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Sample records for cmp-n-acetylneuraminic acid hydroxylase

  1. Fixation of the Human-Specific CMP-N-Acetylneuraminic Acid Hydroxylase Pseudogene and Implications of Haplotype Diversity for Human Evolution

    PubMed Central

    Hayakawa, Toshiyuki; Aki, Ikuko; Varki, Ajit; Satta, Yoko; Takahata, Naoyuki

    2006-01-01

    The human CMP-N-acetylneuraminic acid hydroxylase gene (CMAH) suffered deletion of an exon that encodes an active center for the enzyme ∼3.2 million years ago (MYA). We analyzed a 7.3-kb intronic region of 132 CMAH genes to explore the fixation process of this pseudogene and the demographic implication of its haplotype diversity. Fifty-six variable sites were sorted into 18 different haplotypes with significant linkage disequilibrium. Despite the rather low nucleotide diversity, the most recent common ancestor at CMAH dates to 2.9 MYA. This deep genealogy follows shortly after the original exon deletion, indicating that the deletion has fixed in the population, although whether this fixation was facilitated by natural selection remains to be resolved. Remarkable features are exceptionally long persistence of two lineages and the confinement of one lineage in Africa, implying that some African local populations were in relative isolation while others were directly involved in multiple African exoduses of the genus Homo. Importantly, haplotypes found in Eurasia suggest interbreeding between then-contemporaneous human species. Although population structure within Africa complicates the interpretation of phylogeographic information of haplotypes, the data support a single origin of modern humans, but not with complete replacement of archaic inhabitants by modern humans. PMID:16272417

  2. CMP-N-acetylneuraminic acid synthetase interacts with fragile X related protein 1

    PubMed Central

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-01-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. PMID:27357083

  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. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs

    PubMed Central

    Kwon, Deug-Nam; Lee, Kiho; Kang, Man-Jong; Choi, Yun-Jung; Park, Chankyu; Whyte, Jeffrey J.; Brown, Alana N.; Kim, Jae-Hwan; Samuel, Melissa; Mao, Jiude; Park, Kwang-Wook; Murphy, Clifton N.; Prather, Randall S.; Kim, Jin-Hoi

    2013-01-01

    After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation. PMID:23760311

  5. Loss of N-glycolylneuraminic acid in humans: Mechanisms, consequences, and implications for hominid evolution.

    PubMed

    Varki, A

    2001-01-01

    The surface of all mammalian cells is covered with a dense and complex array of sugar chains, which are frequently terminated by members of a family of molecules called sialic acids. One particular sialic acid called N-glycolylneuraminic acid (Neu5Gc) is widely expressed on most mammalian tissues, but is not easily detectable on human cells. In fact, it provokes an immune response in adult humans. The human deficiency of Neu5Gc is explained by an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase, the rate-limiting enzyme in generating Neu5Gc in cells of other mammals. This deficiency also results in an excess of the precursor sialic acid N-acetylneuraminic acid (Neu5Ac) in humans. This mutation appears universal to modern humans, occurred sometime after our last common ancestor with the great apes, and happens to be one of the first known human-great ape genetic differences with an obvious biochemical readout. While the original selection mechanisms and major biological consequences of this human-specific mutation remain uncertain, several interesting clues are currently being pursued. First, there is evidence that the human condition can explain differences in susceptibility or resistance to certain microbial pathogens. Second, the functions of some endogenous receptors for sialic acids in the immune system may be altered by this difference. Third, despite the lack of any obvious alternate pathway for synthesis, Neu5Gc has been reported in human tumors and possibly in human fetal tissues, and traces have even been detected in normal human tissues. One possible explanation is that this represents accumulation of Neu5Gc from dietary sources of animal origin. Finally, a markedly reduced expression of hydroxylase in the brains of other mammals raises the possibility that the human-specific mutation of this enzyme could have played a role in human brain evolution.

  6. Plant fatty acid hydroxylase

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    2000-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  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. 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

  9. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis

    SciTech Connect

    Leon, J.; Shulaev, V.; Yalpani, N.

    1995-10-24

    Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicyclic acid with {sup 18}O{sub 2} suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation of benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[{sup 35}S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes. 21 refs., 6 figs., 1 tab.

  10. 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

  11. 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

  12. Development of Monoclonal Antibodies against CMP-N-Acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 (ST3Gal-I) Recombinant Protein Expressed in E. coli.

    PubMed

    Gupta, Anuj Kumar; Kaur, Parvinder; Patil, Harshada; Kadam, Pallavi; Bhanushali, Paresh B; Chugh, Manoj

    2015-01-01

    Aberrant glycosylation is one of the major hallmarks of cancer with altered gene expression signatures of sialyltransferases. ST3Gal-I, a sialyltransferase, is known to play a crucial role in sialylation of T antigen in bladder cancer and it has reported elevated expression in breast carcinogenesis with increased tumor progression stages. The aim of the current study is to develop new monoclonal antibodies (mAbs) against human ST3Gal-I and evaluate their diagnostic potential. We developed a repertoire of stable hybridoma cell lines producing high-affinity IgG antibodies against recombinant human ST3Gal-I, expressed in E. coli BL21-DE3 strain. In order to demonstrate the diagnostic value of the mAbs, various clones were employed for the immunohistochemistry analysis of ST3Gal-I expression in cancerous tissues. Antibodies generated by 7E51C83A10 clone demonstrated a strong and specific fluorescence staining in breast cancer tissue sections and did not exhibit significant background in fibroadenoma sections. In conclusion, the mAbs raised against recombinant ST3Gal-I recognize cellular ST3Gal-I and represent a promising diagnostic tool for the immunodetection of ST3Gal-I expressing cells. Specific-reactivity of clone 7E51C83A10 mAbs towards ST3Gal-I was also confirmed by immunoblotting. Therefore, our observations warrant evaluation of ST3Gal-I as a potential marker for cancer diagnosis at larger scale. PMID:26783462

  13. Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium.

    PubMed

    Matson, R S; Hare, R S; Fulco, A J

    1977-06-22

    The fatty acid (omega-2) hydroxylase from Bacillus megaterium ATCC 14581 was examined with respect to some general enzymatic properties attributed to an intact complex isolated in a partially purified state. Hydroxylase specific activity was found to increase with increasing protein concentration in a manner consistent with a reversible association of the components in the complex. There was a substantial kinetic lag phase for palmitate hydroxylation which was abolished by a substrate preincubation in the absence of NADPH. The substrate bound and presumably activated the hydroxylase complex without the formation of a substrate-derived intermediated. The oxidation of NADPH and the hydroxylation of palmitate were found to occur in a one to one molar ration, independent of the protein concentration. Finally, a cytochrome P-450 component of the complex was identified on the basis of its CO-binding difference spectrum. It appears, that this cytochrome P-450 component is not identical to P-450 meg of the steroid hydroxylase system of B. megaterium ATCC 13368, since progesterone, an active substrate for the latter, is not hydroxylated by the preparation from B. megaterium ATCC 14581. PMID:18202

  14. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism.

    PubMed

    Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng

    2013-09-01

    The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence.

  15. Cholesterol 7{alpha}-hydroxylase is phosphorylated at multiple amino acids

    SciTech Connect

    Stroup, D. . E-mail: dstroup1@kent.edu; Ramsaran, J.R.

    2005-04-15

    The activity of cholesterol 7{alpha}-hydroxylase (gpCYP7A1), the rate limiting enzyme in bile acid synthesis, has been postulated to be regulated by phosphorylation/dephosphorylation. This study has found that several kinase activators rapidly reduce the amount of bile acid produced by the human hepatoma cell line, HepG2, and that gpCYP7A1 from HepG2 cell extracts eluted in the phosphoprotein fraction of FeIII columns. After incubating the HepG2 cells with radioactive orthophosphate, the band identified as gpCYP7Al on immunoblots was strongly labeled. Recombinant gpCYP7A was expressed as 6x HIS fusion polypeptides and subjected to kinase assays. The locations of phosphorylation were mapped further by screening synthetic peptides against AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase, protein kinase A, and a panel of nine protein kinase C isoforms. AMPK, also known as 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase, phosphorylated cholesterol 7{alpha}-hydroxylase, suggesting a potential mechanism of coordination of cholesterol synthesis and degradation.

  16. WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds.

    PubMed

    Adhikari, Neil D; Bates, Philip D; Browse, John

    2016-05-01

    Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis. PMID:27208047

  17. WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds1[OPEN

    PubMed Central

    Browse, John

    2016-01-01

    Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis. PMID:27208047

  18. Single Turnover Kinetics of Tryptophan Hydroxylase: Evidence for a New Intermediate in the Reaction of the Aromatic Amino Acid Hydroxylases

    PubMed Central

    Pavon, Jorge Alex; Eser, Bekir; Huynh, Michaela T.; Fitzpatrick, Paul F.

    2010-01-01

    Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH·Fe(II), TrpH·Fe(II)·tryptophan, TrpH·Fe(II)·6MePH4·tryptophan, and TrpH·Fe(II)·6MePH4·phenylalanine complexes with O2 were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH·Fe(II) has a value of 104 M−1s−1 irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH·Fe(II)·6MePH4·tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s−1 of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s−1, matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s−1, and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s−1. All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release. PMID:20687613

  19. Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings.

    PubMed

    Kojima, M; Takeuchi, W

    1989-02-01

    A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The enzyme acted only on p-coumaric acid (Km, 3.0 X 10(-5) M), while its specificity for the electron donor was rather broad. The Km value for NADPH (1.5 X 10(-4) M) was much lower than that for L-ascorbic acid (1.0 X 10(-2) M), although the Vmax value was almost the same with both electron donors. The enzyme was potently inhibited by beta-mercaptoethanol (Ki, 3.5 X 10(-6) M) and diethyldithiocarbamate (Ki, 2.3 X 10(-4) M), but was insensitive to p-chloromercuribenzoate. The enzyme was localized in the cell organelles which sedimented between mitochondria and endplasmic reticulum on sucrose density gradient centrifugation. The enzyme activity in the seedling was changed in response to induction by light in a manner suggesting its involvement in biosynthesis of phenolic compounds in mung bean seedlings.

  20. 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...

  1. Induction of benzoic acid 2-hydroxylase in virus-inoculated tobacco

    SciTech Connect

    Leon, J.; Yalpani, N.; Raskin, I.; Lawton, M.A. )

    1993-10-01

    Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco catalyze the 2-hydroxylation of Ba to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h[sup [minus]1] g[sup [minus]1] fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[degrees]C. TMV induction of BA2H activity and Sa accumulation were inhibited when inoculated tobacco plants were incubated for 4 d at 32[degrees]C and then transferred to 24[degrees]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[degrees]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco. 33 refs., 6 figs., 3 tabs.

  2. Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism.

    PubMed Central

    Günzler, V; Hanauske-Abel, H M; Myllylä, R; Mohr, J; Kivirikko, K I

    1987-01-01

    From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit. PMID:3036081

  3. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  4. A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2014-11-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  5. 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.

  6. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

    PubMed Central

    2016-01-01

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

  7. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

    PubMed

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  8. Lipophilic modification enhances anti-colitic properties of rosmarinic acid by potentiating its HIF-prolyl hydroxylases inhibitory activity.

    PubMed

    Jeong, Seongkeun; Park, Huijeong; Hong, Sungchae; Yum, Soohwan; Kim, Wooseong; Jung, Yunjin

    2015-01-15

    Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway. PMID:25483211

  9. Molecular Docking Study of Catecholamines and [4-(Propan-2-yl) Phenyl]Carbamic acid with Tyrosine Hydroxylase

    PubMed Central

    Parveen, Zahida; Nawaz, Muhammad Sulaman; Shakil, Shazi; Greig, Nigel H.; Kamal, Mohammad A.

    2016-01-01

    Parkinson’s disease is a major age-related neurodegenerative disorder. As the classical disease-related motor symptoms are associated with the loss of dopamine-generating cells within the substantia nigra, tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines has become an important target in the development of Parkinson’s disease drug candidates, with the focus to augment TH levels or its activity. By contrast, TH inhibitors are of relevance in the treatment of conditions associated with catecholamine over-production, as occurs in pheochromocytomas. To aid characterizing new drug candidates, a molecular docking study of catecholamines and a novel hypothetical compound [4-(propan-2-yl) phenyl]carbamic acid (PPCA) with TH is described. Docking was performed using Autodock4.2 and results were analyzed using Chimera1.5.2. All the studied ligands were found to bind within a deep narrow groove lined with polar aromatic and acidic residues within TH. Our results corroborated a ‘hexa interacting amino acids unit’ located in this deep narrow groove crucial to the interaction of PPCA and the studied catecholamines with TH, whereby the ‘His361-His336 dyad’ was found to be even more crucial to these binding interactions. PPCA displayed a binding interaction with human TH that was comparable to the original TH substrate, L-tyrosine. Hence PPCA may warrant in vitro and in vivo characterization with TH to assess its potential as a candidate therapeutic. PMID:22583429

  10. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  11. 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

  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. 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.

  14. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, C.; Loo, F. van de

    1998-09-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds. 35 figs.

  15. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, C.; Loo, F. van de

    1997-09-16

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds. 35 figs.

  16. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    1998-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  17. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    1997-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  18. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    2002-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  19. Substrate Specificity and Ligand Interactions of CYP26A1, the Human Liver Retinoic Acid Hydroxylase

    PubMed Central

    Thatcher, Jayne E.; Buttrick, Brian; Shaffer, Scott A.; Shimshoni, Jakob A.; Goodlett, David R.; Nelson, Wendel L.

    2011-01-01

    All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC50 values for 10 inhibitors were determined. The imidazole- and triazole-containing inhibitors [S-(R*,R*)]-N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine (R116010) and (R)-N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine (R115866) were the most potent inhibitors of CYP26A1 with IC50 values of 4.3 and 5.1 nM, respectively. Liarozole and ketoconazole were significantly less potent with IC50 values of 2100 and 550 nM, respectively. The retinoic acid receptor (RAR) γ agonist CD1530 was as potent an inhibitor of CYP26A1 as ketoconazole with an IC50 of 530 nM, whereas the RARα and RARβ agonists tested did not significantly inhibit CYP26A1. The pan-RAR agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC50 values of 3.7, 4.2, and 8.6 μM, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear

  20. Sialylation Facilitates the Maturation of Mammalian Sperm and Affects Its Survival in Female Uterus.

    PubMed

    Ma, Xue; Pan, Qian; Feng, Ying; Choudhury, Biswa P; Ma, Qianhong; Gagneux, Pascal; Ma, Fang

    2016-06-01

    Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20-50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)-/- transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract.

  1. Incorporation of N-acetylneuraminic acid into Haemophilus somnus lipooligosaccharide (LOS): enhancement of resistance to serum and reduction of LOS antibody binding.

    PubMed

    Inzana, Thomas J; Glindemann, Gretchen; Cox, Andrew D; Wakarchuk, Warren; Howard, Michael D

    2002-09-01

    Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by

  2. Incorporation of N-acetylneuraminic acid into Haemophilus somnus lipooligosaccharide (LOS): enhancement of resistance to serum and reduction of LOS antibody binding.

    PubMed

    Inzana, Thomas J; Glindemann, Gretchen; Cox, Andrew D; Wakarchuk, Warren; Howard, Michael D

    2002-09-01

    Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by

  3. Structural and Functional Characteristics of Oxysterol 7α-Hydroxylase with Amino-Acid Substitution R486C and Their Relation to the Appearance of Neurodegenerative Diseases

    NASA Astrophysics Data System (ADS)

    Dichenko, Ya. V.; Yantsevich, A. V.; Usanov, S. A.

    2015-03-01

    The influence of the amino-acid substitution Arg486Cys on the conformational stability of recombinant cytochrome P450 7B1 (CYP7B1, oxysterol 7α-hydroxylase) was studied. The single base change was shown to decrease the free energy of the transition of the heme-protein from its native state to a denatured one, which pointed to a lower thermodynamic stability for the mutant form of the enzyme. This could be the cause of the metabolic disruption of neurosteroids and, as a consequence, the appearance of neurodegenerative diseases.

  4. Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus.

    PubMed

    González-Flores, Oscar; Gómora-Arrati, Porfirio; García-Juárez, Marcos; Miranda-Martínez, Alfredo; Armengual-Villegas, Alejandra; Camacho-Arroyo, Ignacio; Guerra-Araiza, Christian

    2011-10-01

    Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A+PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A+B antisense (PR A+B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A+B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A+B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the

  5. 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).

  6. Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

    PubMed Central

    Princen, H M; Huijsmans, C M; Kuipers, F; Vonk, R J; Kempen, H J

    1986-01-01

    In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy. PMID:3760182

  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. 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

  9. Dietary fish oil up-regulates cholesterol 7alpha-hydroxylase mRNA in mouse liver leading to an increase in bile acid and cholesterol excretion.

    PubMed

    Bérard, Annie M; Dumon, Marie-France; Darmon, Michel

    2004-02-13

    To investigate the molecular events controlling reverse cholesterol transport, we compared gene expression of normal mouse liver to that of mice fed a long chain (LC) omega-3 fatty acid-enriched diet. Using cDNA microarrays, we assessed expression levels of 1176 genes, and we found that D-site binding protein (DBP) was three-fold increased in mice on a LC omega-3 fatty acid-rich diet compared to controls. DBP is known to increase transcriptional level of cholesterol 7alpha-hydroxylase (C7alpha), the rate-limiting enzyme for bile acid production and cholesterol excretion, and we found that C7alpha mRNA was also up-regulated by LC omega-3 fatty acids. Moreover, liver X receptor-alpha, another transcription factor up-regulating C7alpha, was three- to four-fold increased in liver of treated mice. On the other hand, we demonstrated that bile acid and cholesterol excretion were two-fold increased. These results show that LC omega-3 fatty acids control cholesterol metabolism in mice at a new endpoint.

  10. Increased levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurons after rapid eye movement sleep deprivation in rats.

    PubMed

    Majumdar, S; Mallick, B N

    2003-03-01

    Norepinephrine, acetylcholine and GABA levels alter during rapid eye movement (REM) sleep and its deprivation. Increased synthesis of those neurotransmitters is necessary for their sustained release. Hence, in this study, the concentrations of tyrosine hydroxylase (TH), choline acetyl transferase (ChAT) and glutamic acid decarboxylase (GAD), the enzymes responsible for their synthesis, were immunohistochemically estimated within the neurons in locus coeruleus, laterodorsal tegmentum and pedunculopontine tegmentum and medial preoptic area in REM sleep deprived and control rats. It was observed that as compared to controls, deprivation increased TH and GAD significantly in the locus coeruleus only, while in other areas, they remained unchanged. The findings help explaining the mechanism of increase in neurotransmitter levels in the brain after REM sleep deprivation and their significance has been discussed.

  11. 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.

  12. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  13. Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

    PubMed

    Pu, Gaobin; Wang, Peng; Zhou, Bingqian; Liu, Zhenhua; Xiang, Fengning

    2013-01-01

    Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.

  14. Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

    PubMed

    Pu, Gaobin; Wang, Peng; Zhou, Bingqian; Liu, Zhenhua; Xiang, Fengning

    2013-01-01

    Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance. PMID:23832359

  15. Chlorophenol hydroxylase activity encoded by TfdB from 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Bradyrhizobium sp. strain RD5-C2.

    PubMed

    Huong, Nguyen L; Itoh, Kazuhito; Miyamoto, Masao; Suyama, Kousuke; Yamamoto, Hiroki

    2007-07-01

    The tfdB gene encoding chlorophenol hydroxylase and its homolog were found in 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading strain RD5-C2, which belongs to Bradyrhizobium sp. of alpha-Proteobacteria. The nucleotide and deduced amino acid sequence identities of the two genes, designated tfdBa and tfdBb, were 60% and 57% respectively. Their nucleotide sequences most closely matched those of previously reported tfdB, which consisted of those from 2,4-D-degrading beta- and gamma-Proteobacteria and Sphingomonas sp. in alpha-Proteobacteria, with 61-67% identity. The TfdBa expressed in Escherichia coli showed the highest activity for 2,4-dichlorophenol but a narrower range of activity for the other chlorophenols than previously reported TfdBs. In the case of TfdBb, however, no observable activity for any chlorophenols or phenol was detected, although production of a protein with an appropriate molecular size was observed. Based on codon usage patterns and the GC content of the genes, it probable that the tfdBa genes in the 2,4-D-degrading Bradyrhizobium sp. were obtained through horizontal gene transfer.

