of labeled MMOB (K(D) = 13.5 nM at pH 6.6, 25 degrees C) for the oxidized MMOH decreases substantially with increasing p

  14. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism.

    PubMed

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu; Zhang, Kezhong

    2015-12-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH.

  15. Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

    PubMed

    Neuner, Andreas; Wagner, Ines; Sieker, Tim; Ulber, Roland; Schneider, Konstantin; Peifer, Susanne; Heinzle, Elmar

    2013-01-20

    Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

  16. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

    PubMed Central

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu

    2015-01-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  17. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set.

    PubMed

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  18. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set

    PubMed Central

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  19. Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300

    PubMed Central

    Quinlan, Edward J.; Culleton, Sara P.; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2013-01-01

    The p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an in vitro acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression. PMID:23152516

  20. Lysine fortification: past, present, and future.

    PubMed

    Pellett, Peter L; Ghosh, Shibani

    2004-06-01

    Fortification with lysine to improve the protein value of human diets that are heavily based on cereals has received support from the results of these recent studies [1,2]. Support also comes from examination of average food and nutrient availability data derived from food balance sheets. Whereas nutritional status is influenced by the nutrient content of foods consumed in relation to need, the requirements for protein and amino acids are influenced by many additional factors [10, 12, 14, 28, 29]. These include age, sex, body size, physical activity, growth, pregnancy and lactation, infection, and the efficiency of nutrient utilization. Even if the immune response was influenced by the added lysine, adequate water and basic sanitation would remain essential. Acute and chronic undernutrition and most micronutrient deficiencies primarily affect poor and deprived people who do not have access to food of adequate nutritional value, live in unsanitary environments without access to clean water and basic services, and lack access to appropriate education and information [30]. A further variable is the possible interaction between protein and food energy availability [31]. This could affect the protein value of diets when food energy is limiting to a significant degree. Thus, the additional effects of food energy deficiency on protein utilization could well be superimposed on the very poorest. The improvement of dietary diversity must be the long-term aim, with dietary fortification considered only a short-term solution. The former should take place as wealth improves and the gaps between rich and poor diminish. Although such changes are taking place, they are highly uneven. Over the last several decades, increases have occurred in the availability of food energy, total protein, and animal protein for both developed and developing countries. However, for the very poorest developing countries over the same period, changes have been almost nonexistent, and the values for

  1. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency.

    PubMed

    Singh, Rani H; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B; Vockley, Jerry; Van Calcar, Sandra C

    2014-02-01

    The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency.

  2. Serum Dopamine Beta Hydroxylase and Maltreatment in Psychiatrically Hospitalized Boys.

    ERIC Educational Resources Information Center

    Galvin, Matthew; And Others

    1995-01-01

    Males (ages 7 to 17) in a psychiatric hospital were studied while off psychoactive medication to determine how serum dopamine beta hydroxylase (DBH) activity varies with childhood maltreatment experiences. Lowest DBH levels were found in boys maltreated before 72 months of age or with the principal diagnosis of conduct disorder solitary aggressive…

  3. Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase

    PubMed Central

    2015-01-01

    The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural–mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest. PMID:26368022

  4. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    NASA Astrophysics Data System (ADS)

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-08-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

  5. Current advances in the novel functions of hypoxia-inducible factor and prolyl hydroxylase in invertebrates.

    PubMed

    Wang, L; Cui, S; Ma, L; Kong, L; Geng, X

    2015-12-01

    Oxygen is essential for aerobic life, and hypoxia has very severe consequences. Organisms need to overcome low oxygen levels to maintain biological functions during normal development and in disease states. The mechanism underlying the hypoxic response has been widely investigated in model animals such as Drosophila melanogaster and Caenorhabditis elegans. Hypoxia-inducible factor (HIF), a key gene product in the response to oxygen deprivation, is primarily regulated by prolyl hydroxylase domain enzymes (PHDs). However, recent findings have uncovered novel HIF-independent functions of PHDs. This review provides an overview of how invertebrates are able to sustain hypoxic damages, and highlights some recent discoveries in the regulation of cellular signalling by PHDs. Given that some core genes and major pathways are evolutionarily conserved, these research findings could provide insight into oxygen-sensitive signalling in mammals, and have biomedical implications for human diseases.

  6. To Cheat or Not To Cheat: Tryptophan Hydroxylase 2 SNP Variants Contribute to Dishonest Behavior

    PubMed Central

    Shen, Qiang; Teo, Meijun; Winter, Eyal; Hart, Einav; Chew, Soo H.; Ebstein, Richard P.

    2016-01-01

    Although, lying (bear false witness) is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology, and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Toward addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2) gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior. PMID:27199691

  7. Characterization of Heronamide Biosynthesis Reveals a Tailoring Hydroxylase and Indicates Migrated Double Bonds.

    PubMed

    Zhu, Yiguang; Zhang, Wenjun; Chen, Yaolong; Yuan, Chengshan; Zhang, Haibo; Zhang, Guangtao; Ma, Liang; Zhang, Qingbo; Tian, Xinpeng; Zhang, Si; Zhang, Changsheng

    2015-09-21

    Heronamides belong to a growing family of β-amino acid polyketide macrolactams (βPMs) with an unsaturated side chain. The biosynthetic gene cluster for heronamide F was identified from the deep-sea-derived Streptomyces sp. SCSIO 03032. The involvement of the gene cluster in heronamide biosynthesis was confirmed by the functional characterization of the P450 enzyme HerO as an 8-hydroxylase for tailoring heronamide biosynthesis. The presence of migrated double bonds in the conjugated diene-containing side chain of heronamides was confirmed by feeding experiments with labeled small carboxylic acid molecules. This study is the first demonstration of migrated double bonds in βPMs with an unsaturated side chain.

  8. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    PubMed

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  9. Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites.