  16. Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

    PubMed Central

    Bergfeld, Anne K.; Pearce, Oliver M. T.; Diaz, Sandra L.; Pham, Tho; Varki, Ajit

    2012-01-01

    The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc. PMID:22692205

  17. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius

    PubMed Central

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-01-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance. PMID:24309561

  18. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

    PubMed

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-11-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

  19. Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses.

    PubMed

    Matsumura, Ryuji; Hirakawa, Jotaro; Sato, Kaori; Ikeda, Toshiaki; Nagai, Motoe; Fukuda, Minoru; Imai, Yasuyuki; Kawashima, Hiroto

    2015-06-12

    Sialyl Lewis X (sLe(x)) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLe(x) monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLe(x) mAbs, termed F1 and F2, which react well with both human and mouse sLe(x), by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLe(x)-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLe(x) as well as 6-sulfo-sLe(x), a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLe(x) mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLe(x) antigen under physiological and pathological conditions.

  20. Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1.

    PubMed

    Foti, Robert S; Isoherranen, Nina; Zelter, Alex; Dickmann, Leslie J; Buttrick, Brian R; Diaz, Philippe; Douguet, Dominique

    2016-05-01

    Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics. PMID:26937021

  1. 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

  2. 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.

  3. 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

  4. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  5. 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.

  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. Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.

    PubMed

    Kang, Kee Ryeon; Kim, Yeon Sook; Wolff, Edith C; Park, Myung Hee

    2007-03-16

    Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site. PMID:17213197

  8. 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.

  9. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    SciTech Connect

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  10. Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation.

    PubMed

    Gravot, Antoine; Larbat, Romain; Hehn, Alain; Lièvre, Karine; Gontier, Eric; Goergen, Jean Louis; Bourgaud, Frédéric

    2004-02-01

    Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.

  11. Development and Characterization of Novel and Selective Inhibitors of Cytochrome P450 CYP26A1, the Human Liver Retinoic Acid Hydroxylase.

    PubMed

    Diaz, Philippe; Huang, Weize; Keyari, Charles M; Buttrick, Brian; Price, Lauren; Guilloteau, Nicolas; Tripathy, Sasmita; Sperandio, Vanessa G; Fronczek, Frank R; Astruc-Diaz, Fanny; Isoherranen, Nina

    2016-03-24

    Cytochrome P450 CYP26 enzymes are responsible for all-trans-retinoic acid (atRA) clearance. Inhibition of CYP26 enzymes will increase endogenous atRA concentrations and is an attractive therapeutic target. However, the selectivity and potency of the existing atRA metabolism inhibitors toward CYP26A1 and CYP26B1 is unknown, and no selective CYP26A1 or CYP26B1 inhibitors have been developed. Here the synthesis and potent inhibitory activity of the first CYP26A1 selective inhibitors is reported. A series of nonazole CYP26A1 selective inhibitors was identified with low nM potency. The lead compound 3-{4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl] phenyl}4-propanoic acid (24) had 43-fold selectivity toward CYP26A1 with an IC50 of 340 nM. Compound 24 and its two structural analogues also inhibited atRA metabolism in HepG2 cells, resulting in increased potency of atRA toward RAR activation. The identified compounds have potential to become novel treatments aiming to elevate endogenous atRA concentrations and may be useful as cotreatment with atRA to combat therapy resistance.

  12. 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.

  13. Nitric Oxide and Interleukin-1β Stimulate the Proteasome-Independent Degradation of the Retinoic Acid Hydroxylase CYP2C22 in Primary Rat Hepatocytes

    PubMed Central

    Lee, Choon-myung; Lee, Bang-sub; Arnold, Samuel L.; Isoherranen, Nina

    2014-01-01

    CYP2C22 was recently described as a retinoic acid–metabolizing cytochrome P450 enzyme whose transcription is induced by all-trans-retinoic acid (atRA) in hepatoma cells (Qian L, Zolfaghari R, and Ross AC (2010) J Lipid Res 51:1781–1792). We identified CYP2C22 as a putative nitric oxide (NO)–regulated protein in a proteomic screen and raised specific polyclonal antibodies to CYP2C22 to study its protein expression. We found that CYP2C22 is a liver-specific protein that was not significantly induced by activators of the pregnane X receptor, constitutive androstane receptor, or peroxisome proliferator-activated receptor-α, but was downregulated to <25% of control by the aryl hydrocarbon receptor agonist β-naphthoflavone in cultured rat hepatocytes. CYP2C22 protein and its mRNA both were induced by atRA in hepatocytes, with EC50 of 100–300 nM, whereas the maximal extent of mRNA induction was twice that of the protein. CYP2C22 protein, but not its mRNA, was rapidly downregulated in hepatocytes by interleukin-1 (IL-1) or NO-donating compounds, and the downregulation by IL-1 was blocked by inhibition of NO synthases. The NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate reduced the half-life of CYP2C22 from 8.7 to 3.4 hours in the presence of cycloheximide, demonstrating that NO-dependent downregulation is due to stimulated proteolysis. No intermediate degradation products were detected. However, this degradation was insensitive to inhibitors of calpains or the canonical proteasomal or lysosomal pathways, indicating that NO-dependent degradation of CYP2C22 proceeds via a novel pathway. PMID:24144795

  14. 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

  15. 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

  16. Production of hydroxylated fatty acids in genetically modified plants

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

    2011-08-23

    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.

  17. Production of hydroxylated fatty acids in genetically modified plants

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

    2005-08-30

    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.

  18. 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. ...

  19. Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae

    PubMed Central

    Ambrose, Karen V.; Tian, Zipeng; Wang, Yifei; Smith, Jordan; Zylstra, Gerben; Huang, Bingru; Belanger, Faith C.

    2015-01-01

    Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte. PMID:26055188

  20. CYP4 Enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities

    PubMed Central

    Edson, Katheryne Z.; Rettie, Allan E.

    2014-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20-hydroxyeicosatetraenoic acid or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases. PMID:23688133

  1. CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities.

    PubMed

    Edson, Katheryne Z; Rettie, Allan E

    2013-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20- hydroxyeicosatetraenoic acid, or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases.

  2. 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

  3. Production of hydroxylated fatty acids in genetically modified plants

    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.

  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. 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 ...

  7. Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11 beta-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production.

    PubMed

    Tremblay, A; Parker, K L; Lehoux, J G

    1992-06-01

    Increasing evidence indicates that the adrenal cortex of most mammalian species expresses distinct forms of cytochrome P-450(11 beta), a steroidogenic enzyme that catalyses the terminal steps in the biosynthesis of both glucocorticoids and mineralocorticoids. In the human, mouse, and rat, two genes have been isolated, designated CYP11B1 and CYP11B2. The product of CYP11B2 (aldosterone synthase) is required for the successive 11 beta-, 18-hydroxylations and 18-oxidation of deoxycorticosterone that lead to the production of aldosterone in the zona glomerulosa. In contrast, the product of CYP11B1 (11 beta-hydroxylase) mediates only the 11 beta-hydroxylation of deoxycorticosterone and 11-deoxycortisol. The recent identification of these two P-450(11 beta) isozymes mandates further analysis of their expression in different zones of the adrenal cortex, both under basal conditions and in response to conditions known to alter mineralocorticoid biosynthesis. To evaluate the expression of the two isozymes in different adrenocortical zones, we performed Northern blotting analyses with specific oligonucleotide probes that discriminated between the two forms of rat P-450(11 beta). The transcripts detected by the two probes were of similar size (2.7 kilobase), but differed in their zonal distribution: aldosterone synthase P-450 messenger RNA (mRNA) was detected only in zona glomerulosa, whereas 11 beta-hydroxylase P-450 was expressed in both zona fasciculata-reticularis and zona glomerulosa. Next, we analyzed the response of these two genes to various physiological and pharmacological interventions known to affect aldosterone biosynthesis. High potassium or low sodium diet given to rats for 1 week increased aldosterone synthase P-450 mRNA levels by approximately 5- and 6-fold, respectively. These increases, moreover, were significantly attenuated by treatment with captopril, an inhibitor of angiotensin-converting enzyme. In contrast, neither dietary manipulation significantly

  8. 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.

  9. Allelic variants from Dahlia variabilis encode flavonoid 3'-hydroxylases with functional differences in chalcone 3-hydroxylase activity.

    PubMed

    Schlangen, Karin; Miosic, Silvija; Halbwirth, Heidi

    2010-02-01

    In the petals of Dahlia variabilis, hydroxylation of chalcones at position 3 can be detected, except the well-known flavonoid 3'-hydroxylation. Although the reaction is well characterized at the enzymatic level, it remained unclear whether it is catalyzed by a flavonoid 3'-hydroxylase (F3'H, EC1.14.13.21, CYP75B) with broad substrate specificity. Two novel allelic variants of F3'H were cloned from D. variabilis, which differ only in three amino acids within their 508 residues. The corresponding recombinant enzymes show significant differences in their chalcone 3-hydroxylase (CH3H) activity. A substitution of alanine at position 425 with valine enables CH3H activity, whereas the reciprocal substitution leads to a loss of CH3H activity. Interaction of the valine at position 425 with not yet identified structural properties seems to be decisive for chalcone acceptance. This is the first identification of an F3'H which is able to catalyze chalcone 3-hydroxylation to a physiologically relevant extent from any plant species. PMID:19931222

  10. Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.

    PubMed

    Nowak, Elizabeth C; de Vries, Victor C; Wasiuk, Anna; Ahonen, Cory; Bennett, Kathryn A; Le Mercier, Isabelle; Ha, Dae-Gon; Noelle, Randolph J

    2012-10-22

    Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity. PMID:23008335

  11. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    SciTech Connect

    Van De Loo, F.J.; Broun, P.; Turner, S.; Somerville, C.

    1995-07-18

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major

    PubMed Central

    Lye, Lon-Fye; Kang, Song Ok; Nosanchuk, Joshua D.; Casadevall, Arturo; Beverley, Stephen M.

    2010-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H4B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H4B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H4B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah− null mutant and an episomal complemented overexpressing derivative (pah−/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah− parasites showed reactivity with an antibody to melanin when grown with L-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah− showed an increased sensitivity to Tyr deprivation, while the pah−/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah− showed no alterations in H4B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin. PMID:20887755

  17. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism. PMID:9367532

  18. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  19. Red Clover Coumarate 3'-Hydroxylase (CYP98A44) is Capable of Hydroxylating P-Coumaroyl-Shikimate but not P-Coumaroyl-Malate: Implications for the Biosynthesis of Phaselic Acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense) leaves accumulate several µmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicag...

  20. Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

    PubMed

    Lewis, E J; Allison, S; Fader, D; Claflin, V; Baizer, L

    1990-01-15

    We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.

  1. 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

  2. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    PubMed

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Genetics Home Reference: fatty acid hydroxylase-associated neurodegeneration

    MedlinePlus

    ... nerves ) and difficulties with the muscles that control eye movement. Affected individuals may have a loss of sharp ... look in the same direction (strabismus), rapid involuntary eye movements (nystagmus), or difficulty moving the eyes intentionally (supranuclear ...

  10. L-leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst.

    PubMed

    Hibi, Makoto; Kawashima, Takashi; Sokolov, Pavel M; Smirnov, Sergey V; Kodera, Tomohiro; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-03-01

    L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

  11. Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera.

    PubMed

    Wetzler, L M; Barry, K; Blake, M S; Gotschlich, E C

    1992-01-01

    Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study.

  12. 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

  13. Structural insights into substrate specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    DOE PAGES

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; Zhang, Hua; Yuan, Qipeng; Wang, Bi -Cheng; Rose, John; Yan, Yajun

    2015-05-20

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures,more » we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

  14. 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.

  15. 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

  16. 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.

  17. 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…

  18. 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…

  19. 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

  20. [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.

  1. 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

  2. Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase.

    PubMed

    Abe, I; Kashiwagi, K; Noguchi, H

    2000-10-20

    Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.

  3. 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.

  4. 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

  5. Structural Characterization of the Catalytic Sites of Mononuclear Nonheme Fe Hydroxylases Using ²H-ESEEM.

    PubMed

    McCracken, John

    2015-01-01

    Aromatic amino acid hydroxylases are members of a larger group of enzymes that use a mononuclear nonheme Fe center to catalyze a variety of thermodynamically challenging reactions in which O2 is used in the oxidative transformation of substrates. The hydroxylase enzymes are catalytically active in the ferrous oxidation state and are high-spin. To render the catalytic site EPR-active, we have used nitric oxide (NO) as a surrogate for substrate O2 to form an S=3/2 paramagnetic center. While the continuous-wave (cw)-EPR spectra of NO-enzyme adducts are rather generic, they provide electron spin echo envelope modulation (ESEEM) data that are rich with structural information derived from ligand hyperfine couplings. This chapter will focus on (2)H-ESEEM spectroscopy, an approach that we have taken for assigning these spectra and harvesting the unique information on Fe(II) coordination chemistry that they provide. While these spectroscopic measurements are routine, an emphasis will be placed on the analysis of cw-EPR and (2)H-ESEEM data using an unconstrained nonlinear optimization approach. These analysis methods are based on simple custom "scripts" that run in the MATLAB environment and that use EasySpin, a public-domain EPR simulation package, as their calculation engine. The examples provided here use a strategy that can be adapted for the treatment of most EPR measurements.

  6. Structural Characterization of the Catalytic Sites of Mononuclear Nonheme Fe Hydroxylases Using ²H-ESEEM.

    PubMed

    McCracken, John

    2015-01-01

    Aromatic amino acid hydroxylases are members of a larger group of enzymes that use a mononuclear nonheme Fe center to catalyze a variety of thermodynamically challenging reactions in which O2 is used in the oxidative transformation of substrates. The hydroxylase enzymes are catalytically active in the ferrous oxidation state and are high-spin. To render the catalytic site EPR-active, we have used nitric oxide (NO) as a surrogate for substrate O2 to form an S=3/2 paramagnetic center. While the continuous-wave (cw)-EPR spectra of NO-enzyme adducts are rather generic, they provide electron spin echo envelope modulation (ESEEM) data that are rich with structural information derived from ligand hyperfine couplings. This chapter will focus on (2)H-ESEEM spectroscopy, an approach that we have taken for assigning these spectra and harvesting the unique information on Fe(II) coordination chemistry that they provide. While these spectroscopic measurements are routine, an emphasis will be placed on the analysis of cw-EPR and (2)H-ESEEM data using an unconstrained nonlinear optimization approach. These analysis methods are based on simple custom "scripts" that run in the MATLAB environment and that use EasySpin, a public-domain EPR simulation package, as their calculation engine. The examples provided here use a strategy that can be adapted for the treatment of most EPR measurements. PMID:26478489

  7. 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

  8. Cytochrome P450 ω-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in non-small cell lung cancer

    PubMed Central

    Yu, Wei; Chen, Li; Yang, Yu-Qing; Falck, John R.; Guo, Austin M.; Li, Ying

    2013-01-01

    Purpose Cytochrome P450 (CYP) ω-hydroxylase, mainly consisting of CYP4A and CYP4F, converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) that induces angiogenic responses in vivo and in vitro. The present study examined the role of CYP ω-hydroxylase in angiogenesis and metastasis of human non-small cell lung cancer (NSCLC). Methods The effect of WIT003, a stable 20-HETE analog, on invasion was evaluated using a modified Boyden chamber in three NSCLC cell lines. A549 cells were transfected with CYP4A11 expression vector or exposed to CYP ω-hydroxylase inhibitor (HET0016) or 20-HETE antagonist (WIT002), and then ω-hydroxylation activity toward arachidonic acid and the levels of matrix metalloproteinases (MMPs) and VEGF were detected. The in vivo effects of CYP ω-hydroxylase were tested in established tumor xenografts and an experimental metastasis model in athymic mice. Results Addition of WIT003 or overexpression of CYP4A11 with an associated increase in 20-HETE production significantly induced invasion and expression of VEGF and MMP-9. Treatment of A549 cells with HET0016 or WIT002 inhibited invasion with reduction in VEGF and MMP-9. The PI3 K or ERK inhibitors also attenuated expression of VEGF and MMP-9. Compared with control, CYP4A11 transfection significantly increased tumor weight, microvessel density (MVD), and lung metastasis by 2.5-fold, 2-fold, and 3-fold, respectively. In contrast, WIT002 or HET0016 decreased tumor volume, MVD, and spontaneous pulmonary metastasis occurrences. Conclusion CYP ω-hydroxylase promotes tumor angiogenesis and metastasis by upregulation of VEGF and MMP-9 via PI3 K and ERK1/2 signaling in human NSCLC cells. PMID:21120482

  9. Targeted delivery of the hydroxylase inhibitor DMOG provides enhanced efficacy with reduced systemic exposure in a murine model of colitis.

    PubMed

    Tambuwala, Murtaza M; Manresa, Mario C; Cummins, Eoin P; Aversa, Vincenzo; Coulter, Ivan S; Taylor, Cormac T

    2015-11-10

    Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

  10. Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1

    PubMed Central

    Zhu, XiangDong; Pang, CuiPing; Cao, Yuting; Fan, Dan

    2016-01-01

    The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0. PMID:27340662

  11. Cholesterol 7 alpha-hydroxylase activity is increased by dietary modification with psyllium hydrocolloid, pectin, cholesterol and cholestyramine in rats.

    PubMed

    Matheson, H B; Colón, I S; Story, J A

    1995-03-01

    Sources of dietary fiber known to alter cholesterol metabolism and/or bile acid pool size were fed to rats, and activity of the rate-limiting step in bile acid synthesis, cholesterol 7 alpha-hydroxylase, was measured. In the first experiment, semipurified diets containing 5% cellulose, psyllium hydrocolloid, pectin or oat bran as dietary fiber sources or 2% cholestyramine were fed to groups of 10 male Wistar rats for 4 wk. In the second experiment, groups of six rats were fed diets containing 5% cellulose, rice bran, oat bran or psyllium with and without 0.25% cholesterol. In the first experiment, the activity of cholesterol 7 alpha-hydroxylase (pmol.min-1.mg protein-1) was highest in the cholestyramine-treated group (95.6 +/- 3.6), followed by groups fed psyllium (35.5 +/- 3.5) or pectin (36.0 +/- 4.5), which exhibited more than twice the enzyme activity of groups fed cellulose (16.9 +/- 1.9) or oat bran (12.3 +/- 2.0). In the second experiment, feeding cholesterol resulted in significantly higher enzyme activity when cellulose (65%), oat bran (118%) and rice bran (60%) were fed, but no difference in activity was observed when cholesterol was added to the psyllium-containing diet. Higher activity of cholesterol 7 alpha-hydroxylase when pectin or psyllium rather than cellulose was fed may explain the almost twofold higher bile acid pool sizes previously reported in response to feeding either of these fibers. These data support the hypothesis that the hypocholesterolemic effect of soluble fibers is modulated through increased synthesis and therefore pool size of bile acids.

  12. 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.

  13. 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

  14. 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.

  15. Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

    PubMed

    Foulon, Veerle; Asselberghs, Stanny; Geens, Wendy; Mannaerts, Guy P; Casteels, Minne; Van Veldhoven, Paul P

    2003-12-01

    Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.

  16. 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.

  17. 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.

  18. Conformational analysis of the N-terminal sequence Met1 Val60 of the tyrosine hydroxylase

    NASA Astrophysics Data System (ADS)

    Alieva, Irada N.; Mustafayeva, Narmina N.; Gojayev, Niftali M.

    2006-03-01

    Molecular mechanics method and molecular dynamics (MD) simulation techniques are used to study the behavior and the effect of the amino acids substitution on structure and molecular dynamics of the specific portion of Met1-Val60 amino acid residues from N-terminal regulatory domain of the tyrosine hydroxylase (TH) and its mutants in which the positively charged arginine residues at positions 37 and 38 were replaced by electrically neutral Gly and negatively charged Glu, and serine residue at position 40 was replaced by Ala or Asp residue. Our study allowed us to make the following conclusions: (i) the higher conformational flexibility of the Met1-Arg16 sequence is revealed in comparision to other part of the N-terminus; (ii) the stretch of amino acid residues Met30-Ser40 within the N-terminus forms β-turn so that two α-helices (residues 16-29 and residues 41-60) are paralel one another; (ii) the significant differences that are observed for the Arg37→Gly37, Arg37-Arg38→Glu37-Glu38 mutant segments indicates that the positive charge of the Arg37 and Arg38 residues is one of the main factor that maintains the characteristic of the turn; (ii) no major conformational changes are observed between Ser40→Ala40, and Ser40→Asp40 mutant segments.