    PubMed

    Hong, Yeong H; Lillehoj, Hyun S; Siragusa, Gregory R; Bannerman, Douglas D; Lillehoj, Erik P

    2008-06-01

    NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role in innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we examined its ability to reduce the viability of various bacterial strains and two species of Eimeria parasites. Culture supernatants from COS7 cells transfected with a chicken NK-lysin cDNA and His-tagged purified NK-lysin from the transfected cells both showed high cytotoxic activity against Eimeria acervulina and Eimeria maxima sporozoites. In contrast, no bactericidal activity was observed. Further studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry.

  10. The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

    PubMed

    Escribese, María M; Sierra-Filardi, Elena; Nieto, Concha; Samaniego, Rafael; Sánchez-Torres, Carmen; Matsuyama, Takami; Calderon-Gómez, Elisabeth; Vega, Miguel A; Salas, Azucena; Sánchez-Mateos, Paloma; Corbí, Angel L

    2012-08-15

    Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro. PMID:22778395

  11. Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a

    PubMed Central

    Romano, Andrea; Trip, Hein; Lolkema, Juke S.

    2013-01-01

    Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system. PMID:23316036

  12. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    SciTech Connect

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  13. Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

    PubMed Central

    Choo, K H; Cotton, R G; Danks, D M; Jennings, I G

    1979-01-01

    Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:496890

  14. Global analysis of lysine acetylation in strawberry leaves.

    PubMed

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants. PMID:26442052

  15. Histone lysine methylation: critical regulator of memory and behavior.

    PubMed

    Jarome, Timothy J; Lubin, Farah D

    2013-01-01

    Histone lysine methylation is a well-established transcriptional mechanism for the regulation of gene expression changes in eukaryotic cells and is now believed to function in neurons of the central nervous system to mediate the process of memory formation and behavior. In mature neurons, methylation of histone proteins can serve to both activate and repress gene transcription. This is in stark contrast to other epigenetic modifications, including histone acetylation and DNA methylation, which have largely been associated with one transcriptional state in the brain. In this review, we discuss the evidence for histone methylation mechanisms in the coordination of complex cognitive processes such as long-term memory formation and storage. In addition, we address the current literature highlighting the role of histone methylation in intellectual disability, addiction, schizophrenia, autism, depression, and neurodegeneration. Further, we discuss histone methylation within the context of other epigenetic modifications and the potential advantages of exploring this newly identified mechanism of cognition, emphasizing the possibility that this molecular process may provide an alternative locus for intervention in long-term psychopathologies that cannot be clearly linked to genes or environment alone.

  16. Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism.

    PubMed

    Meneely, Kathleen M; Lamb, Audrey L

    2007-10-23

    Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. PMID:17900176

  17. Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase

    PubMed Central

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-01-01

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. l-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of l-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from l-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L l-lysine in 12 h. Because l-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate. PMID:25012259

  18. Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80a lysin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phage lytic enzymes are promising antimicrobial agents. Lysins of phage phi11 (LysPhi11) and phi80a (LysPhi80a) can lyse (destroy) biofilms and cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The obj...

  19. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat

    PubMed Central

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

  20. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  1. Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB.

    PubMed

    Ma, Weichao; Cao, Weijia; Zhang, Hong; Chen, Kequan; Li, Yan; Ouyang, Pingkai

    2015-04-01

    The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of L-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12%, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of L-lysine to cadaverine was constructed, and three strategies for L-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l(-1) with a molar yield of 92% from lysine was obtained. PMID:25515797

  2. Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

    PubMed

    Zhang, Nan; Han, Zhentai; Sun, Guiling; Hoffman, Angela; Wilson, Iain W; Yang, Yanfang; Gao, Qian; Wu, Jianqiang; Xie, Dan; Dai, Jungui; Qiu, Deyou

    2014-01-17

    Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

  3. A novel profibrotic mechanism mediated by TGF-β-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells

    PubMed Central

    Luo, Yongfeng; Xu, Wei; Chen, Hui; Warburton, David; Dong, Rachel; Qian, Bangping; Selman, Moisés; Gauldie, Jack; Kolb, Martin; Shi, Wei

    2015-01-01

    Idiopathic pulmonary fibrosis is a severe chronic lung disease with a high mortality rate. Excessive TGF-β signaling is recognized as a central player in lung fibrosis. However, the related mechanisms remain unclear. Herein we used a novel Tbx4 lung enhancer-driven Tet-On transgenic system to inhibit TGF-β signaling in mouse lung resident mesenchymal cells at different stages of bleomycin-induced fibrosis by conditionally knocking out TGF-β receptor II or expressing a dominant-negative TGF-β receptor II. Abrogation of mesenchymal TGF-β signaling markedly attenuated bleomycin-induced fibrotic pathology, which was independent of altered early inflammation. Furthermore, a novel TGF-β downstream target gene P4HA3 (an α-subunit of collagen prolyl hydroxylase) was identified, and its expression was significantly increased in fibroblastic foci of both bleomycin-induced fibrotic mouse lungs and idiopathic pulmonary fibrosis patients’ lungs. The relationship between activated TGF-β signaling, upregulation of P4HA3, as well as increased hydroxyproline/collagen production was further verified in cultured lung fibroblasts. Moreover, inhibition of collagen prolyl hydroxylase by pyridine-2,5-dicarboxylate attenuated both TGF-β-stimulated collagen production in cultured fibroblasts and bleomycin-induced mouse lung fibrosis. These data indicate that increased expression and activity of collagen prolyl hydroxylase is one of the important mechanisms underlying TGF-β-mediated profibrotic effects. Inhibition of collagen prolyl hydroxylase may be a new promising approach for preventing and treating pulmonary fibrosis. PMID:25779936

  4. Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase.

    PubMed

    Bursy, Jan; Pierik, Antonio J; Pica, Nathalie; Bremer, Erhard

    2007-10-26

    By using natural abundance (13)C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

  5. HIF hydroxylase pathways in cardiovascular physiology and medicine.

    PubMed

    Bishop, Tammie; Ratcliffe, Peter J

    2015-06-19

    Hypoxia inducible factors (HIFs) are α/β heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.

  6. HIF hydroxylase pathways in cardiovascular physiology and medicine

    PubMed Central

    Bishop, Tammie; Ratcliffe, Peter J.

    2015-01-01

    Hypoxia inducible factors (HIFs) are alpha/beta heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate dependent dioxygenases that catalyse post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFalpha subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease. PMID:26089364

  7. Lysine carboxylation: unveiling a spontaneous post-translational modification

    SciTech Connect

    Jimenez-Morales, David; Adamian, Larisa; Shi, Dashuang; Liang, Jie

    2014-01-01

    A computational method for the prediction of lysine carboxylation (KCX) in protein structures is described. The method accurately identifies misreported KCXs and predicts previously unknown KCX sites. The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown. In this work, the signature microenvironment of lysine-carboxylation sites has been characterized. In addition, a computational method called Predictor of Lysine Carboxylation (PreLysCar) for the detection of lysine carboxylation in proteins with available three-dimensional structures has been developed. The likely prevalence of lysine carboxylation in the proteome was assessed through large-scale computations. The results suggest that about 1.3% of large proteins may contain a carboxylated lysine residue. This unexpected prevalence of lysine carboxylation implies an enrichment of reactions in which it may play functional roles. The results also suggest that by switching enzymes on and off under appropriate physicochemical conditions spontaneous PTMs may serve as an important and widely used efficient biological machinery for regulation.

  8. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  9. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  10. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  11. Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach

    PubMed Central

    Bao, Xiucong; Wang, Yi; Li, Xin; Li, Xiao-Meng; Liu, Zheng; Yang, Tangpo; Wong, Chi Fat; Zhang, Jiangwen; Hao, Quan; Li, Xiang David

    2014-01-01

    Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile ‘eraser’ enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase. DOI: http://dx.doi.org/10.7554/eLife.02999.001 PMID:25369635

  12. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9.

    PubMed

    Mutskov, Vesco; Felsenfeld, Gary

    2004-01-14

    Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system. PMID:14685282

  13. De Novo Assembly and Comparative Transcriptome Analysis Provide Insight into Lysine Biosynthesis in Toona sinensis Roem

    PubMed Central

    Zhang, Xia; Song, Zhenqiao; Liu, Tian; Guo, Linlin; Li, Xingfeng

    2016-01-01

    Toona sinensis Roem is a popular leafy vegetable in Chinese cuisine and is also used as a traditional Chinese medicine. In this study, leaf samples were collected from the same plant on two development stages and then used for high-throughput Illumina RNA-sequencing (RNA-Seq). 125,884 transcripts and 54,628 unigenes were obtained through de novo assembly. A total of 25,570 could be annotated with known biological functions, which indicated that the T. sinensis leaves and shoots were undergoing multiple developmental processes especially for active metabolic processes. Analysis of differentially expressed unigenes between the two libraries showed that the lysine biosynthesis was an enriched KEGG pathway, and candidate genes involved in the lysine biosynthesis pathway in T. sinensis leaves and shoots were identified. Our results provide a primary analysis of the gene expression files of T. sinensis leaf and shoot on different development stages and afford a valuable resource for genetic and genomic research on plant lysine biosynthesis. PMID:27376077

  14. Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

    PubMed

    Widderich, Nils; Höppner, Astrid; Pittelkow, Marco; Heider, Johann; Smits, Sander H J; Bremer, Erhard

    2014-01-01

    Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo

  15. Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

    PubMed

    Widderich, Nils; Höppner, Astrid; Pittelkow, Marco; Heider, Johann; Smits, Sander H J; Bremer, Erhard

    2014-01-01

    Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo

  16. Biochemical Properties of Ectoine Hydroxylases from Extremophiles and Their Wider Taxonomic Distribution among Microorganisms

    PubMed Central

    Pittelkow, Marco; Heider, Johann; Smits, Sander H. J.; Bremer, Erhard

    2014-01-01

    Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo

  17. Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure

    PubMed Central

    Banerjee, Kasturi; Wang, Meng; Cai, Elizabeth; Fujiwara, Nana; Baker, Harriet; Cave, John W.

    2014-01-01

    Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds C-rich single DNA strands within these conserved regions and also associates with double-stranded sequences when proteins, such as CREB, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype. PMID:25493445

  18. Lysine catabolism in Rhizoctonia leguminicola and related fungi.

    PubMed Central

    Guengerich, F P; Broquist, H P

    1976-01-01

    The catabolism of lysine was studied in several yeasts and fungi. Results with cell-free extracts of Rhizoctonia leguminicola support a proposed pathway involving (D- and L-) EPSILON-N-acetyllysine, alpha-keto-epsilon-acetamidohexanoic acid, delta-acetamidovaleric acid, and delta-aminovaleric acid in the conversion of L-lysine to shortchain organic acids. Label from radioactive L-lysine was found to accumulate in D- and L-epsilon-N-acetyllysine, delta-acetamidovaleric acid, delta-aminovaleric acid, and glutaric acid in cultures of R. leguminicola, Neurospora crassa, Saccharomyces cerevisiae, and Hansenula saturnus, suggesting that the proposed omega-acetyl pathway of lysine catabolism is generalized among yeasts and fungi. In N. crassa, as is the case in R. leguminicola, the major precursor of L-pipecolic acid was the L-isomer of lysine; 15N experiments were consistent with delta1-piperideine-2-carboxylic acid as an intermediate in the transformation. PMID:131119