  19. Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

    PubMed Central

    2015-01-01

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  20. 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.

  1. 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.

  2. The Cytochrome P450 CYP86A22 Is a Fatty Acyl-CoA ω-Hydroxylase Essential for Estolide Synthesis in the Stigma of Petunia hybrida*

    PubMed Central

    Han, Jixiang; Clement, Joel M.; Li, Jia; King, Andrew; Ng, Shirley; Jaworski, Jan G.

    2010-01-01

    The stigmatic estolide is a lipid-based polyester constituting the major component of exudate in solanaceous plants. Although the exudate is believed to play important roles in the pollination process, the biosynthetic pathway of stigmatic estolide, including genes encoding the key enzymes, remains unknown. Here we report the cloning and characterization of the cytochrome P450 gene CYP86A22, which encodes a fatty acyl-CoA ω-hydroxylase involved in estolide biosynthesis in the stigma of Petunia hybrida. A CYP86A22 cDNA was isolated from a developing stigma cDNA library, and the corresponding gene was shown to express predominantly in the developing stigma. Among six P450 genes isolated from this library, only CYP86A22 was implicated in ω-hydroxylation following RNA interference (RNAi)-mediated suppression. Unlike wild-type plants in which ω-hydroxy fatty acids (mainly in the form of 18-hydroxy oleic acid and 18-hydroxy linoleic acid) compose 96% of total stigma fatty acids, the ω-hydroxy fatty acids were essentially absent in the stigmas from 18 of 46 CYP86A22-RNAi transgenic plants and had varying levels of suppression in the remaining 28 plants. Furthermore, lipids in the 18 CYP86A22-RNAi stigmas were predominantly triacylglycerols and diacylglycerols instead of the estolides, which characterize the wild-type stigma. Analyses of recombinant CYP86A22 conclusively demonstrated that this P450 is a ω-hydroxylase with a substrate preference for both saturated and unsaturated acyl-CoAs rather than free fatty acids. We conclude that the cytochrome P450 enzyme CYP86A22 is the key fatty acyl-CoA ω-hydroxylase essential for the production of ω-hydroxy fatty acids and the biosynthesis of triacylglycerol-/diacylglycerol-based estolide polyesters in the petunia stigma. PMID:19940120

  3. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues.

    PubMed

    Britsch, L; Dedio, J; Saedler, H; Forkmann, G

    1993-10-15

    A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

  4. Structural insights into substrate specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    SciTech Connect

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; Zhang, Hua; Yuan, Qipeng; Wang, Bi -Cheng; Rose, John; Yan, Yajun

    2015-05-20

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.

  5. Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis

    PubMed Central

    Chung, Hak Suk; Yang, Eun Gyeong; Hwang, Dohyeon; Lee, Ji Eun; Guan, Ziqiang; Raetz, Christian R.H.

    2014-01-01

    The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains D-glycero-D-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe2+/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVA in vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. PMID:25204504

  6. Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

    PubMed

    Chung, Hak Suk; Yang, Eun Gyeong; Hwang, Dohyeon; Lee, Ji Eun; Guan, Ziqiang; Raetz, Christian R H

    2014-09-26

    The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. PMID:25204504

  7. Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

    PubMed

    Yew, Nelson S; Dufour, Emmanuelle; Przybylska, Malgorzata; Putelat, Julie; Crawley, Cristin; Foster, Meta; Gentry, Sarah; Reczek, David; Kloss, Alla; Meyzaud, Aurélien; Horand, Françoise; Cheng, Seng H; Godfrin, Yann

    2013-08-01

    Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

  8. 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

  9. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    PubMed

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments.

  10. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  11. Influence of the thyroid hormone status on tyrosine hydroxylase in central and peripheral catecholaminergic structures.

    PubMed

    Claustre, J; Balende, C; Pujol, J F

    1996-03-01

    We investigated the effect of hyper- and hypothyroidism on tyrosine hydroxylase protein concentration in the locus coeruleus (divided into anterior and posterior parts), the substantia nigra and the adrenals of adult rats. Rats were made hypothyroid with propylthiouracile (PTU, 0.02% in drinking water for 21 days) or hyperthyroid by thyroxine injection (100 or 250 micrograms/kg/day), for 3 or 17 days. PTU treatment resulted in statistically significant decrease of tyrosine hydroxylase in the anterior locus coeruleus (-13%) and the adrenals (-14%). After thyroxine treatment, in the anterior locus coeruleus, tyrosine hydroxylase was significantly higher (2 way ANOVA) after the 3 day treatment than after the 17 day treatment: tyrosine hydroxylase showed a trend to increase the 3 day treatment (+20% with the 250 micrograms/kg dose) and to decrease after the 17 day treatment (-15% with the 250 micrograms/kg dose). In the adrenals, tyrosine hydroxylase was increased by the 3 day treatment (+42% after the 250 micrograms/kg dose), but this increase was not observed after 17 days of treatment. Tyrosine hydroxylase was not altered in the posterior locus coeruleus and the substantia nigra, whatever the treatment. Together, our results support the hypothesis that in the anterior locus coeruleus and in the adrenals tyrosine hydroxylase level is positively modulated by thyroid hormones. After long-term treatment (17 days) this effect is not observed. PMID:8813245

  12. Organ specificity of aryl hydrocarbon hydroxylase induction by cigarette smoke

    SciTech Connect

    Yoshikawa, M.; Arashidani, K.; Kawamoto, T.; Kodama, Y. )

    1990-06-01

    Biotransformation of many chemicals found in cigarette smoke, such as PAHs and nitrosamines, is generally considered essential for the mutagenic, carcinogenic effects of these xenobiotics. In fact, the genotic action of these premutagens or precarcinogens is dependent on metabolic activation catalyzed by microsomal monooxygenases. The first enzymatic reaction of the PAHs metabolic pathway is catalyzed by a cytochrome P-450-dependent monooxygenase, the aryl hydrocarbon hydroxylase (AHH). AHH leads to the formation of reactive arene oxides, which are further metabolized by enzymatic and non-enzymatic reaction into many metabolites. AHH induction in laboratory animals exposed to cigarette smoke has also been reported, and the data show that this response is highly dependent on species and tissues. Exposure of small laboratory animals to cigarette smoke generally induces AHH in the kidney and lung, while the effect of cigarette smoke on the hepatic AHH activity appears variable.

  13. Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

    PubMed

    Fratzl-Zelman, Nadja; Bächinger, Hans-Peter; Vranka, Janice A; Roschger, Paul; Klaushofer, Klaus; Rauch, Frank

    2016-04-01

    Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix. PMID:26808442

  14. Bioavailable affinity label for collagen prolyl 4-hydroxylase

    PubMed Central

    Vasta, James D.; Higgin, Joshua J.; Kersteen, Elizabeth A.

    2013-01-01

    Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the non-heme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases. PMID:23702396

  15. Lysyl Hydroxylase 3-mediated Glucosylation in Type I Collagen

    PubMed Central

    Sricholpech, Marnisa; Perdivara, Irina; Yokoyama, Megumi; Nagaoka, Hideaki; Terajima, Masahiko; Tomer, Kenneth B.; Yamauchi, Mitsuo

    2012-01-01

    Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization. PMID:22573318

  16. Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; de Souza, Robson F.; Aravind, L.

    2010-01-01

    Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth’s history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded β-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily. PMID:20423905

  17. 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

  18. Evaluation of aspirin metabolites as inhibitors of hypoxia-inducible factor hydroxylases.

    PubMed

    Lienard, Benoit M; Conejo-García, Ana; Stolze, Ineke; Loenarz, Christoph; Oldham, Neil J; Ratcliffe, Peter J; Schofield, Christopher J

    2008-12-21

    Known and potential aspirin metabolites were evaluated as inhibitors of oxygen-sensing hypoxia-inducible transcription factor (HIF) hydroxylases; some of the metabolites were found to stabilise HIF-alpha in cells. PMID:19048166

  19. 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.

  20. Genetics Home Reference: congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency

    MedlinePlus

    ... Intersex Society of North America MalaCards: adrenal hyperplasia, congenital, due to 11-beta-hydroxylase deficiency March of Dimes: Genital and Urinary Tract Defects Merck Manual Consumer Version: The Body's Control ...

  1. 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.

  2. Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis

    SciTech Connect

    Ruegger, M.; Meyer, K.; Cusumano, J.C.; Chapple, C.

    1999-01-01

    Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapolymalate, and sinapolycholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here the authors show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3{prime} of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapolycholine accumulation in embryos.

  3. Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

    This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

  4. 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

  5. Overexpression of Tyrosine hydroxylase and Dopa decarboxylase associated with pupal melanization in Spodoptera exigua

    PubMed Central

    Liu, Sisi; Wang, Mo; Li, Xianchun

    2015-01-01

    Melanism has been found in a wide range of species, but the molecular mechanisms involved remain largely elusive. In this study, we studied the molecular mechanisms of the pupal melanism in Spodoptera exigua. The full length cDNA sequences of tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), two key enzymes in the biosynthesis pathway of melanin, were cloned, and their temporal expression patterns in the integument were compared during the larval-pupal metamorphosis process of the S. exigua wild type (SEW) and melanic mutant (SEM) strains. No amino acid change in the protein sequence of TH and DDC was found between the two strains. Both DDC and TH were significantly over-expressed in the integument of the SEM strain at late-prepupa and 0 h pupa, respectively, compared with those of the SEW strain. Feeding 5th instar larvae of SEM with diets incorporated with 1 mg/g of the DDC inhibitor L-α-Methyl-DOPA and 0.75 mg/g of the TH inhibitor 3-iodo-tyrosine (3-IT) resulted in 20% pupae with partially-rescued phenotype and 68.2% of pupae with partially- or fully-rescued phenotype, respectively. These results indicate that overexpressions of TH and DDC are involved in the pupal melanization of S. exigua. PMID:26084938

  6. Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

    PubMed

    Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F

    2013-02-12

    Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

  7. Synthesis of 16 alpha-3H androgen and estrogen substrates for 16 alpha-hydroxylase.

    PubMed

    Cantineau, R; Kremers, P; De Graeve, J; Cornelis, A; Laszlo, P; Gielen, J E; Lambotte, R

    1981-02-01

    The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX). PMID:7013160

  8. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  9. 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

  10. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

    PubMed Central

    Liang, Jian-qing; Wang, Li; He, Jian-cheng; Hua, Xian-dong

    2016-01-01

    Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson's disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson's disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson's disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson's disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson's model group. These findings suggest that the mechanism by which verbascoside treats Parkinson's disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. PMID:26981096

  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. 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.

  13. Loss of FERULATE 5-HYDROXYLASE Leads to Mediator-Dependent Inhibition of Soluble Phenylpropanoid Biosynthesis in Arabidopsis1[OPEN

    PubMed Central

    Anderson, Nickolas A.; Bonawitz, Nicholas D.; Nyffeler, Kayleigh; Chapple, Clint

    2015-01-01

    Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism. PMID:26048881

  14. Serum immunoreactive prolyl hydroxylase in inflammatory rheumatic diseases.

    PubMed Central

    Kuutti-Savolainen, E R; Kivirikko, K I; Laitinen, O

    1980-01-01

    Serum immunoreactive prolyl hydroxylase protein (S-IRPH) was measured in 56 patients with inflammatory rheumatic diseases, and the values were compared with those in 32 control subjects. S-IRPH was above the 95% confidence limit of the controls in about 70% of the patients with active systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Reiter's syndrome, Sjögren's syndrome, polyarteritis nodosa, or polymyositis. Raised values were observed in about half of the patients with an erythrocyte sedimentation rate (ESR) of 21-50 and in about 90% of those with ESR of over 50, whereas only about 10% of the patients with an inactive disease had an S-IRPH concentration exceeding this limit. Only 1 out of 8 patients with active ankylosing spondylitis had a raised S-IRPH value. The results support previous data indicating that significant changes in collagen metabolism occur in active connective tissue diseases. Assays of S-IRPH might be of some value in assessing the activity of these diseases and in monitoring the treatment provided. PMID:6251755

  15. 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.

  16. 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

  17. 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.

  18. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  19. 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

  20. 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

  1. Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells*

    PubMed Central

    Vilcaes, Aldo A.; Demichelis, Vanina Torres; Daniotti, Jose L.

    2011-01-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  2. Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease.

    PubMed

    McDonough, Michael A; Kavanagh, Kathryn L; Butler, Danica; Searls, Timothy; Oppermann, Udo; Schofield, Christopher J

    2005-12-01

    Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

  3. 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

  4. Infarction-induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves

    PubMed Central

    Parrish, Diana C.; Alston, Eric N.; Rohrer, Hermann; Nkadi, Paul; Woodward, William R.; Schütz, Günther; Habecker, Beth A.

    2010-01-01

    Myocardial infarction causes heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischemia-induced alterations in sympathetic transmission include regional variations in cardiac norepinephrine (NE) and in tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NE content in cardiac nerves we used gp130DBH-Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine beta hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle one week after ischemia-reperfusion. Surprisingly, restoring tyrosine hydroxylase in the damaged ventricle did not return neuronal NE content to normal levels. NE uptake into cardiac nerves was significantly lower in gp130 KO mice, contributing to the lack of neuronal NE stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtMAX, or dP/dtMIN between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulating the heart with dobutamine or releasing endogenous NE with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post-infarct depletion of TH in the left ventricle, but does not alter NE content or cardiac function. PMID:19880537

  5. Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency

    PubMed Central

    Turcu, Adina F.; Rege, Juilee; Chomic, Robert; Liu, Jiayan; Nishimoto, Hiromi K.; Else, Tobias; Moraitis, Andreas G.; Palapattu, Ganesh S.; Rainey, William E.

    2015-01-01

    Context: Marked elevations of 17-hydroxyprogesterone (17OHP) are characteristic of classic 21-hydroxylase deficiency (21OHD). Testing of 17OHP provides the basis for 21OHD diagnosis, although it suffers from several pitfalls. False-positive or false-negative results and poor discrimination of nonclassic 21OHD from carriers limit the utility of serum 17OHP and necessitate dynamic testing after cosyntropin stimulation when values are indeterminate. Objective: The objective was to provide a detailed characterization of 21-carbon (C21) steroids in classic 21OHD, which might identify other candidate steroids that could be employed for the diagnosis of 21OHD. Setting and Participants: Patients (11 women, 10 men) with classic 21OHD and 21 sex- and age-matched controls seen in a tertiary referral center were studied. Methods: C21 steroids in the peripheral sera from all subjects, as well as in media from cultured testicular adrenal rest tumor (TART) cells and normal adrenal (NA) cells, were analyzed using liquid chromatography/tandem mass spectrometry (10 steroids). Additionally, the dynamics of C21 steroid metabolism in TART and NA cells were assessed with radiotracer studies. Results: Five C21 steroids were significantly higher in 21OHD patients: 17OHP (67-fold; P < .01), 21-deoxycortisol (21dF; 35-fold; P < .01), 16α-hydroxyprogesterone (16OHP; 28-fold; P < .01), progesterone (2-fold; P < .01), and 11β-hydroxyprogesterone (11OHP; not detected in controls; P < .01). The same steroids were the highest in media from TART cells relative to the NA cells: 11OHP, 58- to 65-fold; 21dF, 30- to 41-fold; 17OHP, 9-fold; progesterone, 9- to 12-fold; and 16OHP, 7-fold. Conclusion: Measurement of 16OHP and 11OHP along with 17OHP and 21dF by liquid chromatography/tandem mass spectrometry might comprise a biomarker panel to accurately diagnose all forms of 21OHD. PMID:25850025

  6. 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

  7. Reduced striatal tyrosine hydroxylase in incidental Lewy body disease

    PubMed Central

    Adler, Charles H.; Sue, Lucia I.; Peirce, Jeffrey B.; Bachalakuri, Jyothi; Dalsing-Hernandez, Jessica E.; Lue, Lih Fen; Caviness, John N.; Connor, Donald J.; Sabbagh, Marwan N.; Walker, Douglas G.

    2009-01-01

    Incidental Lewy body disease (ILBD) is the term used when Lewy bodies are found in the nervous system of subjects without clinically documented parkinsonism or dementia. The prevalence of ILBD in the elderly population has been estimated at between 3.8 and 30%, depending on subject age and anatomical site of sampling. It has been speculated that ILBD represents the preclinical stage of Parkinson’s disease (PD) and/or dementia with Lewy bodies (DLB). Studies of ILBD could potentially identify early diagnostic signs of these disorders. At present, however, it is impossible to know whether ILBD is a precursor to PD or DLB or is just a benign finding of normal aging. We hypothesized that, if ILBD represents an early stage of PD or DLB, it should be associated with depletion of striatal dopaminergic markers. Eleven subjects with ILBD and 27 control subjects were studied. The ILBD subjects ranged in age from 74 to 96 years (mean 86.5) while the control subjects’ age ranged from 75 to 102 years (mean 86.7). Controls and subjects did not differ in terms of age, postmortem interval, gender distribution, medical history conditions, brain weight, neuritic plaque density or Braak neurofibrillary stage. Quantitative ELISA measurement of striatal tyrosine hydroxylase (TH), the principal enzyme for dopamine synthesis, showed a 49.8% (P = 0.01) reduction in ILBD cases, as compared with control cases. The finding suggests that ILBD is not a benign condition but is likely a precursor to PD and/or DLB. PMID:17985144

  8. 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

  9. Monoamine Oxidase and Dopamine β-Hydroxylase Inhibitors from the Fruits of Gardenia jasminoides

    PubMed Central

    Kim, Ji Ho; Kim, Gun Hee; Hwang, Keum Hee

    2012-01-01

    This research was designed to determine what components of Gardenia jasminoides play a major role in inhibiting the enzymes related antidepressant activity of this plant. In our previous research, the ethyl acetate fraction of G. jasminosides fruits inhibited the activities of both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B), and oral administration of the ethanolic extract slightly increased serotonin concentrations in the brain tissues of rats and decreased MAO-B activity. In addition, we found through in vitro screening test that the ethyl acetate fraction showed modest inhibitory activity on dopamine-β hydroxylase (DBH). The bioassay-guided fractionation led to the isolation of five bio-active compounds, protocatechuic acid (1), geniposide (2), 6'-O-trans-p-coumaroylgeniposide (3), 3,5-d-ihydroxy-1,7-bis (4-hydroxyphenyl) heptanes (4), and ursolic acid (5), from the ethyl acetate fraction of G. jasminoides fruits. The isolated compounds showed different inhibitory potentials against MAO-A, -B, and DBH. Protocatechuic acid showed potent inhibition against MAO-B (IC50 300 μmol/L) and DBH (334 μmol/L), exhibiting weak MAO-A inhibition (2.41 mmol/L). Two iridoid glycosides, geniposide (223 μmol/L) and 6'-O-trans-p-coumaroylgeniposide (127μmol/L), were selective MAO-B inhibitor. Especially, 6'-O-trans-p-coumaroylgeniposide exhibited more selective MAO-B inhibition than deprenyl, well-known MAO-B inhibitor for the treatment of early-stage Parkinson’s disease. The inhibitory activity of 3,5-di-hydroxy-1,7-bis (4-hydroxyphenyl) heptane was strong for MAO-B (196 μmol/L), modest for MAO-A (400 μmol/L), and weak for DBH (941 μmol/L). Ursolic acid exhibited significant inhibition of DBH (214 μmol/L), weak inhibition of MAO-B (780 μmol/L), and no inhibition against MAO-A. Consequently, G. jasminoides fruits are considerable for development of biofunctional food materials for the combination treatment of depression and neurodegenerative disorders

  10. Purification and properties of gamma-butyrobetaine hydroxylase from Pseudomonas sp AK 1.

    PubMed

    Lindstedt, G; Lindstedt, S; Nordin, I

    1977-05-17

    gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) has been isolated from Pseudomonas sp AK 1 by ion-exchange, adsorption, and molecular-sieving chromatography. The preparation was homogeneous as judged from electrophoresis in agarose and polyacrylamide gels, isoelectric focusing, and equilibrium sedimentation. The molecular mass was 95 kdaltons as determined by sedimentation equilibrium centrifugation. From electrophoresis in polyacrylamide gel the molecular mass was estimated to 92 kdaltons, from gel filtration through columns of Sephadex G-200 to 86 kdaltons, and from gel filtration through thin layers of Sephadex G-150 and G-200 to 82 kdaltons. Calculation of molecular mass from Stokes radius, sedimentation coefficient, and partial specific volume gave a value of 96 kdaltons, and from the sedimentation coefficient, 93 kdaltons. Gel filtration through Sephadex G-200 in 6 M guanidinium chloride and electrophoresis in polyacrylamide gel containing 3.5 mM sodium dodecyl sulfate resulted in single bands with mobilities corresponding to molecular masses of 39 and 37 kdaltons, respectively, indicating that the enzyme is composed of two polypeptides chains with similar size. NH2-terminal amino acid sequencing in three cycles resulted in two amino acids in each cycle (Ala + Asn, Ala + Ile, Ala + Ile). The Stokes radius was 3.8 nm, corresponding to a diffusion coefficient of 5.7 X 10(-7) cm2/s. A sedimentation coefficient of 5.8 X 10(-13) s and a frictional ratio of 1.26 was found. The partial specific volume was 0.729 mL/g at 20 degrees C as calculated from amino acid analysis. The isoelectric point was 5.1, as determined by isoelectric focusing analysis. The light absorption in the ultraviolet and visible regions was that of a protein without light-absorbing prosthetic groups. The absorption coefficient at 280 nm of a 1.0% solution at pH 6.5 was 12.6. Amino acid analysis by ion

  11. Effect of sialylation of lipopolysaccharide of Neisseria gonorrhoeae on recognition and complement-mediated killing by monoclonal antibodies directed against different outer-membrane antigens.