  19. Creative lysins: Listeria and the engineering of antimicrobial enzymes.

    PubMed

    Van Tassell, Maxwell L; Angela Daum, M; Kim, Jun-Seob; Miller, Michael J

    2016-02-01

    Cell wall lytic enzymes have been of increasing interest as antimicrobials for targeting Gram-positive spoilage and pathogenic bacteria, largely due to the development of strains resistant to antibiotics and bacteriophage therapy. Such lysins show considerable promise against Listeria monocytogenes, a primary concern in food-processing environments, but there is room for improvement via protein engineering. Advances in antilisterial applications could benefit from recent developments in lysin biotechnology that have largely targeted other organisms. Herein we present various considerations for the future development of lysins, including environmental factors, cell physiology concerns, and dynamics of protein architecture. Our goal is to review key developments in lysin biotechnology to provide a contextual framework for the current models of lysin-cell interactions and highlight key considerations for the characterization and design of novel lytic enzymes. PMID:26710271

  20. Creative lysins: Listeria and the engineering of antimicrobial enzymes.

    PubMed

    Van Tassell, Maxwell L; Angela Daum, M; Kim, Jun-Seob; Miller, Michael J

    2016-02-01

    Cell wall lytic enzymes have been of increasing interest as antimicrobials for targeting Gram-positive spoilage and pathogenic bacteria, largely due to the development of strains resistant to antibiotics and bacteriophage therapy. Such lysins show considerable promise against Listeria monocytogenes, a primary concern in food-processing environments, but there is room for improvement via protein engineering. Advances in antilisterial applications could benefit from recent developments in lysin biotechnology that have largely targeted other organisms. Herein we present various considerations for the future development of lysins, including environmental factors, cell physiology concerns, and dynamics of protein architecture. Our goal is to review key developments in lysin biotechnology to provide a contextual framework for the current models of lysin-cell interactions and highlight key considerations for the characterization and design of novel lytic enzymes.

  1. Identification of N(α)-acetyl-α-lysine as a probable thermolyte and its accumulation mechanism in Salinicoccus halodurans H3B36.

    PubMed

    Jiang, Kai; Xue, Yanfen; Ma, Yanhe

    2015-01-01

    Salinicoccus halodurans H3B36 is a moderate halophile that was isolated from a 3.2-m-deep sediment sample in Qaidam Basin, China. Our results suggest that N(α)-acetyl-α-lysine can accumulate and act as a probable thermolyte in this strain. The accumulation mechanism and biosynthetic pathway for this rare compatible solute were also elucidated. We confirmed that the de novo synthesis pathway of N(α)-acetyl-α-lysine in this strain starts from aspartate and passes through lysine. Through RNA sequencing, we also found an 8-gene cluster (orf_1582-1589) and another gene (orf_2472) that might encode the biosynthesis of N(α)-acetyl-α-lysine in S. halodurans H3B36. Orf_192, orf_193, and orf_1259 might participate in the transportation of precursors for generating N(α)-acetyl-α-lysine under the heat stress. The transcriptome reported here also generated a global view of heat-induced changes and yielded clues for studying the regulation of N(α)-acetyl-α-lysine accumulation. Heat stress triggered a global transcriptional disturbance and generated a series of actions to adapt the strain to heat stress. Furthermore, the transcriptomic results showed that the regulon of RpoN (orf_2534) may be critical to conferring heat stress tolerance and survival to S. halodurans. PMID:26687465

  2. Identification of Nα-acetyl-α-lysine as a probable thermolyte and its accumulation mechanism in Salinicoccus halodurans H3B36

    PubMed Central

    Jiang, Kai; Xue, Yanfen; Ma, Yanhe

    2015-01-01

    Salinicoccus halodurans H3B36 is a moderate halophile that was isolated from a 3.2-m-deep sediment sample in Qaidam Basin, China. Our results suggest that Nα-acetyl-α-lysine can accumulate and act as a probable thermolyte in this strain. The accumulation mechanism and biosynthetic pathway for this rare compatible solute were also elucidated. We confirmed that the de novo synthesis pathway of Nα-acetyl-α-lysine in this strain starts from aspartate and passes through lysine. Through RNA sequencing, we also found an 8-gene cluster (orf_1582–1589) and another gene (orf_2472) that might encode the biosynthesis of Nα-acetyl-α-lysine in S. halodurans H3B36. Orf_192, orf_193, and orf_1259 might participate in the transportation of precursors for generating Nα-acetyl-α-lysine under the heat stress. The transcriptome reported here also generated a global view of heat-induced changes and yielded clues for studying the regulation of Nα-acetyl-α-lysine accumulation. Heat stress triggered a global transcriptional disturbance and generated a series of actions to adapt the strain to heat stress. Furthermore, the transcriptomic results showed that the regulon of RpoN (orf_2534) may be critical to conferring heat stress tolerance and survival to S. halodurans. PMID:26687465

  3. Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta

    PubMed Central

    Cleys, Ellane R.; Halleran, Jennifer L.; Enriquez, Vanessa A.; da Silveira, Juliano C.; West, Rachel C.; Winger, Quinton A.; Anthony, Russell V.; Bruemmer, Jason E.; Clay, Colin M.; Bouma, Gerrit J.

    2015-01-01

    Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders. PMID:25675430

  4. Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum

    PubMed Central

    2010-01-01

    Background Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. Results The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. Conclusions CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism. PMID:20128892

  5. Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme.

    PubMed

    Xiong, Hai; Reynolds, Noah M; Fan, Chenguang; Englert, Markus; Hoyer, Denton; Miller, Scott J; Söll, Dieter

    2016-03-14

    Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications. PMID:26914285

  6. Restriction fragment length polymorphisms for growth hormone, prolactin, osteonectin, alpha crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase in cattle.