    PubMed

    de la Paz, H; Cooke, S J; Heckels, J E

    1995-04-01

    Growth of gonococci in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) has previously been shown to induce resistance to the bactericidal effect of normal human serum and is accompanied by sialylation of the gonococcal lipopolysaccharide (LPS). We have used monoclonal antibodies (mAbs) to compare the effect of LPS sialylation on recognition of gonococci and complement-mediated killing by antibodies directed either against LPS or against defined epitopes on outer-membrane protein PI. Despite differences in binding to sialylated LPS on Western blots, all three mAbs directed against LPS showed considerably reduced binding to gonococci grown in the presence of CMP-NANA and a concomitant reduction in ability to promote complement-mediated killing. In contrast, mAbs directed against previously defined epitopes on a surface exposed loop of PI showed little difference in binding between sialylated and non-sialylated gonococci and promoted killing of the sialylated gonococci. Similarly a mAb directed against an epitope on a loop of the outer-membrane Rmp protein, which had previously been shown to block killing by antibodies directed against other surface antigens, also exerted a blocking effect with sialylated gonococci. Thus in the present study the continued biological effect of mAbs was correlated with the ability of the antibody to recognize surface-exposed epitopes on sialylated gonococci. Despite the presence of the sialylation which is likely to occur in vivo, it should be possible to induce complement-mediated killing by focusing the immune response to those surface-exposed epitopes which are least susceptible to the potential inhibitory effect of LPS sialylation.

  12. Estradiol-induced synaptic remodeling of tyrosine hydroxylase immunopositive neurons in the rat arcuate nucleus.

    PubMed

    Csakvari, Eszter; Kurunczi, Anita; Hoyk, Zsofia; Gyenes, Andrea; Naftolin, Frederick; Parducz, Arpad

    2008-08-01

    Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.

  13. 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.

  14. 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

  15. 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

  16. Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain.

    PubMed

    Gerginova, Maria; Manasiev, Jordan; Shivarova, Nedka; Alexieva, Zlatka

    2007-01-01

    The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.

  17. Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-beta-hydroxylase.

    PubMed

    Laduron, P M

    1984-01-01

    Presynaptic muscarinic receptors labeled with [3H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [3H]quinuclidinyl benzilate or [3H]dexetimide binding revealed a distribution profile similar to that of dopamine-beta-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-beta-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-beta-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle. PMID:6198205

  18. Guidelines for diagnosis and treatment of 21-hydroxylase deficiency (2014 revision)

    PubMed Central

    Ishii, Tomohiro; Anzo, Makoto; Adachi, Masanori; Onigata, Kazumichi; Kusuda, Satoshi; Nagasaki, Keisuke; Harada, Shohei; Horikawa, Reiko; Minagawa, Masanori; Minamitani, Kanshi; Mizuno, Haruo; Yamakami, Yuji; Fukushi, Masaru; Tajima, Toshihiro

    2015-01-01

    Purpose of developing the guidelines: The first guidelines for diagnosis and treatment of 21-hydroxylase deficiency (21-OHD) were published as a diagnostic handbook in Japan in 1989, with a focus on patients with severe disease. The “Guidelines for Treatment of Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency) Found in Neonatal Mass Screening (1999 revision)” published in 1999 were revised to include 21-OHD patients with very mild or no clinical symptoms. Accumulation of cases and experience has subsequently improved diagnosis and treatment of the disease. Based on these findings, the Mass Screening Committee of the Japanese Society for Pediatric Endocrinology further revised the guidelines for diagnosis and treatment. Target disease/conditions: 21-hydroxylase deficiency. Users of the guidelines: Physician specialists in pediatric endocrinology, pediatric specialists, referring pediatric practitioners, general physicians; and patients. PMID:26594092

  19. How to optimize vitamin D supplementation to prevent cancer, based on cellular adaptation and hydroxylase enzymology.

    PubMed

    Vieth, Reinhold

    2009-09-01

    The question of what makes an 'optimal' vitamin D intake is usually equivalent to, 'what serum 25-hydroxyvitamin D [25(OH)D] do we need to stay above to minimize risk of disease?'. This is a simplistic question that ignores the evidence that fluctuating concentrations of 25(OH)D may in themselves be a problem, even if concentrations do exceed a minimum desirable level. Vitamin D metabolism poses unique problems for the regulation of 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in the tissues outside the kidney that possess 25(OH)D-1-hydroxylase [CYP27B1] and the catabolic enzyme, 1,25(OH)2D-24-hydroxylase [CYP24]. These enzymes behave according to first-order reaction kinetics. When 25(OH)D declines, the ratio of 1-hydroxylase/24-hydroxylase must increase to maintain tissue 1,25(OH)2D at its set-point level. The mechanisms that regulate this paracrine metabolism are poorly understood. I propose that delay in cellular adaptation, or lag time, in response to fluctuating 25(OH)D concentrations can explain why higher 25(OH)D in regions at high latitude or with low environmental ultraviolet light can be associated with the greater risks reported for prostate and pancreatic cancers. At temperate latitudes, higher summertime 25(OH)D levels are followed by sharper declines in 25(OH)D, causing inappropriately low 1-hydroxylase and high 24-hydroxylase, resulting in tissue 1,25(OH)2D below its ideal set-point. This hypothesis can answer concerns raised by the World Health Organization's International Agency for Research on Cancer about vitamin D and cancer risk. It also explains why higher 25(OH)D concentrations are not good if they fluctuate, and that desirable 25(OH)D concentrations are ones that are both high and stable. PMID:19667164

  20. Inactivation of tyrosine hydroxylase activity by ascorbate in vitro and in rat PC12 cells.

    PubMed

    Wilgus, H; Roskoski, R

    1988-10-01

    Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 microM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 microM) and dehydroascorbate (EC50, 970 microM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant proteolysis of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25-50%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2901463

  1. Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration.

    PubMed

    Suttle, Jeffrey C; Abrams, Suzanne R; De Stefano-Beltrán, Luis; Huckle, Linda L

    2012-09-01

    The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.

  2. Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids ▿ †

    PubMed Central

    Petrusma, Mirjan; Hessels, Gerda; Dijkhuizen, Lubbert; van der Geize, Robert

    2011-01-01

    The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9α-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshADSM43269 homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshADSM43269 homologues are C-9 α-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 α-hydroxylation occurring at different levels during microbial steroid degradation. PMID:21642460

  3. 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),...

  4. A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.

    PubMed

    Soskel, N T; Kuby, S A

    1981-01-01

    Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

  5. Design and synthesis of a series of novel pyrazolopyridines as HIF-1alpha prolyl hydroxylase inhibitors.

    PubMed

    Warshakoon, Namal C; Wu, Shengde; Boyer, Angelique; Kawamoto, Richard; Renock, Sean; Xu, Kevin; Pokross, Matthew; Evdokimov, Artem G; Zhou, Songtao; Winter, Carol; Walter, Richard; Mekel, Marlene

    2006-11-01

    Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.

  6. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.

    PubMed

    Zhang, Shengnan; Roberts, Kenneth M; Fitzpatrick, Paul F

    2014-10-28

    Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer-dimer equilibrium with a dissociation constant of ~46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.

  7. Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase.

    PubMed

    Yasutake, Yoshiaki; Fujii, Yoshikazu; Nishioka, Taiki; Cheon, Woo-Kwang; Arisawa, Akira; Tamura, Tomohiro

    2010-10-01

    Vitamin D(3) hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D(3) (VD(3)) to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)VD(3)) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD(3), i.e. the conversion of VD(3) to 25-hydroxyvitamin D(3) (25(OH)VD(3)) and then of 25(OH)VD(3) to 1α,25(OH)(2)VD(3), a hormonal form of VD(3). Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD(3)-bound, and 25(OH)VD(3)-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD(3) and 25(OH)VD(3) but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD(3) hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD(3) 25-hydroxylase and 25(OH)VD(3) 1α-hydroxylase activities.

  8. A putative role of micro RNA in regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes.

    PubMed

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Chiang, John Y L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3'-untranslated region (3'-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or fibroblast growth factor (FGF)19. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3'-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3'-UTR reporter plasmids, and mutations of miRNA binding sites in 3'-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF19 inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

  9. 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

  10. Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly.

    PubMed

    Bates, Philip D; Johnson, Sean R; Cao, Xia; Li, Jia; Nam, Jeong-Won; Jaworski, Jan G; Ohlrogge, John B; Browse, John

    2014-01-21

    Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [(14)C]acetate and [(3)H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [(14)C]acetate and [(14)C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl-CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl-CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.

  11. Mechanism-based inactivation of benzo(a)pyrene hydroxylase by aryl acetylenes and aryl olefins

    SciTech Connect

    Gan, L.S.; Lu, J.Y.L.; Alworth, W.L.

    1986-05-01

    A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo(a)pyrene hydroxylase. The mechanism-based loss of benzo(a)pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenes therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, /sup 3/H-labeled 1-ethynylpyrene becomes covalently attached to the cytochrome P-450 protein; the measured stoichiometry of binding is one 1-ethynylpyrene per P-450 heme unit. The authors conclude that the inhibition of benzo(a)pyrene hydroxylase produced by 1-ethynylpyrene may be related to the mechanism of suicide inhibition of P-450 activity by chloramphenicol rather than the mechanism of suicide destruction of P-450 previously described for acetylene and propyne.

  12. 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

  13. Cholesterol 26-hydroxylase activity of hamster liver mitochondria: Isotope ratio analysis using deuterated 26-hydroxycholesterol

    SciTech Connect

    Kok, E.; Javitt, N.B. )

    1990-04-01

    Deuterated 26-hydroxycholesterol prepared from diosgenin by modifications of existing methods permitted the determination of mitochondrial cholesterol 26-hydroxylase using endogenous cholesterol as the substrate. Enzyme activity in a group of Syrian hamsters was found to be 10.3 +/- 3.7 pmol.min-1.mg protein-1.

  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. Gallate, the component of HIF-inducing catechins, inhibits HIF prolyl hydroxylase

    SciTech Connect

    Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka; Tokuhara, Takahiro; Hirota, Kiichi; Sakai, Akiko; Hayashi, Hideyuki . E-mail: hayashi@art.osaka-med.ac.jp; Katsumata, Takahiro

    2006-12-08

    Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completely at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.

  16. 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...

  17. 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 ...

  18. Adrenal scan in 17-alpha-hydroxylase deficiency: false indication of adrenal adenoma

    SciTech Connect

    Shore, R.M.; Lieberman, L.M.; Newman, T.J.; Friedman, A.; Bargman, G.J.

    1981-07-01

    A patient who was thought to have testicular feminization syndrome and primary aldosteronism had an adrenal scan that suggested an adrenal adenoma. After later diagnosis of 17-alpha-hydroxylase deficiency, she was treated with glucocorticoids rather than surgery. Her clinical course and a repeat adrenal scan confirmed she did not have a tumor.

  19. The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease.

    PubMed

    Townley-Tilson, W H Davin; Pi, Xinchun; Xie, Liang

    2015-01-01

    Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease.

  20. 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.

  1. The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease

    PubMed Central

    Townley-Tilson, W. H. Davin; Pi, Xinchun; Xie, Liang

    2015-01-01

    Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease. PMID:26491535

  2. Crystallographic and catalytic studies of the peroxide-shunt reaction in a diiron hydroxylase.

    PubMed

    Bailey, Lucas J; Fox, Brian G

    2009-09-29

    A diiron hydroxylase reaction typically begins by combination of O2 with a diferrous center to form reactive intermediates capable of hydrocarbon hydroxylation. In this natural cycle, reducing equivalents are provided by specific interactions with electron transfer proteins. The biological process can be bypassed by combining H2O2 with a diferric center, i.e., peroxide-shunt catalysis. Here we show that toluene 4-monooxygenase has a peroxide-shunt reaction that is approximately 600-fold slower than catalysis driven by biological electron transfer. However, the toluene 4-monooxygenase hydroxylase-effector protein complex was stable in the presence of 300 mM H2O2, suggesting overall benign effects of the exogenous oxidant on active site structure and function. The X-ray structure of the toluene 4-monooxygenase hydroxylase-effector protein complex determined from crystals soaked in H2O2 revealed a bound diatomic molecule, assigned to a cis-mu-1,2-peroxo bridge. This peroxo species resides in an active site position adjacent to the hydrogen-bonding substructure established by effector protein binding and faces into the distal cavity where substrate must bind during regiospecific aromatic ring hydroxylation catalysis. These results provide a new structural benchmark for how activated intermediates may be formed and dispatched during diiron hydroxylase catalysis. PMID:19705873

  3. 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

  4. Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).

    PubMed Central

    Funk, C.; Croteau, R.

    1993-01-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. PMID:12231778

  5. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    SciTech Connect

    Funk, C.; Croteau, R. )

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

    NASA Technical Reports Server (NTRS)

    Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.

    2001-01-01

    A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

  11. 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

  12. 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

  13. HIF prolyl 4-hydroxylase-2 inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction.

    PubMed

    Rahtu-Korpela, Lea; Karsikas, Sara; Hörkkö, Sohvi; Blanco Sequeiros, Roberto; Lammentausta, Eveliina; Mäkelä, Kari A; Herzig, Karl-Heinz; Walkinshaw, Gail; Kivirikko, Kari I; Myllyharju, Johanna; Serpi, Raisa; Koivunen, Peppi

    2014-10-01

    Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2-deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.

  14. Cellular oxygen sensing: Importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2

    SciTech Connect

    Steinhoff, Amrei; Pientka, Friederike Katharina; Moeckel, Sylvia; Kettelhake, Antje; Hartmann, Enno; Koehler, Matthias; Depping, Reinhard

    2009-10-02

    Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-{alpha} subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin {alpha}/{beta} dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of 'classical' importin {alpha}/{beta} receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.

  15. Synthesis of 16 alpha-/sub 3/H androgen and estrogen substrates for 16 alpha-hydroxylase

    SciTech Connect

    Cantineau, R.; Kremers, P.; De Graeve, J.; Cornelis, A.; Laszlo, P.; Gielen, J.E.; Lambotte, R.

    1981-02-01

    The synthesis of 16 alpha-/sup 3/H androgens and estrogens is described. 1-(/sup 3/H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-/sup 3/H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-/sup 3/H-dehydroepiandrosterone (I) and 16 alpha-/sub 4/H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-/sup 3/H-androstenedione (II) and 16 alpha-/sup 3/H-estradiol-17 beta (VII). 16 alpha-/sup 3/H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-/sup 3/H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

  16. 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.

  17. Selective inhibition of the hypoxia-inducible factor prolyl hydroxylase PHD3 by Zn(II).

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Choo, Hyunah; Chung, Hak Suk; Yang, Eun Gyeong

    2015-07-01

    We report herein that Zn(II) selectively inhibits the hypoxia-inducible factor prolyl hydroxylase PHD3 over PHD2, and does not compete with Fe(II). Independent of the oligomer formation induced by Zn(II), inhibition of the activity of PHD3 by Zn(II) involves Cys42 and Cys52 residues distantly located from the active site. PMID:26051901

  18. 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.

  19. Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

    PubMed Central

    Ji, Yurui; Mao, Guannan; Wang, Yingying; Bartlam, Mark

    2013-01-01

    Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments. PMID:23519435

  20. Identification of multiple steroid hydroxylases in Daphnia magna and their modulation by xenobiotics

    SciTech Connect

    Baldwin, W.S.; LeBlanc, G.A. . Dept. of Toxicology)

    1994-07-01

    Steroid hydroxylase activities were characterized in Daphnia magna and evaluated for potential use as biomarkers of xenobiotic exposure. Microsomes prepared from Daphnia magna generated as single NADPH-dependent metabolite of [[sup 14]C] testosterone. However, intact daphnids excreted at least 10 polar metabolites of [[sup 14]C] testosterone into the test medium. Six of these metabolites were identified as 2[alpha]-, 16[beta]-, 6[beta]-, 6[alpha]-, 7[alpha]-, and 15[alpha]-[[sup 14]C]hydroxytestosterone. The unidentified metabolites are also presumed to be hydroxylated products of testosterone, based on their relative migrations during TLC. The inefficient metabolism of [[sup 14]C] testosterone during the in vitro microsomal incubations may have been due to the release of P450 inhibitors during microsome preparation. Exposure of daphnids to the P450 modulators phenobarbital, [beta]-naphthoflavone, piperonyl butoxide, and malathion differentially inhibited the steroid hydroxylase activities. Results from this study indicate that Daphnia magna expresses several P450 enzymes and that these enzymes are differentially modulated by xenobiotic exposure. Steroid hydroxylase activities may serve not only as a biomarker of toxicant exposure, but also as a predictor of toxicant effects involving perturbations of steroid hormone homeostasis.

  1. Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.

    1995-01-01

    Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

  2. 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.

  3. New tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase

    SciTech Connect

    Brown, A.J.; Perlman, K.; Schnoes, H.K.; DeLuca, H.F.

    1987-08-01

    A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-(1 alpha-/sup 3/H)vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This /sup 3/H/sub 2/O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of /sup 3/H/sub 2/O formed when 25-hydroxy-(1 beta-/sup 3/H)vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-(26,27-/sup 3/H)vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of /sup 3/H/sub 2/O produced from 25-hydroxy-(1 alpha-/sup 3/H)vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.

  4. 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

  5. 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

  6. Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress.

    PubMed

    Mapanao, Ratchaneegorn; Cheng, Winton

    2016-09-01

    Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0-61.2% and 45.0-47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 10(5) cfu shrimp(-1) revealed significant increase of LvTH mRNA expression in haemocytes within 30-120 min and in brain within 15-30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30-60 and 15-60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30-60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation.

  7. Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress.

    PubMed

    Mapanao, Ratchaneegorn; Cheng, Winton

    2016-09-01

    Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0-61.2% and 45.0-47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 10(5) cfu shrimp(-1) revealed significant increase of LvTH mRNA expression in haemocytes within 30-120 min and in brain within 15-30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30-60 and 15-60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30-60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation. PMID:27514780

  8. A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants.

    PubMed

    Vialart, Guilhem; Hehn, Alain; Olry, Alexandre; Ito, Kyoko; Krieger, Celia; Larbat, Romain; Paris, Cedric; Shimizu, Bun-Ichi; Sugimoto, Yukihiro; Mizutani, Masaharu; Bourgaud, Frederic

    2012-05-01

    Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.

  9. Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Ferrando, Sara; Gallus, Lorenzo; Giovine, Marco

    2015-08-01

    Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

  10. Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.

    PubMed

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-07-01

    Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).

  11. 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.