    PubMed

    Theilmann, J L; Skow, L C; Baker, J F; Womack, J E

    1989-01-01

    Genomic DNAs from animals representing six breeds of cattle (Angus, Brahman, Hereford, Holstein, Jersey and Texas Longhorn) were screened with cloned gene probes in a search for restriction fragment length polymorphisms (RFLPs). Eleven RFLPs were identified using seven different probes: growth hormone, prolactin, osteonectin, alpha A-crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase. The frequencies of the alleles identified by each probe were calculated and compared in a limited sampling of the six bovine breeds. These polymorphisms greatly enhance the pool of immunogenetic, biochemical and molecular markers available in cattle for linkage analysis, testing of parentage, and distinction of breeds. PMID:2575360

  7. CYP287A1 is a carotenoid 2-β-hydroxylase required for deinoxanthin biosynthesis in Deinococcus radiodurans R1.

    PubMed

    Zhou, Zhengfu; Zhang, Wei; Su, Shiyou; Chen, Ming; Lu, Wei; Lin, Min; Molnár, István; Xu, Yuquan

    2015-12-01

    The carotenoid deinoxanthin is a crucial resistance factor against various stresses in the radiation-resistant bacterium Deinococcus radiodurans. Disruption of the gene dr2473 encoding the cytochrome P450 CYP287A1 led to the accumulation of 2-deoxydeinoxanthin in D. radiodurans, demonstrating that CYP287A1 is a novel β-carotene 2-hydroxylase. The dr2473 knockout mutant was shown to be more sensitive to UV radiation and oxidative stress than the wild-type strain D. radiodurans R1, indicating that the C2 alcohol of deinoxanthin is important for antioxidant activity.

  8. Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants

    PubMed Central

    Jiang, Shu-Ye; Ma, Ali; Xie, Lifen; Ramachandran, Srinivasan

    2016-01-01

    Rice grains are rich in starch but low in protein with very low level of both lysine and threonine. Thus, it is important to further improve protein quality and quantity, especially to increase lysine and threonine content in rice grains. We artificially synthesized two new genes by fusing endogenous rice genes with lysine (K)/threonine (T) motif (TKTKK) coding sequences. They were designated as TKTKK1 and TKTKK2 and their encoded proteins consist of 73.1% and 83.5% of lysine/threonine, respectively. These two genes were under the control of 35S promoter and were independently introduced into the rice genome to generate transgenic plants. Our data showed that overexpression of TKTKK1 generated stable proteins with expected molecular weight and the transgenic rice seeds significantly increased lysine, threonine, total amino acids and crude protein content by 33.87%, 21.21%, 19.43% and 20.45%, respectively when compared with wild type control; significant improvement was also observed in transgenic rice seeds overexpressing TKTKK2. However, limited improvement in protein quality and quantity was observed in transgenic seeds carrying tandom array of these two new genes. Our data provide the basis and alternative strategy on further improving protein quality and quantity in other crops or vegetable plants by synthetic biology. PMID:27677708

  9. The dietary lysine requirement of juvenile hybrid striped bass.

    PubMed

    Griffin, M E; Brown, P B; Grant, A L

    1992-06-01

    Two experiments were conducted to determine the dietary lysine requirement of juvenile hybrid striped bass (Morone saxatilis x M. chrysops). In both experiments the diets contained 35 g crude protein/100 g diet (10 g crude protein supplied by casein and gelatin and 25 g crude protein supplied by crystalline L-amino acids) and contained graded levels of L-lysine.HCl resulting in eight dietary treatments. Diets were fed to triplicate groups of fish and ranged in dietary lysine concentration from 1.2 to 2.6 g/100 g of the dry diet in Experiment 1 and from 0.8 to 2.2 g/100 g of the dry diet in Experiment 2. Weight gain and food efficiency data from Experiment 1 indicated the dietary lysine requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Weight gain, food efficiency and serum lysine data from Experiment 2 confirmed the requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Broken-line analysis of weight gain and food efficiency data from Experiment 2 indicated the dietary lysine requirement to be 1.4 +/- 0.2% of the dry diet, or 4.0 g/100 g of the dietary protein. Changes in the relative proportions of dietary lipid and carbohydrate between the two experiments, although maintaining similar gross energy levels, did not alter the lysine requirement estimate of juvenile hybrid striped bass.

  10. Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

    PubMed Central

    Lavaissiere, L; Jia, S; Nishiyama, M; de la Monte, S; Stern, A M; Wands, J R; Friedman, P A

    1996-01-01

    To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the

  11. Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase.

    PubMed

    Chen, Dawei; Tanem, Justinn; Frey, Perry A

    2007-02-01

    l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.

  12. Cadaverine: a lysine catabolite involved in plant growth and development.

    PubMed

    Tomar, Pushpa C; Lakra, Nita; Mishra, S N

    2013-10-01

    The cadaverine (Cad) a diamine, imino compound produced as a lysine catabolite is also implicated in growth and development of plants depending on environmental condition. This lysine catabolism is catalyzed by lysine decarboxylase, which is developmentally regulated. However, the limited role of Cad in plants is reported, this review is tempted to focus the metabolism and its regulation, transport and responses, interaction and cross talks in higher plants. The Cad varied presence in plant parts/products suggests it as a potential candidate for taxonomic marker as well as for commercial exploitation along with growth and development.

  13. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

    PubMed Central

    2013-01-01

    Background Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Results Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Conclusions Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of

  14. Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

    PubMed

    Flachowsky, Henryk; Halbwirth, Heidi; Treutter, Dieter; Richter, Klaus; Hanke, Magda-Viola; Szankowski, Iris; Gosch, Christian; Stich, Karl; Fischer, Thilo C

    2012-02-01

    Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.

  15. Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    PubMed Central

    Lee, Jae-Young; Lee, Se-Hwan; Heo, Sun-Hee; Kim, Kwang-Soo; Kim, Changhoon; Kim, Dae-Kwan; Ko, Jeong-Jae; Park, Kyung-Soon

    2015-01-01

    G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner. PMID:26492085

  16. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    PubMed

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  17. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    PubMed Central

    Pathak, Ravi; Philizaire, Marc; Mujtaba, Shiraz

    2015-01-01

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets. PMID:26295410

  18. Utilization of potato starch processing wastes to produce animal feed with high lysine content.

    PubMed

    Li, Ying; Liu, Bingnan; Song, Jinzhu; Jiang, Cheng; Yang, Qian

    2015-02-01

    This work aims to utilize wastes from the potato starch industry to produce single-cell protein (SCP) with high lysine content as animal feed. In this work, S-(2-aminoethyl)-L-cysteine hydrochloride-resistant Bacillus pumilus E1 was used to produce SCP with high lysine content, whereas Aspergillus niger was used to degrade cellulose biomass and Candida utilis was used to improve the smell and palatability of the feed. An orthogonal design was used to optimize the process of fermentation for maximal lysine content. The optimum fermentation conditions were as follows: temperature of 40°C, substrate concentration of 3%, and natural pH of about 7.0. For unsterilized potato starch wastes, the microbial communities in the fermentation process were determined by terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes. Results showed that the dominant population was Bacillus sp. The protein quality as well as the amino acid profile of the final product was found to be significantly higher compared with the untreated waste product at day 0. Additionally, acute toxicity test showed that the SCP product was non-toxic, indicating that it can be used for commercial processing.

  19. Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima

    2014-01-01

    Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and 13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

  20. Multi-technique characterization of poly-L-lysine dendrigrafts-Cu(II) complexes for biocatalysis.

    PubMed

    Rossi, Jean-Christophe; Maret, Barbara; Vidot, Kevin; Francoia, Jean-Patrick; Cangiotti, Michela; Lucchi, Susanna; Coppola, Concetta; Ottaviani, Maria Francesca

    2015-02-01

    Poly-L-lysine is a biocompatible polymer used for drug or gene delivery, for transport through cellular membranes, and as nanosized magnetic resonance imaging contrast agents. Cu(II)-poly-L-lysine complexes are of particular interest for their role in biocatalysis. In this study, poly-L-lysine dendrigrafts (DGLs) at different generations (G2, G3, and G4) are synthesized and characterized in absence and presence of Cu(II) by means of electron paramagnetic resonance (EPR), UV-Vis, potentiometric titration and circular dichroism (CD). The analysis is performed as a function of the [Cu(II)]/[Lys] (=R) molar ratio, pH and generation by identifying differently flexible complexes in different dendrimer regions. The amine sites in the lateral chains become increasingly involved with the increase of pH. The good agreement and complementarity of the results from the different techniques provide an integrate view of the structural and dynamic properties of Cu(II)-DGL complexes implementing their use as biocatalysts.

  1. The Antimicrobial Activity of Marinocine, Synthesized by Marinomonas mediterranea, Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity

    PubMed Central

    Lucas-Elío, Patricia; Gómez, Daniel; Solano, Francisco; Sanchez-Amat, Antonio

    2006-01-01

    Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned. PMID:16547036

  2. Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity

    PubMed Central

    Sartor, Gregory C.; Powell, Samuel K.; Brothers, Shaun P.

    2015-01-01

    Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic “reader” proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction. SIGNIFICANCE STATEMENT Proteins involved in the “readout” of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and

  3. Dopamine-beta-hydroxylase, monoamine oxidase, and schizophrenia.

    PubMed

    DeLisi, L E; Wise, C D; Potkin, S G; Zalcman, S; Phelps, B H; Lovenberg, W; Wyatt, R J

    1980-12-01

    Plasma dopamine-beta-hydroxylase (DBH) activity was studied in two different populations of chronic schizophrenic patients and assayed by two independent laboratories. No significant difference between schizophrenic patients and normal controls was found although in both groups chronic undifferentiated schizophrenics with paranoid features had a trend towards lower DBH activity than the other patients and controls. In addition, DBH and monoamine oxidase (MAO) activities were studied in 13 schizophrenic patients and available first degree-relatives. There was no association of low MAO and low DBH activities within the schizophrenic families.

  4. The catecholamines up (Catsup) protein of Drosophila melanogaster functions as a negative regulator of tyrosine hydroxylase activity.

    PubMed Central

    Stathakis, D G; Burton, D Y; McIvor, W E; Krishnakumar, S; Wright, T R; O'Donnell, J M

    1999-01-01

    We report the genetic, phenotypic, and biochemical analyses of Catecholamines up (Catsup), a gene that encodes a negative regulator of tyrosine hydroxylase (TH) activity. Mutations within this locus are semidominant lethals of variable penetrance that result in three broad, overlapping effective lethal phases (ELPs), indicating that the Catsup gene product is essential throughout development. Mutants from each ELP exhibit either cuticle defects or catecholamine-related abnormalities, such as melanotic salivary glands or pseudotumors. Additionally, Catsup mutants have significantly elevated TH activity that may arise from a post-translational modification of the enzyme. The hyperactivation of TH in Catsup mutants results in abnormally high levels of catecholamines, which can account for the lethality, visible phenotypes, and female sterility observed in these mutants. We propose that Catsup is a component of a novel system that downregulates TH activity, making Catsup the fourth locus found within the Dopa decarboxylase (Ddc) gene cluster that functions in catecholamine metabolism. PMID:10471719

  5. Lysine fatty acylation promotes lysosomal targeting of TNF-α

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation. PMID:27079798

  6. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

    Aslan, Joseph E.; David, Larry L.; McCarty, Owen J.T.

    2015-01-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification – mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  7. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  8. Human dopamine {beta}-hydroxylase locus and the chromosome 9q34 region in alcoholism

    SciTech Connect

    Parsian. A.; Suarez, B.K.; Hampe, C.

    1994-09-01

    Human dopamine {beta}-hydroxylase (DBH) is responsible for conversion of dopamine to norepinephrine in catecholamine neurons. Potential inhibitors of this enzyme do exist, but they are generally not effective in vivo in reducing tissue concentrations of catecholamines. The gene for DBH has been localized to 9q34 by linkage analysis and in situ hybridization. Recently there have been reports indicating a suggestive evidence of linkage between DNA markers in 9q34 region and alcoholism. In order to test for this suggestive linkage, we have genotyped a sample of 134 subjects with alcoholism, 30 alcoholic families (n=302) and 92 normal controls. The alcoholic subjects are probands of multiple incidence families. The normal controls are an epidemiologically ascertained samples of middle-aged, unrelated individuals. The two groups were matched for sex and ethnic background. The markers used in this study were dinucleotide repeats in the DBH gene, and two highly informative (CA) markers (D9S64, D9S66) flanking the DBH gene. A preliminary affected-sib-pair analysis was carried out under two diagnostic schemes. Regardless of whether `probable` alcoholics are classified as unaffected (t=0.63) or affected (t=1.50), these data do not reveal a significant excess in DBH marker sharing among affected-sib-pairs. However, the comparison of the DBH marker allele frequencies between the unrelated alcoholic panel and the unrelated normal control panel was significant at the p=0.04 level.