  12. Characterisation of the FAD2 gene family from Hiptage benghalensis: a ricinoleic acid accumulating plant.

    PubMed

    Zhou, Xue-Rong; Singh, Surinder P; Green, Allan G

    2013-08-01

    We have characterised the FAD2 gene family from Hiptage benghalensis, a tropical plant that accumulates high levels of ricinoleic acid in its seeds. Functional characterisation of six FAD2 gene family members showed that two of them were capable of functioning as Δ12-hydroxylases while the other FAD2 members were confirmed to be Δ12-desaturases. The Δ12-hydroxylation function of these two genes was confirmed in yeast cells, using C16:1(Δ9) and C18:1(Δ9) monounsaturated fatty acids as substrates. These Δ12-hydroxylases, like the other Δ12-hydroxylases previously cloned from plants Ricinus communis (castor), Physaria fendleri and fungus Claviceps purpurea, also showed some Δ12-desaturase activity. The hydroxylation activity of the two Hiptage hydroxylases was further confirmed by their expression in the Arabidopsis fad2/fae1 double mutant where they were able to produce equivalent or higher levels hydroxylated fatty acids in the seed oil when compared with the other known hydroxylases.

  13. 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

  14. 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

  15. Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography-electrospray tandem mass spectrometry.

    PubMed

    Vacchina, Paola; Tripodi, Karina E J; Escalante, Andrea M; Uttaro, Antonio D

    2012-07-01

    Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time. PMID:22542487

  16. Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site*

    PubMed Central

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H. J.

    2014-01-01

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner. PMID:25172507

  17. Evidence for the Slow Reaction of Hypoxia-Inducible Factor Prolyl Hydroxylase 2 with Oxygen

    PubMed Central

    Flashman, Emily; Hoffart, Lee M.; Hamed, Refaat B.; Bollinger, J. Martin; Krebs, Carsten; Schofield, Christopher J.

    2010-01-01

    SUMMARY The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. PMID:20840591

  18. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    PubMed

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.

  19. Concurrence of Meningomyelocele and Salt-Wasting Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

    PubMed Central

    Kırmızıbekmez, Heves; Yesiltepe Mutlu, Rahime Gül; Moralıoğlu, Serdar; Tellioğlu, Ahmet; Cerrah Celayir, Ayşenur

    2015-01-01

    Congenital adrenal hyperplasia (CAH) is a group of inherited defects of cortisol biosynthesis. A case of classical CAH due to 21-hydroxylase deficiency (21-OHD) with early onset of salt waste and concurrence of meningomyelocele (MMC) was presented here. The management of salt-wasting crisis which is complicated by a postrenal dysfunction due to neurogenic bladder was described. Possible reasons of growth retardation in the one-year follow-up period were discussed. A significant regression of the phallus with proper medical treatment was also mentioned. PMID:25685584

  20. Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study

    SciTech Connect

    Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S. )

    1991-01-01

    Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

  1. Functional heterogeneity of rat hepatocytes: predominance of aryl hydrocarbon hydroxylase activity in perivenular zone.

    PubMed

    Tazawa, J; Endou, H; Sato, A; Hasumura, Y; Takeuchi, J

    1988-06-01

    To elucidate the hepatic intralobular distribution of aryl hydrocarbon hydroxylase (AHH) activity biochemically, periportal (PP) and perivenular hepatocytes (PV) from male Sprague-Dawley rats were separated by a fluorescence-activated cell sorter after labeling the PP zone with fluorescein diacetate and the perivenular zone with fluorescein isothiocyanate. AHH activity was higher in PV than in PP. The enzyme activity was induced about 6-fold in hepatocytes of rats pretreated with 3-methyl-cholanthrene, and the induction was more prominent in PP than in PV. Neither phenobarbital pretreatment nor altered lipid content of the diet induced the change in the enzyme activity.

  2. 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.

  3. Molecular and Biochemical Analysis of Two Rice Flavonoid 3'-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain.

    PubMed

    Park, Sangkyu; Choi, Min Ji; Lee, Jong Yeol; Kim, Jae Kwang; Ha, Sun-Hwa; Lim, Sun-Hyung

    2016-01-01

    Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3',4'-dihydroxylated leucocyanidin. 3'-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3'H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3'H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice. PMID:27649148

  4. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice

    PubMed Central

    Cubells, Joseph F.; Schroeder, Jason P.; Barrie, Elizabeth S.; Manvich, Daniel F.; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A.; Liles, L. Cameron; Squires, Katherine E.; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L.; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a “C” or a “T” at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh

  5. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    PubMed

    Cubells, Joseph F; Schroeder, Jason P; Barrie, Elizabeth S; Manvich, Daniel F; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A; Liles, L Cameron; Squires, Katherine E; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency

  6. Characterization of the interaction of the novel antihypertensive etamicastat with human dopamine-β-hydroxylase: comparison with nepicastat.

    PubMed

    Bonifácio, Maria João; Sousa, Filipa; Neves, Marco; Palma, Nuno; Igreja, Bruno; Pires, Nuno Miguel; Wright, Lyndon C; Soares-da-Silva, Patrício

    2015-03-15

    The interaction of etamicastat, a novel peripherally acting dopamine-β-hydroxylase (DBH) inhibitor, with the enzyme was studied using a classical kinetic approach and the pharmacodynamics effect of the compound upon administration to rats was also evaluated. SK-N-SH cell homogenates convert tyramine into octopamine with a Km value of 9 mM, and a Vmax of 1747 nmol/mg protein/h. The K(m) value for ascorbate was 3 mM. The inhibition of DBH by etamicastat and nepicastat, a known centrally acting DBH inhibitor, with IC50 values of 107 and 40 nM, respectively, was fully reversed by dilution. Non-linear fitting of the velocities, determined at various concentrations of substrate (tyramine) and co-substrate (ascorbic acid), and of etamicastat and nepicastat, indicated that the inhibition of DBH by both compounds follows a mixed-model inhibition mechanism, approaching competitive behavior with regards to the substrate tyramine, with K(i) values of 34 and 11 nM, respectively. Relatively to ascorbate, both compounds followed a mixed-model inhibition mechanism, approaching uncompetitive behavior. Oral administration of both compounds (at 30 mg/kg) inhibited adrenal DBH activity over time and significantly decreased noradrenaline levels in the heart. Nepicastat also decreased noradrenaline levels in the parietal cortex, but not etamicastat. Both compounds significantly decreased systolic and diastolic blood pressure in spontaneously hypertensive rats. In conclusion, etamicastat and nepicastat behave as multisubstrate DBH inhibitors, binding reversibly and preferentially to the reduced form of the enzyme, and simultaneously at the substrate and oxygen binding sites. Etamicastat, in contrast to nepicastat, offers the advantage of peripheral selectivity without central effects. PMID:25641750

  7. 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

  8. 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.

  9. Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.

    PubMed

    Pramod, K K; Singh, Satpal; Jayabaskaran, C

    2010-12-01

    Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K(m) values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14M(-1) and 0.56M(-1), respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.

  10. Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix*

    PubMed Central

    Risteli, Maija; Ruotsalainen, Heli; Salo, Antti M.; Sormunen, Raija; Sipilä, Laura; Baker, Naomi L.; Lamandé, Shireen R.; Vimpari-Kauppinen, Leena; Myllylä, Raili

    2009-01-01

    Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF+/−) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF+/−, may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family. PMID:19696018

  11. [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.

  12. 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.

  13. Decreased melatonin secretion in a phenotypically male 46,XX patient with classic 21-hydroxylase deficiency.

    PubMed

    Luboshitzky, R; Qupti, G; Shen-Orr, Z; Hardoff, R

    2000-01-01

    The possible role of gonadal steroids and gonadotropins in regulating melatonin secretion has been suggested in clinical syndromes of the hypothalamic-pituitary-gonadal axis. We describe the results of melatonin secretion in a 37-year old male patient who presented with azoospermia. The patient was an XX male, had classic simple virilizing form of 21-hydroxylase deficiency, which led to a masculine phenotype. He was ovariectomized at the age of three years and reared as a male. Melatonin production (aMT6s) was determined at baseline and during 12 months of replacement therapy. Results were compared with those obtained in age-matched male controls. Pretreatment aMT6s values were decreased (14.3 microg/24 h vs. 29.0+/-5.5 in controls). Dexamethasone replacement was associated with an increase in aMT6s values (19.3-20.9 microg/24 h). The addition of testosterone to dexamethasone replacement resulted in normalization of aMT6s (27.6-33.1 microg/24 h) and serum 17OH progesterone, testosterone and estradiol levels. The present data indicate that androgen excess due to 21 hydroxylase deficiency is associated with decreased melatonin secretion. These results support the hypothesis that sex steroids modulate melatonin secretion.

  14. 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

  15. 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

  16. 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.

  17. Embryotoxicity, teratogenicity and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    USGS Publications Warehouse

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.E.; Kubiak, T.J.

    1987-01-01

    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at (i) a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and (ii) an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  18. Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    USGS Publications Warehouse

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.; Kubiak, T.J.

    1987-01-01

    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  19. Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center.

    PubMed

    Vasta, James D; Raines, Ronald T

    2016-06-14

    Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits. PMID:27183028

  20. Lack of tryptophan hydroxylase-1 in mice results in gait abnormalities.

    PubMed

    Suidan, Georgette L; Duerschmied, Daniel; Dillon, Gregory M; Vanderhorst, Veronique; Hampton, Thomas G; Wong, Siu Ling; Voorhees, Jaymie R; Wagner, Denisa D

    2013-01-01

    The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (-/-) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system. PMID:23516593

  1. Involvement of molybdenum hydroxylases in reductive metabolism of nitro polycyclic aromatic hydrocarbons in mammalian skin.

    PubMed

    Ueda, Osamu; Sugihara, Kazumi; Ohta, Shigeru; Kitamura, Shigeyuki

    2005-09-01

    Molybdenum hydroxylases, aldehyde oxidase and xanthine oxidoreductase, were shown to be involved in the nitroreduction of 2-nitrofluorene (NF), 1-nitropyrene, and 4-nitrobiphenyl, environmental pollutants, in the skin of various mammalian species. NF was reduced to 2-aminofluorene by hamster skin cytosol in the presence of 2-hydroxypyrimidine, 4-hydroxypyrimidine, N(1)-methylnicotinamide, or benzaldehyde, but not hypoxanthine or xanthine. Inhibitors of aldehyde oxidase markedly inhibited these nitroreductase activities, but oxypurinol, an inhibitor of xanthine oxidoreductase, had little effect. In DEAE column chromatography of hamster skin cytosol, the major fraction exhibiting nitroreductase activity also showed aldehyde oxidase activity. 2-Hydroxypyrimidine-linked nitroreductase activities of skin cytosol from rabbits and guinea pigs were also inhibited by an inhibitor of aldehyde oxidase. In contrast, nitroreductase activities of skin cytosols of rats and mice were markedly inhibited by oxypurinol. When aldehyde oxidase activity was estimated in skin cytosol of various mammals using benzaldehyde oxidase activity as a marker, considerable variability of the activity was found. The highest activity was observed with hamsters, and the lowest activity with rats. On the other hand, the highest xanthine oxidoreductase activity was observed with rats, and the lowest activity with rabbits. These skin cytosols of various mammals also exhibited significant 2-hydroxypyrimidine-linked nitroreductase activities toward 1-nitropyrene and 4-nitrobiphenyl catalyzed by aldehyde oxidase and xanthine oxidoreductase. Thus, NF was mainly reduced by aldehyde oxidase and xanthine oxidoreductase in skins of animals. However, the contributions of these two molybdenum hydroxylases were considerably different among animal species. PMID:15932950

  2. A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

    SciTech Connect

    Burgos-Trinidad, M.; Brown, A.J.; DeLuca, H.F. )

    1990-10-01

    A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy(26,27-3H)vitamin D3 and 1,25-dihydroxy(26,27-3H)vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.

  3. Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

    PubMed Central

    Chung, Waihong; de la Monte, Suzanne; Thomas, John-Michael; Olsen, Mark; Carlson, Rolf; Yu, Tunan; Dong, Xiaoqun; Wands, Jack

    2016-01-01

    Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo. PMID:26954680

  4. 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.

  5. Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation.

    PubMed

    Kaufmann, Martin; Prosser, David E; Jones, Glenville

    2011-08-19

    CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.

  6. Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase

    SciTech Connect

    Hundahl, C.A.; Fahrenkrug, J.; Luuk, H.; Hay-Schmidt, A.; Hannibal, J.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Restricted Neuroglobin expression in the mouse retina. Black-Right-Pointing-Pointer Antibody validation using Neuroglobin-null mice. Black-Right-Pointing-Pointer Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. Black-Right-Pointing-Pointer No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

  7. 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...

  8. Short-term effects of endothelins on tyrosine hydroxylase activity and expression in the olfactory bulb of normotensive rats.

    PubMed

    Nabhen, Sabrina L; Perfume, Guadalupe; Battistone, María A; Rossi, Andrés; Abramoff, Tamara; Bianciotti, Liliana G; Vatta, Marcelo S

    2009-05-01

    The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca(2+)/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Postnatal changes in sialylation of glycoproteins in rat liver.

    PubMed Central

    Oda-Tamai, S; Kato, S; Akamatsu, N

    1991-01-01

    Glycoproteins containing N-linked oligosaccharides were prepared from plasma and liver microsomes of rats aged 0-5 weeks, and galactose and sialic acid content were determined. The sialic acid/galactose ratios in plasma membrane N-glycans remained at about 1 throughout the postnatal period, suggesting that most of the galactose residues are sialylated. In the same way, it was suggested that most of the galactose residues of microsomal N-glycans were sialylated at 0, 4 and 5 weeks of age, but that the degree of sialylation was lower at the other ages, with a minimum at 2 weeks. When the activities of sialyltransferase and galactosyltransferase in liver Golgi membranes were determined, age-dependent changes were found, not only in the specific activities of the enzymes, but also in the Golgi membrane content per g of liver. The activity of galactosyltransferase per g of liver increased immediately after birth, whereas that of sialyltransferase remained at a low level for 2 weeks and then increased to a constant level at 4 weeks. It is probable that this delayed increase in the activity of sialyltransferase results in the decreased sialylation of microsomal N-glycans at 1, 2 and 3 weeks. Sialyltransferase was solubilized from the liver microsomes of rats aged 2, 3 and 4 weeks and characterized. Phosphocellulose column chromatography separated the activity into two subfractions, designated transferase I and transferase II in the order of elution. The increase in total sialyltransferase activity during this period was caused mainly by an increase in transferase I. Rechromatography of each transferase from 3-week-old rats after neuraminidase treatment showed that transferase I but not transferase II contained sialic acid residue(s) and that desialylated transferase I was eluted in a similar way as transferase II. Although the apparent Km value for CMP-N-acetylneuraminic acid and the heat stability of transferase I were different from those of transferase II, the

  15. Mating increases neuronal tyrosine hydroxylase expression and selectively gates transmission of male chemosensory information in female mice.

    PubMed

    Matthews, Gillian A; Patel, Ronak; Walsh, Alison; Davies, Owain; Martínez-Ricós, Joana; Brennan, Peter A

    2013-01-01

    Exposure to chemosensory signals from unfamiliar males can terminate pregnancy in recently mated female mice. The number of tyrosine hydroxylase-positive neurons in the main olfactory bulb has been found to increase following mating and has been implicated in preventing male-induced pregnancy block during the post-implantation period. In contrast, pre-implantation pregnancy block is mediated by the vomeronasal system, and is thought to be prevented by selective inhibition of the mate's pregnancy blocking chemosignals, at the level of the accessory olfactory bulb. The objectives of this study were firstly to identify the level of the vomeronasal pathway at which selective inhibition of the mate's pregnancy blocking chemosignals occurs. Secondly, to determine whether a post-mating increase in tyrosine hydroxylase-positive neurons is observed in the vomeronasal system, which could play a role in preventing pre-implantation pregnancy block. Immunohistochemical staining revealed that mating induced an increase in tyrosine-hydroxylase positive neurons in the arcuate hypothalamus of BALB/c females, and suppressed c-Fos expression in these neurons in response to mating male chemosignals. This selective suppression of c-Fos response to mating male chemosignals was not apparent at earlier levels of the pregnancy-blocking neural pathway in the accessory olfactory bulb or corticomedial amygdala. Immunohistochemical staining revealed an increase in the number of tyrosine hydroxylase-positive neurons in the accessory olfactory bulb of BALB/c female mice following mating. However, increased dopamine-mediated inhibition in the accessory olfactory bulb is unlikely to account for the prevention of pregnancy block to the mating male, as tyrosine hydroxylase expression did not increase in females of the C57BL/6 strain, which show normal mate recognition. These findings reveal an association of mating with increased dopaminergic modulation in the pregnancy block pathway and support the

  16. 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.

  17. Completion of Tricin Biosynthesis Pathway in Rice: Cytochrome P450 75B4 Is a Unique Chrysoeriol 5'-Hydroxylase.

    PubMed

    Lam, Pui Ying; Liu, Hongjia; Lo, Clive

    2015-08-01

    Flavones are ubiquitously accumulated in land plants, but their biosynthesis in monocots remained largely elusive until recent years. Recently, we demonstrated that the rice (Oryza sativa) cytochrome P450 enzymes CYP93G1 and CYP93G2 channel flavanones en route to flavone O-linked conjugates and C-glycosides, respectively. In tricin, the 3',5'-dimethoxyflavone nucleus is formed before O-linked conjugations. Previously, flavonoid 3',5'-hydroxylases belonging to the CYP75A subfamily were believed to generate tricetin from apigenin for 3',5'-O-methylation to form tricin. However, we report here that CYP75B4 a unique flavonoid B-ring hydroxylase indispensable for tricin formation in rice. A CYP75B4 knockout mutant is tricin deficient, with unusual accumulation of chrysoeriol (a 3'-methoxylated flavone). CYP75B4 functions as a bona fide flavonoid 3'-hydroxylase by restoring the accumulation of 3'-hydroxylated flavonoids in Arabidopsis (Arabidopsis thaliana) transparent testa7 mutants and catalyzing in vitro 3'-hydroxylation of different flavonoids. In addition, overexpression of both CYP75B4 and CYP93G1 (a flavone synthase II) in Arabidopsis resulted in tricin accumulation. Specific 5'-hydroxylation of chrysoeriol to selgin by CYP75B4 was further demonstrated in vitro. The reaction steps leading to tricin biosynthesis are then reconstructed as naringenin → apigenin → luteolin → chrysoeriol → selgin → tricin. Hence, chrysoeriol, instead of tricetin, is an intermediate in tricin biosynthesis. CYP75B4 homologous sequences are highly conserved in Poaceae, and they are phylogenetically distinct from the canonical CYP75B flavonoid 3'-hydroxylase sequences. Recruitment of chrysoeriol-specific 5'-hydroxylase activity by an ancestral CYP75B sequence may represent a key event leading to the prevalence of tricin-derived metabolites in grasses and other monocots today. PMID:26082402

  18. 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.

  19. An exploratory evaluation of tyrosine hydroxylase inhibition in planaria as a model for parkinsonism.

    PubMed

    Prokai, David; Nguyen, Thinh; Kamrowski, Kurt; Chandra, Ashwin; Talamantes, Tatjana; Baxter, Lewis R; Prokai, Laszlo

    2013-11-26

    Planaria are the simplest organisms with bilateral symmetry and a central nervous system (CNS) with cephalization; therefore, they could be useful as model organisms to investigate mechanistic aspects of parkinsonism and to screen potential therapeutic agents. Taking advantage of the organism's anti-tropism towards light, we measured a significantly reduced locomotor velocity in planaria after exposure to 3-iodo-L-tyrosine, an inhibitor of tyrosine hydroxylase that is an enzyme catalyzing the first and rate-limiting step in the biosynthesis of catecholamines. A simple semi-automatic assay using videotaped experiments and subsequent evaluation by tracking software was also implemented to increase throughput. The dopaminergic regulation of locomotor velocity was confirmed by bromocriptine, a drug whose mechanisms of action to treat Parkinson's disease is believed to be through the stimulation of nerves that control movement.

  20. Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

    PubMed Central

    Kamps, Jos J. A. G.; Khan, Amjad; Choi, Hwanho; Lesniak, Robert K.; Brem, Jürgen; Rydzik, Anna M.; McDonough, Michael A.; Schofield, Christopher J.; Claridge, Timothy D. W.