  9. A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (-)-limonene hydroxylase from a C6- to a C3-hydroxylase.

    PubMed

    Schalk, M; Croteau, R

    2000-10-24

    The essential oils of peppermint and spearmint are distinguished by the position of oxygenation on the constituent p-menthane monoterpenes. Peppermint produces monoterpenes bearing an oxygen at C3, whereas spearmint produces monoterpenes bearing an oxygen at C6. Branching of the monoterpene biosynthetic pathways in these species is determined by two distinct cytochrome P450s that catalyze the regiospecific hydroxylation of (-)-4S-limonene at C3 or C6 exclusively. cDNAs encoding the limonene-3-hydroxylase from peppermint and the limonene-6-hydroxylase from spearmint have been isolated, shown to be 70% identical at the amino acid level, and functionally expressed. A combination of domain swapping and reciprocal site-directed mutagenesis between these two enzymes demonstrated that the exchange of a single residue (F363I) in the spearmint limonene-6-hydroxylase led to complete conversion to the regiospecificity and catalytic efficiency of the peppermint limonene-3-hydroxylase. PMID:11050228

  10. Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

    PubMed

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids). PMID:21673887

  11. Histone lysine crotonylation during acute kidney injury in mice

    PubMed Central

    Ruiz-Andres, Olga; Sanchez-Niño, Maria Dolores; Cannata-Ortiz, Pablo; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belen

    2016-01-01

    ABSTRACT Acute kidney injury (AKI) is a potentially lethal condition for which no therapy is available beyond replacement of renal function. Post-translational histone modifications modulate gene expression and kidney injury. Histone crotonylation is a recently described post-translational modification. We hypothesized that histone crotonylation might modulate kidney injury. Histone crotonylation was studied in cultured murine proximal tubular cells and in kidneys from mice with AKI induced by folic acid or cisplatin. Histone lysine crotonylation was observed in tubular cells from healthy murine and human kidney tissue. Kidney tissue histone crotonylation increased during AKI. This was reproduced by exposure to the protein TWEAK in cultured tubular cells. Specifically, ChIP-seq revealed enrichment of histone crotonylation at the genes encoding the mitochondrial biogenesis regulator PGC-1α and the sirtuin-3 decrotonylase in both TWEAK-stimulated tubular cells and in AKI kidney tissue. To assess the role of crotonylation in kidney injury, crotonate was used to increase histone crotonylation in cultured tubular cells or in the kidneys in vivo. Crotonate increased the expression of PGC-1α and sirtuin-3, and decreased CCL2 expression in cultured tubular cells and healthy kidneys. Systemic crotonate administration protected from experimental AKI, preventing the decrease in renal function and in kidney PGC-1α and sirtuin-3 levels as well as the increase in CCL2 expression. For the first time, we have identified factors such as cell stress and crotonate availability that increase histone crotonylation in vivo. Overall, increasing histone crotonylation might have a beneficial effect on AKI. This is the first observation of the in vivo potential of the therapeutic manipulation of histone crotonylation in a disease state. PMID:27125278

  12. Folding simulations of alanine-based peptides with lysine residues.

    PubMed Central

    Sung, S S

    1995-01-01

    The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies. PMID:7756550

  13. Degradation signals in the lysine-asparagine sequence space.

    PubMed

    Suzuki, T; Varshavsky, A

    1999-11-01

    The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding. PMID:10545113

  14. Water reuse in the L-lysine fermentation process.

    PubMed

    Hsiao, T Y; Glatz, C E

    1996-02-01

    L-Lysine is produced commercially by fermentation. As is typical for fermentation processes, a large amount of liquid waste is generated. To minimize the waste, which is mostly the broth effluent from the cation exchange column used for l-lysine recovery, we investigated a strategy of recycling a large fraction of this broth effluent to the subsequent fermentation. This was done on a labscale process with Corynebacterium glutamicum ATCC 21253 as the l-lysine-producing organism. Broth effluent from a fermentation in a defined medium was able to replace 75% of the water for the subsequent batch; this recycle ratio was maintained for three sequential batches without affecting cell mass and l-lysine production. Broth effluent was recycled at 50% recycle ratio in a fermentation in a complex medium containing beet molasses. The first recycle batch had an 8% lower final l-lysine level, but 8% higher maximum cell mass. In addition to reducing the volume of liquid waste, this recycle strategy has the additional advantage of utilizing the ammonium desorbed from the ion-exchange column as a nitrogen source in the recycle fermentation. The major problem of recycling the effluent from the complex medium was in the cation-exchange operation, where column capacity was 17% lower for the recycle batch. The loss of column capacity probably results from the buildup of cations competing with l-lysine for binding. (c) 1996 John Wiley & Sons, Inc.

  15. Biofortification of rice with lysine using endogenous histones.

    PubMed

    Wong, H W; Liu, Q; Sun, S S M

    2015-02-01

    Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.

  16. Fibroblast growth factor 23 impairs phosphorus and vitamin D metabolism in vivo and suppresses 25-hydroxyvitamin D-1alpha-hydroxylase expression in vitro.