    2015-01-01

    Abstract γ‐Butyrobetaine hydroxylase (BBOX) is a non‐heme FeII‐ and 2‐oxoglutarate‐dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C−H bond of γ‐butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N+>P+>As+. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation–π interactions in productive substrate recognition. PMID:26660433

  1. Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Kim, So Yeon; Yang, Eun Gyeong

    2016-04-01

    Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 μM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α. PMID:26940742

  2. 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.

  3. 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

  4. 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

  5. Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lappi, D. A.; Robertson, D.

    1994-01-01

    Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.

  6. Distribution of neurons expressing tyrosine hydroxylase in the human cerebral cortex

    PubMed Central

    Benavides-Piccione, Ruth; DeFelipe, Javier

    2007-01-01

    Since the very first detailed description of the different types of cortical interneurons by Cajal, the tremendous variation in the morphology, physiology and neurochemical properties of these cells has become apparent. However, it still remains unclear whether all types of interneurons are present in all cortical areas and species. Here we have focused on tyrosine hydroxylase (TH)-immunoreactive cortical interneurons, which although only present in certain species, are particularly abundant in the human neocortex. We argue that this type of interneuron is more widespread in the human neocortex than in any other species examined so far and that, therefore, it is probably involved in a larger variety of cortical circuits. In addition, notable regional variation can be seen in relation to these interneurons. These differences further emphasize the variability in the design of microcircuits between cortical areas and species, and they probably reflect an evolutionary adaptation of cortical circuits to particular functions. PMID:17593221

  7. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase from Sphingopyxis alaskensis.

    PubMed

    Hoeppner, Astrid; Widderich, Nils; Bremer, Erhard; Smits, Sander H J

    2014-04-01

    The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme from Sphingopyxis alaskensis was developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme.

  8. 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.

  9. 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

  10. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase from Sphingopyxis alaskensis

    PubMed Central

    Hoeppner, Astrid; Widderich, Nils; Bremer, Erhard; Smits, Sander H. J.

    2014-01-01

    The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme from Sphingopyxis alaskensis was developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme. PMID:24699747

  11. Cigarette Smoking and Tryptophan Hydroxylase 2 mRNA in the Dorsal Raphe Nucleus in Suicides

    PubMed Central

    Bach, Helene; Arango, Victoria; Kassir, Suham A.; Dwork, Andrew J.; Mann, J. John; Underwood, Mark D.

    2016-01-01

    Cigarette smoking is associated with suicide and mood disorders and stimulates serotonin release. Tryptophan hydroxylase (TPH2) synthesizes serotonin and is over-expressed in suicides. We determined whether smoking is associated with TPH2 mRNA in suicides and controls. TPH2 mRNA was measured postmortem in the dorsal raphe nucleus (DRN) of controls (N=26, 17 nonsmokers and nine smokers) and suicides (N=23, 5 nonsmokers and 18 smokers). Psychiatric history was obtained by psychological autopsy. TPH2 mRNA was greater in suicide nonsmokers than suicide smokers, control smokers and control nonsmokers (p=0.006). There was more TPH2 mRNA throughout the DRN. Smoking interferes with the TPH2 mRNA increase observed in suicide nonsmokers. The absence of altered TPH2 expression in non-suicide smokers suggests no pharmacological effect of smoking. PMID:26954509

  12. 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

  13. Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia

    PubMed Central

    Witsch, Thilo J.; Turowski, Paweł; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D.; Seeger, Werner

    2013-01-01

    Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD. PMID:24285264

  14. 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.

  15. Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

    PubMed Central

    Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

  16. Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

    SciTech Connect

    Greiner, J.W.; Malan-Shibley, L.B.; Janss, D.H.

    1980-01-28

    Studies were conducted to determine whether normal and/or neoplastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the in vitro addition of 10 ..mu..M DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 ..mu..M DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 ..mu..M BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, ..cap alpha..-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.

  17. 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).

  18. FOX-2 PROTEIN REGULATES THE ALTERNATE SPLICING OF SCLERODERMA -ASSOCIATED LYSYL HYDROXYLASE 2 mRNA

    PubMed Central

    Seth, Puneet; Yeowell, Heather N.

    2010-01-01

    Objective Scleroderma is a complex connective tissue disorder characterized by hardening and thickening of skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen cross-links derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternately-spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively-spliced LH2 exon 13A thereby increasing levels of the long transcript of LH2 [LH2(long)], are linked to scleroderma pathology. In this study we have examined the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion that leads to the generation of scleroderma-associated LH2(long) mRNA. Methods and Results We report that over-expression of Fox-2 enhances inclusion of exon 13A and increases the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreases the LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that two of the four Fox binding motifs flanking LH2 exon 13A are required for its inclusion. In early passage fibroblasts derived from patients with systemic scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Conclusions Fox-2 appears to play an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease the levels of fibrosis-associated LH2(long) mRNA in primary scleroderma cells may suggest a novel approach to strategies directed against scleroderma. PMID:20131247

  19. Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors.

    PubMed

    Thirstrup, Kenneth; Christensen, Søren; Møller, Henriette Aaris; Ritzén, Andreas; Bergström, Ann-Louise; Sager, Thomas Nikolaj; Jensen, Henrik Sindal

    2011-09-01

    The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel-Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC(50) values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed.

  20. Clinical, biochemical, and genetic features of non-classical 21-hydroxylase deficiency in Japanese children.

    PubMed

    Kashimada, Kenichi; Ishii, Tomohiro; Nagasaki, Keisuke; Ono, Makoto; Tajima, Toshihiro; Yokota, Ichiro; Hasegawa, Yukihiro

    2015-01-01

    Non-classical 21-hydroxylase deficiency (NC21-OHD) is a mild form of 21-hydroxylase deficiency lacking apparent symptoms of androgen excess at birth. Most NC21-OHD cases are diagnosed after the onset of puberty, while a substantial number of patients are not diagnosed during childhood. Previous studies have reported ethnic differences in the prevalence of NC21-OHD. To date, the clinical features of NC21-OHD in Japanese children have not been systemically reported. Thus, we performed 3 independent analyses: retrospective analyses of newborn screening in 2 major Japanese cities (Sapporo and Niigata) and a national surveillance collecting clinical information from pediatric endocrinologists throughout the country. During the last 10 years, one case of NC21-OHD was diagnosed by newborn screening in each city, resulting in incidences of 2.0 (95% confidence interval = 0.0-5.9) and 2.1 (0.0-6.2) per 1,000,000 in Sapporo and Niigata, respectively. We collected information from 85% of the 135 Councilors of Japanese Society of Pediatric Endocrinology. Fifteen NC21-OHD patients were diagnosed during childhood, resulting in the estimated prevalence of 0.58 (0.28-1.1) per 1,000,000. Eleven patients were discovered by newborn screening, 7 patients developed hyperandrogenism symptoms (2-8 years of age, median 7), and 9 patients were treated with hydrocortisone at the time of the survey. Ten out of 13 patients showed compound heterozygosity for the P30L mutation of CYP21A2. Our study suggests that the prevalence/incidence of NC21-OHD is lower than that in Western countries, and that the age for initial onset of androgen excess symptoms varies during the prepubertal period.

  1. Classroom Demonstration of a Spot Test for Pbenylpyruvic Acid and Its Relationship to Phenylketonuria

    ERIC Educational Resources Information Center

    Halkides, Christopher J.

    2004-01-01

    Classical phenylketonuria (PKU) is caused by a lack activity in the enzyme phenylalanine hydroxylase, leading to elevated concentrations of phenylalanine in the blood. A simple demonstration and three advanced demonstrations of a spot test for phenylpyruvic acid and its relationship to phenylketonuria are given.

  2. Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

    PubMed

    Thill, Jana; Miosic, Silvija; Gotame, Tek Prasad; Mikulic-Petkovsek, Maja; Gosch, Christian; Veberic, Robert; Preuss, Anja; Schwab, Wilfried; Stampar, Franci; Stich, Karl; Halbwirth, Heidi

    2013-11-01

    Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background.

  3. Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering.

    PubMed

    Meisburger, Steve P; Taylor, Alexander B; Khan, Crystal A; Zhang, Shengnan; Fitzpatrick, Paul F; Ando, Nozomi

    2016-05-25

    Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity. PMID:27145334

  4. Hypocholesterolemic effects of curcumin via up-regulation of cholesterol 7a-hydroxylase in rats fed a high fat diet.

    PubMed

    Kim, Minji; Kim, Yangha

    2010-06-01

    There is an increasing interest in curcumin (Curcuma longa L.) as a cardiovascular disease (CVD) protective agent via decreased blood total cholesterol and low-density lipoprotein-cholesterol (LDL-cholesterol) level. The aim of this study was to investigate further the potential mechanism in the hypocholesterolemic effect of curcumin by measuring cholesterol 7a-hydroxylase (CYP7A1), a rate limiting enzyme in the biosynthesis of bile acid from cholesterol, at the mRNA level. Male Sprague-Dawley rats were fed a 45% high fat diet or same diet supplemented with curcumin (0.1% wt/wt) for 8 weeks. The curcumin diet significantly decreased serum triglyceride (TG) by 27%, total cholesterol (TC) by 33.8%, and LDL-cholesterol by 56%, respectively as compared to control group. The curcumin-supplemented diet also significantly lowered the atherogenic index (AI) by 48% as compared to control group. Hepatic TG level was significantly reduced by 41% in rats fed with curcumin-supplemented diet in comparison with control group (P < 0.05). Conversely, the curcumin diet significantly increased fecal TG and TC. The curcumin diet up-regulated hepatic CYP7A1 mRNA level by 2.16-fold, compared to control group p (P < 0.05). These findings suggested that the increases in the CYP7A1 gene expression may partially account for the hypocholesterolemic effect of curcumin.

  5. 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

  6. Alternative splicing transcription of Megalobrama amblycephala HIF prolyl hydroxylase PHD3 and up-regulation of PHD3 by HIF-1α.

    PubMed

    Chen, Nan; Huang, Cui-Hong; Chen, Bo-Xiang; Liu, Hong; Wang, Wei-Min; Gul, Yasmeen; Wang, Huan-Ling

    2016-01-15

    PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala. PMID:26697748

  7. 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

  8. 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

  9. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    PubMed

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  10. Insulin-like growth factor I enhances proenkephalin synthesis and dopamine. beta. -hydroxylase activity in adrenal chromaffin cells

    SciTech Connect

    Wilson, S.P. )

    1991-01-01

    Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin derived enkephalin-containing peptides and the activity of dopamine {beta}-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine {beta}-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was {approximately} 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1,000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of ({sup 35}S)proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.

  11. 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.

  12. New, tritium-release assay for 25-hydroxyvitamin D-1. cap alpha. -hydroxylase

    SciTech Connect

    Brown, A.J.; Perlman, K.; DeLuca, H.F.

    1986-05-01

    A new, rapid assay for 25-hydroxyvitamin D (25-OH-D)-1..cap alpha..-hydroxylase has been developed using 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ as substrate. This compound was prepared by reduction of 1-oxo-25-hydroxycyclovitamin D/sub 3/ with (/sup 3/H)NaBH/sub 4/, separation of the 1..cap alpha..- and 1..beta..-hydroxy products by HPLC, subsequent treatments with methylsulfonylchloride and lithium aluminum hydride, cycloreversion, and saponification. The 1..cap alpha..- and 1..beta..-tritiated substrates were tested in the solubilized and reconstituted chick 1..cap alpha..-hydroxylase system. After incubation, the reaction mixture was passed through a reversed phase silica cartridge to separate (/sup 3/H)H/sub 2/O from the labeled substrate. The cartridges were then washed with methanol to elute all vitamin D metabolites, and the amount of 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ was measured by HPLC. In addition, identical reaction mixtures using 25-OH-(26,27-/sup 3/H)D/sub 3/ as substrate were extracted and analyzed by HPLC for 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/. Reactions with 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ produced (/sup 3/H)H/sub 2/O comparable to the amount of 1,25-(OH)/sub 2/(26,27-/sup 3/H)D/sub 3/ and negligible (/sup 3/H) in 1,25-(OH)/sub 2/D/sub 3/. Conversely, reactions with 25-OH-(1..beta..-/sup 3/H)D/sub 3/ produced negligible (/sup 3/H)H/sub 2/O but produced 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ comparable to that from reactions with 25-OH-(26,27-/sup 3/H)D/sub 3/. The results indicate that 1..cap alpha..-hydroxylation specifically displaces the 1..cap alpha..-hydrogen of 25-OH-D/sub 3/ and that the release of the 1..cap alpha..-/sup 3/H provides an accurate measure of vitamin D 1..cap alpha..-hydroxylation.

  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. 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.

  15. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  16. Monoclonal antibody-directed phenotyping of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues

    SciTech Connect

    Fujino, T.; West, D.; Park, S.S.; Gelboin, H.V.

    1984-07-25

    The distribution of cytochromes P-450 that catalyze aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase were studied with monoclonal antibody (MAb) 1-7-1 which completely inhibits these activities of a purified 3-methylcholanthrene-induced rat liver cytochrome P-450. The degree of inhibition by MAb 1-7-1 quantitatively assesses the contribution of different cytochromes P-450 in the liver, lung, and kidney microsomes from untreated, 3-methylcholanthrene- and phenobarbital (PB)-treated rats, mice, guinea pigs, and hamsters. Enzyme sensitivity to MAb 1-7-1 inhibition defines two types of cytochrome P-450 contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The MAb 1-7-1 sensitive cytochrome P-450 is a major contributor to aryl hydrocarbonhydroxylase in rat liver, lung, and kidney of 3-methylcholanthrene-treated rats, C57BL/6 mice, guinea pigs, and hamsters. 7-Ethoxycoumarin 0-deethylase is also a function of both the MAb 1-7-1-sensitive and insensitive classes of cytochromeP-450. The ratio of the classes contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase differs in the various tissues and species and after inducer treatment. All of the 7-ethoxycoumarin O-deethylase activity in guinea pigs and hamsters is a function of cytochromes P-450 different than the MAb 1-7-1-sensitive cytochrome P-450 responsible for aryl hydrocarbon hydroxylase activity. Thus, the MAb 1-7-1 antigenically defines the type of cytochromes P-450 contributing to each reaction.

  17. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

  18. Organization of tyrosine hydroxylase-immunoreactive neurons in the di- and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis.

    PubMed

    Carr, J A; Norris, D O; Samora, A

    1991-01-01

    We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di- and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.

  19. 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

  20. 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

  1. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  2. 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.

  3. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2.

    PubMed

    Yoshimoto, Francis K; Desilets, Melissa C; Auchus, Richard J

    2012-01-01

    The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Influence of some anti-inflammatory drugs on the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content

    SciTech Connect

    Mostafa, M.H.; Sheweita, S.A.; Abdel-Moneam, N.M. )

    1990-06-01

    The metabolism of benzo({alpha})pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text.

  9. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

    PubMed Central

    Yoshimoto, Francis K.; Desilets, Melissa C.; Auchus, Richard J.

    2011-01-01

    The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases. PMID:22001566

  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. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

  12. Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

    PubMed Central

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M.; Huttner, Wieland; Weidemann, Alexander

    2013-01-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds. PMID:23798557

  13. Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna.

    PubMed

    Li, Jing; van Belkum, Marco J; Vederas, John C

    2012-07-15

    (-)-Hyoscyamine, the enantiomerically pure form of atropine, and its derivative scopolamine are tropane alkaloids that are extensively used in medicine. Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), a monomeric α-ketoglutarate dependent dioxygenase, converts (-)-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. In this study, H6H of Atropa belladonna (AbH6H) was cloned, heterologously expressed in Escherichia coli, purified and characterized. The catalytic efficiency of AbH6H, especially for the second oxidation, was found to be low, and this may be one of the reasons why Atropa belladonna produces less scopolamine than other species in the same family. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, was shown not to be an obligatory intermediate in the biosynthesis of scopolamine using purified AbH6H with an in vitro (18)O labeling experiment. Moreover, the nitrogen atom in the tropane ring of (-)-hyoscyamine was found to play an important role in substrate recognition.

  14. 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.

  15. High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2

    PubMed Central

    Mordechai, Shikma; Gradstein, Libe; Pasanen, Annika; Ofir, Rivka; El Amour, Khalil; Levy, Jaime; Belfair, Nadav; Lifshitz, Tova; Joshua, Sara; Narkis, Ginat; Elbedour, Khalil; Myllyharju, Johanna; Birk, Ohad S.

    2011-01-01

    Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. PMID:21885030

  16. Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations.

    PubMed Central

    Wang, J; Freeman, D J; Grundy, S M; Levine, D M; Guerra, R; Cohen, J C

    1998-01-01

    Interindividual differences in plasma low-density lipoprotein cholesterol (LDL-C) levels reflect both environmental variation and genetic polymorphism, but the specific genes involved and their relative contributions to the variance in LDL-C are not known. In this study we investigated the relationship between plasma LDL-C concentrations and three genes with pivotal roles in LDL metabolism: the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and cholesterol 7alpha-hydroxylase (CYP7). Analysis of 150 nuclear families indicated statistically significant linkage between plasma LDL-C concentrations and CYP7, but not LDLR or APOB. Further sibling pair analyses using individuals with high plasma LDL-C concentrations as probands indicated that the CYP7 locus was linked to high plasma LDL-C, but not to low plasma LDL-C concentrations. This finding was replicated in an independent sample. DNA sequencing revealed two linked polymorphisms in the 5' flanking region of CYP7. The allele defined by these polymorphisms was associated with increased plasma LDL-C concentrations, both in sibling pairs and in unrelated individuals. Taken together, these findings indicate that polymorphism in CYP7 contributes to heritable variation in plasma LDL-C concentrations. Common polymorphisms in LDLR and APOB account for little of the heritable variation in plasma LDL-C concentrations in the general population. PMID:9502769

  17. The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution

    PubMed Central

    Vendelboe, Trine V.; Harris, Pernille; Zhao, Yuguang; Walter, Thomas S.; Harlos, Karl; El Omari, Kamel; Christensen, Hans E. M.

    2016-01-01

    The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson’s disease, schizophrenia, Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system. PMID:27152332

  18. 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

  19. Cinnamate-4-hydroxylase expression in Arabidopsis. Regulation in response to development and the environment.

    PubMed Central

    Bell-Lelong, D A; Cusumano, J C; Meyer, K; Chapple, C

    1997-01-01

    Cinnamate-4-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-beta-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven beta-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. PMID:9085570

  20. HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.

    PubMed

    Maxwell, Patrick H; Eckardt, Kai-Uwe

    2016-03-01

    Small-molecule stabilizers of hypoxia inducible factor (HIF) are being developed for the treatment of renal anaemia. These molecules inhibit prolyl hydroxylase domain-containing (PHD) enzymes, resulting in HIF activation and increased production of erythropoietin. Currently, renal anaemia is treated with recombinant human erythropoietin or related analogues, referred to as conventional erythropoiesis stimulating agents (ESAs). Advantages of PHD enzyme inhibitors over conventional ESAs include their oral administration and their simpler - and potentially cheaper - production. Importantly, inhibition of PHD enzymes is likely to have a range of consequences other than increasing levels of erythropoietin, and these effects could be beneficial - for instance by reducing the need for parenteral iron - but might in some instances be harmful. Several companies are currently testing PHD enzyme inhibitors in patients with renal anaemia and have reported clear evidence of efficacy without serious safety concerns. A central question that current studies are beginning to address is whether using PHD enzyme inhibitors will influence hard end points, including mortality and the rate of cardiovascular events. In terms of approaches to therapy, the exquisite specificity of conventional ESAs is a striking contrast to the pleiotropic effects of activating HIF. Excitingly, PHD inhibitors could also be useful for conditions besides renal anaemia, such as protection from ischaemic injury. PMID:26656456

  1. Tyrosine Hydroxylase is crucial for maintaining pupal tanning and immunity in Anopheles sinensis

    PubMed Central

    Qiao, Liang; Du, Minghui; Liang, Xin; Hao, Youjin; He, Xiu; Si, Fengling; Mei, Ting; Chen, Bin

    2016-01-01

    Tyrosine hydroxylase (TH), the initial enzyme in the melanin pathway, catalyzes tyrosine conversion into Dopa. Although expression and regulation of TH have been shown to affect cuticle pigmentation in insects, no direct functional studies to date have focused on the specific physiological processes involving the enzyme during mosquito development. In the current study, silencing of AsTH during the time period of continuous high expression in Anopheles sinensis pupae led to significant impairment of cuticle tanning and thickness, imposing a severe obstacle to eclosion in adults. Meanwhile, deficiency of melanin in interference individuals led to suppression of melanization, compared to control individuals. Consequently, the ability to defend exogenous microorganisms declined sharply. Accompanying down-regulation of the basal expression of five antimicrobial peptide genes resulted in further significant weakening of immunity. TH homologs as well as the composition of upstream transcription factor binding sites at the pupal stage are highly conserved in the Anopheles genus, implying that the TH-mediated functions are crucial in Anopheles. The collective evidence strongly suggests that TH is essential for Anopheles pupae tanning and immunity and provides a reference for further studies to validate the utility of the key genes involved in the melanization pathway in controlling mosquito development. PMID:27416870

  2. Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea

    PubMed Central

    Lambrus, Bramwell G.; Cochet-Escartin, Olivier; Gao, Jiarong; Newmark, Phillip A.; Collins, Eva-Maria S.; Collins, James J.