    PubMed

    Perwad, Farzana; Zhang, Martin Y H; Tenenhouse, Harriet S; Portale, Anthony A

    2007-11-01

    Fibroblast growth factor-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/P(i)) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 microg/g body wt) induced significant hypophosphatemia, 20 and 29% decreases (P < 0.001) in brush-border membrane (BBM) Na/Pi cotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/P(i) cotransporter protein in BBM. Multiple injections (6, 12, and 24 mug/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1alpha-hydroxylase mRNA (P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1alpha-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1alpha-hydroxylase mRNA with a maximum suppression of 37% (P < 0.05). Suppression was detectable after 6 h of exposure and maximal after 21 h. In MCT cells, FGF-23(R176Q) suppressed 1alpha-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1alpha-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1alpha-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.

  17. Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways.

    PubMed

    Naik, Rangeetha J; Chandra, Pallavi; Mann, Anita; Ganguli, Munia

    2011-05-27

    Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA.

  18. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

    PubMed

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L

    1988-09-01

    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  19. Histone lysine methyltransferases as anti-cancer targets for drug discovery

    PubMed Central

    Liu, Qing; Wang, Ming-wei

    2016-01-01

    Post-translational epigenetic modification of histones is controlled by a number of histone-modifying enzymes. Such modification regulates the accessibility of DNA and the subsequent expression or silencing of a gene. Human histone methyltransferases (HMTs)constitute a large family that includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). There is increasing evidence showing a correlation between HKMTs and cancer pathogenesis. Here, we present an overview of representative HKMTs, including their biological and biochemical properties as well as the profiles of small molecule inhibitors for a comprehensive understanding of HKMTs in drug discovery. PMID:27397541

  20. Engineering Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid.

    PubMed

    Pérez-García, Fernando; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-09-01

    The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

  1. Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2.

    PubMed

    Verma, Sharad K; Tian, Xinrong; LaFrance, Louis V; Duquenne, Céline; Suarez, Dominic P; Newlander, Kenneth A; Romeril, Stuart P; Burgess, Joelle L; Grant, Seth W; Brackley, James A; Graves, Alan P; Scherzer, Daryl A; Shu, Art; Thompson, Christine; Ott, Heidi M; Aller, Glenn S Van; Machutta, Carl A; Diaz, Elsie; Jiang, Yong; Johnson, Neil W; Knight, Steven D; Kruger, Ryan G; McCabe, Michael T; Dhanak, Dashyant; Tummino, Peter J; Creasy, Caretha L; Miller, William H

    2012-12-13

    The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2. PMID:24900432

  2. Molecular cloning of the. alpha. -subunit of human prolyl 4-hydroxylase: The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts

    SciTech Connect

    Helaakoski, T.; Vuori, K.; Myllylae, R.; Kivirikko, K.I.; Pihlajaniemi, T. )

    1989-06-01

    Prolyl 4-hydroxylase an {alpha}{sub 2}{beta}{sub 2} tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. The authors report here on the isolation of cDNA clones encoding the {alpha}-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3,000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the {beta}-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Gly-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The {alpha}-subunit does not have this C-terminal sequence, and thus one function of the {beta}-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Southern blot analyses of human genomic DNA with a cDNA probe for the {alpha}-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

  3. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  4. Purification, characterization, and directed evolution study of a vitamin D{sub 3} hydroxylase from Pseudonocardia autotrophica

    SciTech Connect

    Fujii, Yoshikazu; Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi; Takeda, Koji; Tamura, Noriko; Arisawa, Akira; Tamura, Tomohiro

    2009-07-24

    Vitamin D{sub 3} (VD{sub 3}) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD{sub 3} into its physiologically active forms, namely, 25(OH)VD{sub 3} or 1{alpha},25(OH){sub 2}VD{sub 3}. In this study, we isolated and characterized Vdh (vitamin D{sub 3} hydroxylase), which hydroxylates VD{sub 3} from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V{sub max} values for VD{sub 3} 25-hydroxylation and 25(OH)VD{sub 3} 1{alpha}-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD{sub 3}. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD{sub 3} 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD{sub 3} into 25(OH)VD{sub 3} was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD{sub 3} by a single cytochrome P450.

  5. The draft genome and transcriptome of Amaranthus hypochondriacus: a C4 dicot producing high-lysine edible pseudo-cereal.

    PubMed

    Sunil, Meeta; Hariharan, Arun K; Nayak, Soumya; Gupta, Saurabh; Nambisan, Suran R; Gupta, Ravi P; Panda, Binay; Choudhary, Bibha; Srinivasan, Subhashini

    2014-12-01

    Grain amaranths, edible C4 dicots, produce pseudo-cereals high in lysine. Lysine being one of the most limiting essential amino acids in cereals and C4 photosynthesis being one of the most sought-after phenotypes in protein-rich legume crops, the genome of one of the grain amaranths is likely to play a critical role in crop research. We have sequenced the genome and transcriptome of Amaranthus hypochondriacus, a diploid (2n = 32) belonging to the order Caryophyllales with an estimated genome size of 466 Mb. Of the 411 linkage single-nucleotide polymorphisms (SNPs) reported for grain amaranths, 355 SNPs (86%) are represented in the scaffolds and 74% of the 8.6 billion bases of the sequenced transcriptome map to the genomic scaffolds. The genome of A. hypochondriacus, codes for at least 24,829 proteins, shares the paleohexaploidy event with species under the superorders Rosids and Asterids, harbours 1 SNP in 1,000 bases, and contains 13.76% of repeat elements. Annotation of all the genes in the lysine biosynthetic pathway using comparative genomics and expression analysis offers insights into the high-lysine phenotype. As the first grain species under Caryophyllales and the first C4 dicot genome reported, the work presented here will be beneficial in improving crops and in expanding our understanding of angiosperm evolution.

  6. The Draft Genome and Transcriptome of Amaranthus hypochondriacus: A C4 Dicot Producing High-Lysine Edible Pseudo-Cereal

    PubMed Central

    Sunil, Meeta; Hariharan, Arun K.; Nayak, Soumya; Gupta, Saurabh; Nambisan, Suran R.; Gupta, Ravi P.; Panda, Binay; Choudhary, Bibha; Srinivasan, Subhashini

    2014-01-01

    Grain amaranths, edible