    2015-01-01

    Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems. PMID:26017970

  3. The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution.

    PubMed

    Vendelboe, Trine V; Harris, Pernille; Zhao, Yuguang; Walter, Thomas S; Harlos, Karl; El Omari, Kamel; Christensen, Hans E M

    2016-04-01

    The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system. PMID:27152332

  4. Hypoxia. Regulation of NFκB signalling during inflammation: the role of hydroxylases

    PubMed Central

    Oliver, Kathryn M; Taylor, Cormac T; Cummins, Eoin P

    2009-01-01

    NFκB is a master regulator of innate immunity and inflammatory signalling. Microenvironmental hypoxia has long been identified as being coincident with chronic inflammation. The contribution of microenvironmental hypoxia to NFκB-induced inflammation has more recently been appreciated. Identification of the co-regulation of NFκB and hypoxia inducible factor (HIF) pathways by 2-oxo-glutarate-dependent hydroxylase family members has highlighted an intimate relationship between NFκB inflammatory signalling and HIF-mediated hypoxic signalling pathways. Adding another layer of complexity to our understanding of the role of NFκB inflammatory signalling by hypoxia is the recent recognition of the contribution of basal NFκB activity to HIF-1α transcription. This observation implicates an important and previously unappreciated role for NFκB in inflammatory disease where HIF-1α is activated. The present review will discuss recent literature pertaining to the regulation of NFκB inflammatory signalling by hypoxia and some of the inflammatory diseases where this may play an important role. Furthermore, we will discuss the potential for prolylhydroxylase inhibitors in inflammatory disease. PMID:19291263

  5. Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

    PubMed Central

    Rolland, Anne-Sophie; Kareva, Tatyana; Yarygina, Olga; Kholodilov, Nikolai; Burke, Robert E

    2016-01-01

    The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson’s disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications. PMID:27689101

  6. FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase

    PubMed Central

    Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo

    2016-01-01

    Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312

  7. 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

  8. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    PubMed Central

    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.

    2016-01-01

    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. PMID:27561929

  9. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

    PubMed

    Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2014-07-18

    5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

  10. Evaluation of impact of steroid replacement treatment on bone health in children with 21-hydroxylase deficiency.

    PubMed

    Delvecchio, M; Soldano, L; Lonero, A; Ventura, A; Giordano, P; Cavallo, L; Grano, M; Brunetti, G; Faienza, M F

    2015-04-01

    There are conflicting data regarding the potential impact of chronic glucocorticoid (GC) therapy on the bone mineral density of patients with congenital adrenal hyperplasia (CAH). Previous studies performed by dual-energy X-ray absorptiometry reported conflicting results. The purpose of this study was to assess the impact of chronic GC replacement treatment in children with classical and non classical CAH due to 21-hydroxylase deficiency (21-OHD) by quantitative ultrasonometry (QUS), an easy, cheap, and radiation-free technique. The study population consisted of nineteen 21-OHD patients (nine males) on lifelong GC treatment. Anthropometric, hormonal, and treatment data were recorded for each patient, and bone quality was assessed by QUS measurements. QUS findings (amplitude-dependent speed of sound and bone transmission time) were normal in 21-OHD patients and did not correlate with duration of treatment, daily, total, and yearly hydrocortisone dose. Furthermore, no significant correlation was found between QUS findings and 17α-hydroxy progesterone, Δ4-androstenedione, and testosterone levels. In conclusion, our results provide reassurance that currently used replacement doses of GC do not have a major impact on bone in patients with CAH. QUS seems to be a reliable tool for screening of bone health in children with 21-OHD.

  11. Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

    PubMed

    Huang, Mei; Paglialunga, Sabina; Wong, Julia M-K; Hoang, Monica; Pillai, Renjitha; Joseph, Jamie W

    2016-03-01

    Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic β-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion. PMID:26997627

  12. Gibberellin (GA) biosynthesis in elongating internodes Zea mays (maize): The 3. beta. -hydroxylase

    SciTech Connect

    Spray, C.R.; Phinney, B.O. ); Gaskin, P.; MacMillan, J. )

    1989-04-01

    The early-13-hydroxylation pathway for GA biosynthesis in maize leads to GA{sub 1}, the main GA responsible for shoot elongation. Growth response data, double-labeled feeding studies and endogenous GA levels suggest that the dwarf-1 mutant blocks the step GA{sub 20}to GA{sub 1}. We are screening for a system from maize from which we can purify the 3{beta}-hydroxylase and study its physical and biological properties. We find that diced internodal tissues will metabolize ({sup 13}C, {sup 3}H)-GA{sub 20} to ({sup 13}C, {sup 3}H)-GA{sub 1} and ({sup 13}C, {sup 3}H)-GA{sub 29} with metabolism as high as 80%. A cell free system from this material will give 5% metabolism. In a typical experiment 1g of internodal tissue is frozen in liquid nitrogen and macerated in 0.1M Tris plus cofactors. The homogenate is centrifuged at 15000 x g for 30 min at 4{degrees}C and the supernatant used for metabolic studies.

  13. 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.

  14. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

    PubMed Central

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

    2016-01-01

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

  15. The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability.

    PubMed

    Neira, José L; Hornos, Felipe; Bacarizo, Julio; Cámara-Artigás, Ana; Gómez, Javier

    2016-06-21

    Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements. PMID:27224548

  16. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  17. Detection of methyl salicylate using bi-enzyme electrochemical sensor consisting salicylate hydroxylase and tyrosinase.

    PubMed

    Fang, Yi; Bullock, Hannah; Lee, Sarah A; Sekar, Narendran; Eiteman, Mark A; Whitman, William B; Ramasamy, Ramaraja P

    2016-11-15

    Volatile organic compounds have been recognized as important marker chemicals to detect plant diseases caused by pathogens. Methyl salicylate has been identified as one of the most important volatile organic compounds released by plants during a biotic stress event such as fungal pathogen infection. Advanced detection of these marker chemicals could help in early identification of plant diseases and has huge significance for agricultural industry. This work describes the development of a novel bi-enzyme based electrochemical biosensor consisting of salicylate hydroxylase and tyrosinase enzymes immobilized on carbon nanotube modified electrodes. The amperometric detection using the bi-enzyme platform was realized through a series of cascade reactions that terminate in an electrochemical reduction reaction. Electrochemical measurements revealed that the sensitivity of the bi-enzyme sensor was 30.6±2.7µAcm(-2)µM(-1) and the limit of detection and limit of quantification were 13nM (1.80ppb) and 39nM (5.39ppb) respectively. Interference studies showed no significant interference from the other common plant volatile compounds. Synthetic analyte studies revealed that the bi-enzyme based biosensor can be used to reliably detect methyl salicylate released by unhealthy plants. PMID:27236726

  18. Aspartate-β-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma.

    PubMed

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P; Martin, William; De Groot, Anne S; Gregory, Stephen H; Wands, Jack R

    2015-03-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA class I and II peptides to generate antigen specific CD4(+) and CD8(+) immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4(+) T cells and CD8(+) cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4(+) and CD8(+) T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity.

  19. Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.

    PubMed

    Fornalé, Silvia; Rencoret, Jorge; Garcia-Calvo, Laura; Capellades, Montserrat; Encina, Antonio; Santiago, Rogelio; Rigau, Joan; Gutiérrez, Ana; Del Río, José-Carlos; Caparros-Ruiz, David

    2015-07-01

    Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves.

  20. Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo.

    PubMed

    Rolland, Anne-Sophie; Kareva, Tatyana; Yarygina, Olga; Kholodilov, Nikolai; Burke, Robert E

    2016-01-01

    The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications. PMID:27689101

  1. Inhibition of human natural killer cell functional activity by human aspartyl β-hydroxylase.

    PubMed

    Huyan, Ting; Li, Qi; Ye, Lin-Jie; Yang, Hui; Xue, Xiao-Ping; Zhang, Ming-Jie; Huang, Qing-Sheng; Yin, Da-Chuan; Shang, Peng

    2014-12-01

    Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl β-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.

  2. Molecular docking of bacosides with tryptophan hydroxylase: a model to understand the bacosides mechanism.

    PubMed

    Rajathei, David Mary; Preethi, Jayakumar; Singh, Hemant K; Rajan, Koilmani Emmanuvel

    2014-08-01

    Tryptophan hydroxylase (TPH) catalyses l-tryptophan into 5-hydroxy-l-tryptophan, which is the first and rate-limiting step of serotonin (5-HT) biosynthesis. Earlier, we found that TPH2 up-regulated in the hippocampus of postnatal rats after the oral treatment of Bacopa monniera leaf extract containing the active compound bacosides. However, the knowledge about the interactions between bacosides with TPH is limited. In this study, we take advantage of in silico approach to understand the interaction of bacoside-TPH complex using three different docking algorithms such as HexDock, PatchDock and AutoDock. All these three algorithms showed that bacoside A and A3 well fit into the cavity consists of active sites. Further, our analysis revealed that major active compounds bacoside A3 and A interact with different residues of TPH through hydrogen bond. Interestingly, Tyr235, Thr265 and Glu317 are the key residues among them, but none of them are either at tryptophan or BH4 binding region. However, its note worthy to mention that Tyr 235 is a catalytic sensitive residue, Thr265 is present in the flexible loop region and Glu317 is known to interacts with Fe. Interactions with these residues may critically regulate TPH function and thus serotonin synthesis. Our study suggested that the interaction of bacosides (A3/A) with TPH might up-regulate its activity to elevate the biosynthesis of 5-HT, thereby enhances learning and memory formation.

  3. 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.

  4. A unique case of female pseudohermaphroditism with 21-hydroxylase deficiency and small supernumerary marker chromosome 7.

    PubMed

    Al-Achkar, Walid; Wafa, Abdulsamad; Assaad, Manar; Ehlers, Christian; Liehr, Thomas

    2013-05-01

    Small supernumerary marker chromosomes (sSMCs) are present in ~2.6x10⁶ individuals worldwide. Concerning their clinical consequences as well as their chromosomal origin and shape, sSMCs are a heterogeneous group of derivative chromosomes; 70% of sSMC carriers are clinically normal. In the present study, we report on a female with mosaicism (45%) of a de novo sSMC derived from chromosome 7, in which the observed clinical signs do not correspond to comparable cases in the literature. She is clinically normal apart from problems in gender determination, a uterus without ovaries and an external penis, pointing overall towards an adrenogenital syndrome (AGS). 21-Hydroxylase deficiency (21-OHD) is the most common cause of AGS. A corresponding analysis for underlying mutations in the CYP21A2 gene revealed a homozygous mutation c.518T>A (p.Ile173Asn) inherited from both non-related parents. Overall, in this study, we report a unique case of female pseudohermaphroditism, classified as a simple virilization form of 21-OHD having an additional minute-shaped chromosome 7 [min(7)(:p11.1->q11.23:)]. Notably, AGS was due to a mutation in the CYP21A2 gene located on chromosome 6. This is a further example that detection of an sSMC does not always resolve the clinical case. PMID:23450434

  5. Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

    SciTech Connect

    Goodman, R.; Slater, E.; Herschman, H.R.

    1980-03-01

    We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EFG has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3 to 4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.

  6. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

    PubMed

    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

    2016-01-01

    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. PMID:27561929

  7. Tyrosine Hydroxylase is crucial for maintaining pupal tanning and immunity in Anopheles sinensis.

    PubMed

    Qiao, Liang; Du, Minghui; Liang, Xin; Hao, Youjin; He, Xiu; Si, Fengling; Mei, Ting; Chen, Bin

    2016-01-01

    Tyrosine hydroxylase (TH), the initial enzyme in the melanin pathway, catalyzes tyrosine conversion into Dopa. Although expression and regulation of TH have been shown to affect cuticle pigmentation in insects, no direct functional studies to date have focused on the specific physiological processes involving the enzyme during mosquito development. In the current study, silencing of AsTH during the time period of continuous high expression in Anopheles sinensis pupae led to significant impairment of cuticle tanning and thickness, imposing a severe obstacle to eclosion in adults. Meanwhile, deficiency of melanin in interference individuals led to suppression of melanization, compared to control individuals. Consequently, the ability to defend exogenous microorganisms declined sharply. Accompanying down-regulation of the basal expression of five antimicrobial peptide genes resulted in further significant weakening of immunity. TH homologs as well as the composition of upstream transcription factor binding sites at the pupal stage are highly conserved in the Anopheles genus, implying that the TH-mediated functions are crucial in Anopheles. The collective evidence strongly suggests that TH is essential for Anopheles pupae tanning and immunity and provides a reference for further studies to validate the utility of the key genes involved in the melanization pathway in controlling mosquito development. PMID:27416870

  8. Improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate

    SciTech Connect

    Junker, L.H.; Story, J.A.

    1985-10-01

    A persistent problem in measurement of cholesterol 7 alpha-hydroxylase (7 alpha-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7 alpha-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 microM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7 alpha-hydroxycholesterol formed. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.

  9. 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

  10. Putaminal Mosaic Visualized by Tyrosine Hydroxylase Immunohistochemistry in the Human Neostriatum.

    PubMed

    Morigaki, Ryoma; Goto, Satoshi

    2016-01-01

    Among the basal ganglia-thalamocortical circuits, the putamen plays a critical role in the "motor" circuits that control voluntary movements and motor learning. The human neostriatum comprises two functional subdivisions known as the striosome (patch) and matrix compartments. Accumulating evidence suggests that compartment-specific dysregulations of dopamine activity might be involved in the disease-specific pathology and symptoms of human striatal diseases including movement disorders. This study was undertaken to examine whether or how striatal dopaminergic innervations are organized into the compartmentalized architecture found in the putamen of adult human brains. For this purpose, we used a highly sensitive immunohistochemistry (IHC) technique to identify tyrosine hydroxylase (TH; EC 1.14.16.2), a marker for striatal dopaminergic axons and terminals, in formalin-fixed paraffin-embedded (FFPE) tissues obtained from autopsied human brains. Herein, we report that discrete compartmentalization of TH-labeled innervations occurs in the putamen, as in the caudate nucleus (CN), with a higher density of TH labeling in the matrix compared to the striosomes. Our results provide anatomical evidence to support the hypothesis that compartment-specific dysfunction of the striosome-matrix dopaminergic systems might contribute to the genesis of movement disorders. PMID:27092059

  11. Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.

    PubMed

    Fornalé, Silvia; Rencoret, Jorge; Garcia-Calvo, Laura; Capellades, Montserrat; Encina, Antonio; Santiago, Rogelio; Rigau, Joan; Gutiérrez, Ana; Del Río, José-Carlos; Caparros-Ruiz, David

    2015-07-01

    Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves. PMID:26025540

  12. Molecular docking of bacosides with tryptophan hydroxylase: a model to understand the bacosides mechanism.

    PubMed

    Rajathei, David Mary; Preethi, Jayakumar; Singh, Hemant K; Rajan, Koilmani Emmanuvel

    2014-08-01

    Tryptophan hydroxylase (TPH) catalyses l-tryptophan into 5-hydroxy-l-tryptophan, which is the first and rate-limiting step of serotonin (5-HT) biosynthesis. Earlier, we found that TPH2 up-regulated in the hippocampus of postnatal rats after the oral treatment of Bacopa monniera leaf extract containing the active compound bacosides. However, the knowledge about the interactions between bacosides with TPH is limited. In this study, we take advantage of in silico approach to understand the interaction of bacoside-TPH complex using three different docking algorithms such as HexDock, PatchDock and AutoDock. All these three algorithms showed that bacoside A and A3 well fit into the cavity consists of active sites. Further, our analysis revealed that major active compounds bacoside A3 and A interact with different residues of TPH through hydrogen bond. Interestingly, Tyr235, Thr265 and Glu317 are the key residues among them, but none of them are either at tryptophan or BH4 binding region. However, its note worthy to mention that Tyr 235 is a catalytic sensitive residue, Thr265 is present in the flexible loop region and Glu317 is known to interacts with Fe. Interactions with these residues may critically regulate TPH function and thus serotonin synthesis. Our study suggested that the interaction of bacosides (A3/A) with TPH might up-regulate its activity to elevate the biosynthesis of 5-HT, thereby enhances learning and memory formation. PMID:25089244

  13. Overexpression of beta-carotene hydroxylase enhances stress tolerance in Arabidopsis.

    PubMed

    Davison, P A; Hunter, C N; Horton, P

    2002-07-11

    Plant stress caused by extreme environmental conditions is already a principal reason for yield reduction in crops. The threat of global environment change makes it increasingly important to generate crop plants that will withstand such conditions. Stress, particularly stress caused by increased sunlight, leads to the production of reactive oxygen species that cause photo-oxidative cell damage. Carotenoids, which are present in the membranes of all photosynthetic organisms, help protect against such light-dependent oxidative damage. In plants, the xanthophyll cycle (the reversible interconversion of two carotenoids, violaxanthin and zeaxanthin) has a key photoprotective role and is therefore a promising target for genetic engineering to enhance stress tolerance. Here we show that in Arabidopsis thaliana overexpression of the chyB gene that encodes beta-carotene hydroxylase--an enzyme in the zeaxanthin biosynthetic pathway--causes a specific twofold increase in the size of the xanthophyll cycle pool. The plants are more tolerant to conditions of high light and high temperature, as shown by reduced leaf necrosis, reduced production of the stress indicator anthocyanin and reduced lipid peroxidation. Stress protection is probably due to the function of zeaxanthin in preventing oxidative damage of membranes. PMID:12110893

  14. 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

  15. 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

  16. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    PubMed

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  17. Measurement of the intramolecular isotope effect on aliphatic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.

    PubMed

    Panay, Aram J; Fitzpatrick, Paul F

    2010-04-28

    The non-heme iron enzyme phenylalanine hydroxylase from Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[(2)H(11)-cyclohexyl]alanine as the substrate, the isotope effect on the k(cat) value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-(2)H(6)-cyclohexyl]alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.

  18. Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.

    PubMed

    Zhang, Shengnan; Fitzpatrick, Paul F

    2016-04-01

    Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

  19. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.

    PubMed

    Bezem, Maria T; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  20. Human phenylalanine hydroxylase mutations and hyperphenylalaninemia phenotypes: a metanalysis of genotype-phenotype correlations.

    PubMed Central

    Kayaalp, E; Treacy, E; Waters, P J; Byck, S; Nowacki, P; Scriver, C R

    1997-01-01

    We analyzed correlations between mutant genotypes at the human phenylalanine hydroxylase locus (gene symbol PAH) and the corresponding hyperphenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of "functionally hemizygous" heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus. PMID:9399896

  1. CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase.

    PubMed

    Ly, Thuy T B; Khatri, Yogan; Zapp, Josef; Hutter, Michael C; Bernhardt, Rita

    2012-07-01

    Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations. PMID:22223101

  2. IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

    PubMed Central

    Kawaai, Katsuhiro; Mizutani, Akihiro; Shoji, Hirotaka; Ogawa, Naoko; Ebisui, Etsuko; Kuroda, Yukiko; Wakana, Shigeharu; Hisatsune, Chihiro; Mikoshiba, Katsuhiko

    2015-01-01

    Inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with IP3 (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation. PMID:25922519

  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. High-Dose Hook Effect in 17-Hydroxyprogesterone Assay in a Patient with 21-Hydroxylase Deficiency

    PubMed Central

    Parlak, Mesut; Ellidağ, Hamit Yaşar; Türkkahraman, Doğa

    2015-01-01

    Congenital adrenal hyperplasia (CAH) describes a group of disorders characterized by enzyme defects in adrenal steroidogenesis. 21-hydroxylase deficiency (21-OHD) is the most commonly encountered form. The analysis of steroids in pediatric cases requires high-sensitivity assays. A 14-year-old Syrian girl was referred for evaluation of short stature, amenorrhea, and hirsutism. On physical examination, breast development was Tanner stage 1. She had a phallic clitoris with a single urogenital orifice. Laboratory findings revealed primary adrenal deficiency with high androgen levels and low levels of 17-hydroxyprogesterone (17-OHP), (<0.05 ng/mL) and estrogen. This unexpected result led to suspicion of a high-dose hook effect. The measurement was repeated after 1/10 dilution of serum, and a high level of 17-OHP (115.4 ng/mL) was detected with the same test-enzyme-linked immunosorbent assay (ELISA). Simple virilizing form of CAH (21-OHD) was suspected and confirmed with genetic analysis. After initiation of glucocorticoid therapy, breast development was noted along with a decrease in testosterone level and an increase in estrogen level. To our knowledge, this is the first case report of hook effect for 17-OHP immunoassay in a patient with 21-OHD. High-dose hook effect should be suspected in patients with CAH when the test results are incompatible with one another. Additionally, this case demonstrates that a high testosterone level can block aromatase activity and consequently also estrogen production and breast development. PMID:26777045

  5. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons

    PubMed Central

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease. PMID:26886559

  6. Identification of tyrosine hydroxylase as a physiological substrate for Cdk5.

    PubMed

    Kansy, Janice W; Daubner, S Colette; Nishi, Akinori; Sotogaku, Naoki; Lloyd, Michael D; Nguyen, Chan; Lu, Lin; Haycock, John W; Hope, Bruce T; Fitzpatrick, Paul F; Bibb, James A

    2004-10-01

    Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.

  7. 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

  8. Parathyroidectomy reduces 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the hypocalcemic vitamin D-deficient chick.

    PubMed Central

    Booth, B E; Tsai, H C; Morris, R C

    1977-01-01

    To test the hypothesis that in the vitamin D-deficient state the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase (25-OHD3-1 alpha-hydroxylase) is modulated by parathyroid hormone and the plasma concentration of phosphate only in the presence of small amounts of 1,25-dihydroxyvitamin D3 (or some other metabolite of vitamin D), we measured the activity of this enzyme 24 h after parathyroidectomy (PTX) in frankly hypocalcemic, vitamin D-deficient chicks that were not supplemented with vitamin D or one of its metabolites. The otherwise predictable complications of PTX in this metabolic setting (hypocalcemia of increasing severity, tetany, moribundity, and death) were prevented by continuous intravenous administration of calcium (as a solution of calcium chloride/calcium gluconate 1:1) through a catheter in the external jugular vein placed at the time of PTX. The findings were as follows: (a) The activity of 25-OHD3-1 alpha-hydroxylase was significantly less in the parathyroidectomized group than in the sham-operated control chicks (P less than 0.001). (b) The reductive effect of PTX on the activity of this enzyme was significantly attenuated when hypophosphatemia was increased in severity by administration of glucose. (c) In the post-PTX state the activity of 25-OHD3-1 alpha-hydroxylase and plasma concentration of phosphate were significantly, inversely related (P less than 0.001). (d) In the sham-operated control group the activity of this enzyme and the plasma concentration of phosphate were not significantly correlated. These findings indicate that in the vitamin D-deficient state, both circulating parathyroid hormone and the plasma concentration of phosphate can significantly modulate the activity of 25-OHD3-1 alpha-hydroxylase in the absence of vitamin D or its metabolites. The findings also suggest that in the vitamin D-deficient state the plasma concentration of phosphate modulates the activity of this enzyme only when the concentration of circulating

  9. Biosynthesis and metabolism of salicylic acid

    SciTech Connect

    Lee, H.; Leon, J.; Raskin, I.

    1995-05-09

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-{beta}-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. 62 refs., 1 fig.

  10. [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.

  11. Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis*

    PubMed Central

    Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.

    2011-01-01

    Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574

  12. [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

  13. Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis1[W][OA

    PubMed Central

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-01-01

    Flavanone 3β-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the β-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction). PMID:17535823

  14. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration. PMID:19560175

  15. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  16. 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

  17. Estrogen receptor β sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription.

    PubMed

    Mak, Paul; Chang, Cheng; Pursell, Bryan; Mercurio, Arthur M

    2013-03-19

    Estrogen receptor β (ERβ) promotes the degradation of hypoxia inducible factor 1α (HIF-1α), which contributes to the ability of this hormone receptor to sustain the differentiation of epithelial and carcinoma cells. Although the loss of ERβ and consequent HIF-1 activation occur in prostate cancer with profound consequences, the mechanism by which ERβ promotes the degradation of HIF-1α is unknown. We report that ERβ regulates the ligand (3β-adiol)-dependent transcription of prolyl hydroxylase 2 (PHD2) also known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-dependent dioxygenase that hydroxylates HIF-1α and targets it for recognition by the von Hippel-Lindau tumor suppressor and consequent degradation. ERβ promotes PHD2 transcription by interacting with a unique estrogen response element in the 5' UTR of the PHD2 gene that functions as an enhancer. PHD2 itself is critical for maintaining epithelial differentiation. Loss of PHD2 expression or inhibition of its function results in dedifferentiation with characteristics of an epithelial-mesenchymal transition, and exogenous PHD2 expression in dedifferentiated cells can restore an epithelial phenotype. Moreover, expression of HIF-1α in cells that express PHD2 does not induce dedifferentiation but expression of HIF-1α containing mutations in the proline residues that are hydroxylated by PHD2 induces dedifferentiation. These data describe a unique mechanism for the regulation of HIF-1α stability that involves ERβ-mediated transcriptional regulation of PHD2 and they highlight an unexpected role for PHD2 in maintaining epithelial differentiation.

  18. A Synopsis on the Role of Tyrosine Hydroxylase in Parkinson's Disease

    PubMed Central

    Tabrez, Shams; Jabir, Nasimudeen R.; Shakil, Shazi; Greig, Nigel H.; Alam, Qamre; Abuzenadah, Adel M.; Damanhouri, Ghazi A.; Kamal, Mohammad A.

    2016-01-01

    Parkinson's disease (PD) is a common chronic progressive neurodegenerative disorder in elderly people. A consistent neurochemical abnormality in PD is degeneration of dopaminergic neurons in substantia nigra pars compacta, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation of L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Problems related to PD usually build up when vesicular storage of DA is altered by the presence of either α-synuclein protofibrils or oxidative stress. Phosphorylation of three physiologically-regulated specific sites of N-terminal domain of TH is vital in regulating its kinetic and protein interaction. The concept of physiological significance of TH isoforms is another interesting aspect to be explored further for a comprehensive understanding of its role in PD. Thus, a logical and efficient strategy for PD treatment is based on correcting or bypassing the enzyme deficiency by the treatment with L-DOPA, DA agonists, inhibitors of DA metabolism or brain grafts with cells expressing a high level of TH. Neurotrophic factors are also attracting the attention of neuroscientists because they provide the essential neuroprotective and neurorestorative properties to the nigrostriatal DA system. PPAR-γ, a key regulator of immune responses, is likewise a promising target for the treatment of PD, which can be achieved by the use of agonists with the potential to impact the expression of pro- and anti-inflammatory cytokines at the transcriptional level in immune cells via expression of TH. Herein, we review the primary biochemical and pathological features of PD, and describe both classical and developing approaches aimed to ameliorate disease symptoms and its progression. PMID:22483313

  19. Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases

    PubMed Central

    Bochevarov, Arteum D.; Li, Jianing; Song, Woon Ju; Friesner, Richard A.; Lippard, Stephen J.

    2010-01-01

    The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites yet the physical and chemical properties of their oxygenated intermediates, designated P*, Hperoxo, Q and Q* in MMOH and ToMOHperoxo in ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of Hperoxo and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotation energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T201 and T213 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and the protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOHperoxo intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. PMID:21517016

  20. 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

  1. Functional Implication of β-Carotene Hydroxylases in Soybean Nodulation1[C][W][OA

    PubMed Central

    Kim, Yun-Kyoung; Kim, Sunghan; Um, Ji-Hyun; Kim, Kyunga; Choi, Sun-Kang; Um, Byung-Hun; Kang, Suk-Woo; Kim, Jee-Woong; Takaichi, Shinichi; Song, Seok-Bo; Lee, Choon-Hwan; Kim, Ho-Seung; Kim, Ki Woo; Nam, Kyoung Hee; Lee, Suk-Ha; Kim, Yul-Ho; Park, Hyang-Mi; Ha, Sun-Hwa; Verma, Desh Pal S.; Cheon, Choong-Ill

    2013-01-01

    Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative β-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation. PMID:23700351

  2. 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

  3. Alzheimer disease and type 2 diabetes mellitus: the link to tyrosine hydroxylase and probable nutritional strategies.

    PubMed

    Aliev, Gjumrakch; Shahida, Khan; Gan, Siew Hua; Firoz, Ck; Khan, Aziz; Abuzenadah, Adel M; Kamal, Warda; Kamal, Mohammad A; Tan, Yi; Qu, Xianqin; Reale, Marcella

    2014-04-01

    Alzheimer disease (AD) and type 2 diabetes mellitus (T2DM) are chronic health disorders that affect millions of people around the world. According to recent studies, there are molecular similarities in the inflammatory pathways involved in both AD and T2DM, which opens a new avenue for researchers with different perspectives to target the cause of these diseases rather than their obvious symptoms. Several links between inflammation, cardiovascular disease, T2DM and central nervous system disorders such as AD and Parkinson's disease have been elucidated. Mutations in the hippocampal-β-amyloid precursor protein gene in genetically high-risk individuals have been shown to cause the early onset of AD symptoms. The overexpression of β-amyloid protein in the hippocampal region and the synaptotoxicity that occurs as a result have been considered a typical feature of AD and leads to neuronal loss and cognitive decline. However, the identity of the cellular components that cause the late onset of the disease seen in the majority of the cases is still unknown. Synaptic insults associated with neuronal dysfunction may involve several cascades and molecules, one of which has been hypothesized to be tyrosine hydroxylase (TH). The axons of the noradrenergic cells that project to the hippocampus appear to be affected by the β-amyloid protein, which subsequently contributes to TH loss in Alzheimer brain cells. In this review, we attempt to shed light on the important mechanisms involved in AD as well as T2DM such as inflammatory factors, abnormalities in the insulin signaling system and the possible role of the endocrine enzyme TH.

  4. Dopamine β-Hydroxylase Inhibitors Enhance the Discriminative Stimulus Effects of Cocaine in Rats

    PubMed Central

    Manvich, Daniel F.; DePoy, Lauren M.

    2013-01-01

    Inhibitors of dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic cells, have shown promise for the treatment of cocaine abuse disorders. However, the mechanisms underlying the beneficial effects of these compounds have not been fully elucidated. We used the drug discrimination paradigm to determine the impact of DBH inhibitors on the interoceptive stimulus properties of cocaine. Sprague-Dawley rats were trained to discriminate cocaine (5.6 mg/kg) from saline using a multicomponent, food-reinforced discrimination procedure. On test days, subjects were pretreated with the nonselective DBH inhibitor disulfiram (0–100.0 mg/kg i.p.) or the selective DBH inhibitor nepicastat (0–56.0 mg/kg i.p.) 2 hours prior to a test session either alone or in combination with cumulatively administered cocaine (0–5.6 mg/kg i.p.). Neither disulfiram nor nepicastat substituted for the cocaine stimulus when tested up to doses that nonspecifically reduced responding. However, in combination studies, pretreatment with either disulfiram or nepicastat produced leftward shifts in the cocaine dose-response function and also conferred cocaine-like stimulus effects to the selective NE transporter inhibitor, reboxetine (0.3–5.6 mg/kg i.p.). These results indicate that pharmacological inhibition of DBH does not produce cocaine-like interoceptive stimulus effects alone, but functionally enhances the interoceptive stimulus effects of cocaine, possibly due to facilitated increases in DA released from noradrenergic terminals. These findings suggest that DBH inhibitors have low abuse liability and provide support to clinical reports that some subjective effects produced by cocaine, particularly aversive effects, are enhanced after DBH inhibition. PMID:24068832

  5. Chronic Inhibition of Dopamine β-Hydroxylase Facilitates Behavioral Responses to Cocaine in Mice

    PubMed Central

    Gaval-Cruz, Meriem; Liles, Larry Cameron; Iuvone, Paul Michael; Weinshenker, David

    2012-01-01

    The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh −/−) mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/−) and Dbh −/− mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh −/− mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/− mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/− mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh −/− mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor) enhance qualitatively different cocaine-induced behaviors. PMID:23209785

  6. 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

  7. A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity.

    PubMed

    Huebener, Nicole; Fest, Stefan; Strandsby, Anne; Michalsky, Elke; Preissner, Robert; Zeng, Yan; Gaedicke, Gerhard; Lode, Holger N

    2008-07-01

    Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.

  8. Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma.

    PubMed

    Huebener, Nicole; Fest, Stefan; Hilt, Kerstin; Schramm, Alexander; Eggert, Angelika; Durmus, Tahir; Woehler, Anja; Stermann, Alexander; Bleeke, Matthias; Baykan, Bianca; Weixler, Silke; Gaedicke, Gerhard; Lode, Holger N

    2009-08-01

    Neuroblastoma (NB) is a challenging malignancy of the sympathetic nervous tissue characterized by a very poor prognosis. One important marker for NB is the expression of tyrosine hydroxylase (TH), the first-step enzyme of catecholamine biosynthesis. We could show stable and high TH gene expression in 67 NB samples independent of the clinical stage. Based on this observation, we addressed the question of whether xenogeneic TH DNA vaccination is effective in inducing an anti-NB immune response. For this purpose, we generated three DNA vaccines based on pCMV-F3Ub and pBUD-CE4.1 plasmids encoding for human (h)THcDNA (A), hTH minigene (B), and hTHcDNA in combination with the proinflammatory cytokine interleukin 12 (C), and tested prophylactic and therapeutic efficacy to suppress primary tumor growth and spontaneous metastasis. Here we report that xenogeneic TH DNA vaccination was effective in eradicating established primary tumors and inhibiting metastasis. Interestingly, this effect could not be enhanced by adding the Th1 cytokine interleukin 12. However, increased IFN-gamma production and NB cytotoxicity of effector cells harvested from vaccinated mice suggested the participation of tumor-specific CTLs in the immune response. The depletion of CD8(+)T cells completely abrogated the hTH vaccine-mediated anti-NB immune response. Furthermore, rechallenging of surviving mice resulted in reduced primary tumor growth, indicating the induction of a memory immune response. In conclusion, xenogeneic immunization with TH-derived DNA vaccines is effective against NB, and may open a new venue for a novel and effective immunotherapeutic strategy against this challenging childhood tumor.

  9. 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

  10. Molecular genetic analysis of the cytochrome P450-debrisoquine hydroxylase locus and association with cancer susceptibility.

    PubMed Central

    Smith, C A; Moss, J E; Gough, A C; Spurr, N K; Wolf, C R

    1992-01-01

    The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including beta-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population. PMID:1486838

  11. Dopamine- and Tyrosine Hydroxylase-Immunoreactive Neurons in the Brain of the American Cockroach, Periplaneta americana

    PubMed Central

    Hamanaka, Yoshitaka; Minoura, Run; Nishino, Hiroshi; Miura, Toru; Mizunami, Makoto

    2016-01-01

    The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine’s actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of “dopaminergic” neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory

  12. Association of Dopamine Beta-Hydroxylase (DBH) Polymorphisms with Susceptibility to Parkinson’s Disease

    PubMed Central

    Shao, Peng; Yu, Yun-xia; Bao, Jing-xi

    2016-01-01

    Background The purpose of this study was to explore the association between 2 single-nucleotide polymorphisms (SNPs) in the dopamine β-hydroxylase (DBH) gene (rs1611115 and rs732833) and the susceptibility to Parkinson’s disease (PD). Material/Methods Polymerase chain reaction direct sequencing (PCR-DS) was used to test the genotypes of DBH polymorphisms in 95 PD patients and 100 healthy examinees frequency-matched with the former by age and sex. The genotype and allele distribution differences between the case and control groups were analyzed by chi-square test, and the relative risk of PD in southern Chinese populations was expressed by odds ratio (OR) and 95% confidence interval (CI). Hardy-Weinberg equilibrium (HWE) was also checked by chi-square test. Results The genotype and allele distribution frequencies in rs1611115 were obviously different between PD patients and the healthy control group (P<0.05). The TT genotype may lead to a 2.95 times higher risk of PD occurrence compared with the common genotype CC (OR=2.95, 95%CI=1.02–8.51), and the C allele increased risk of onset of PD (OR=1.81, 95%CI=1.17–2.82). Cognition of the PD patients was different between CC and CT+TT genotypes of rs1611115 (P=0.047). Conclusions DBH rs1611115 polymorphism was likely to be associated with the susceptibility to PD, but we did not find that rs732833 is a susceptibility marker for PD. PMID:27177268

  13. Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in Asian families with phenylketonuria (PKU).

    PubMed

    Daiger, S P; Reed, L; Huang, S S; Zeng, Y T; Wang, T; Lo, W H; Okano, Y; Hase, Y; Fukuda, Y; Oura, T

    1989-08-01

    DNA polymorphisms at the phenylalanine hydroxylase (PAH) locus have proved highly effective in linkage diagnosis of phenylketonuria (PKU) in Caucasian families. More than 10 RFLP sites have been reported within the PAH structural locus in Caucasians. With information from affected and unaffected offspring in PKU families it is often possible to reconstruct complete RFLP haplotypes in parents and to use these haplotypes to follow the segregation of PKU within families and to determine the distribution of PKU chromosomes within populations. To establish the utility of these RFLPs in characterizing Asian families with PKU, we typed eight DNA sites in 21 Chinese families and 12 Japanese families with classical PKU. The eight RFLPs were chosen for their informativeness in Caucasians. From these families we reconstructed a total of 91 complete PAH haplotypes, 44 from non-PKU chromosomes and 47 from PKU-bearing chromosomes. Although all eight marker sites are polymorphic in both Chinese and Japanese, there is much less haplotypic variation in Asians than in Caucasians. In particular, one haplotype alone, haplotype 4, accounts for more than 77% of non-PKU chromosomes and for more than 80% of PKU-bearing chromosomes. Haplotype 4 is also relatively common in Caucasians. The next most common Asian haplotype is 10 times less frequent than haplotype 4. By contrast, in many Caucasian populations the sum of the frequencies of the five most common haplotypes is still less than 80%, and several of the most common haplotypes are equally frequent. Even though the extent of haplotypic variation in Asians is severely limited, the few haplotypes that are found often differ at a number of RFLP sites.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in European families with phenylketonuria (PKU).

    PubMed

    Daiger, S P; Chakraborty, R; Reed, L; Fekete, G; Schuler, D; Berenssi, G; Nasz, I; Brdicka, R; Kamarýt, J; Pijácková, A

    1989-08-01

    DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Ethanol impaired neuronal migration is associated with reduced aspartyl-asparaginyl-beta-hydroxylase expression.

    PubMed

    Carter, Jade J; Tong, Ming; Silbermann, Elizabeth; Lahousse, Stephanie A; Ding, Fei Fei; Longato, Lisa; Roper, Nitin; Wands, Jack R; de la Monte, Suzanne M

    2008-09-01

    Cerebellar hypoplasia in fetal alcohol spectrum disorders (FASD) is associated with inhibition of insulin and insulin-like growth factor (IGF) signaling in the brain. Aspartyl (asparaginyl)-beta-hydroxylase (AAH) is a mediator of neuronal motility, and stimulated by insulin and IGF activation of PI3 kinase-Akt, or inhibition of GSK-3beta. Since ethanol inhibits PI3 Kinase-Akt and increases GSK-3beta activity in brain, we examined the effects of ethanol and GSK-3beta on AAH expression and directional motility in neuronal cells. Control and ethanol-exposed (100 mM x 48 h) human PNET2 cerebellar neuronal cells were stimulated with IGF-1 and used to measure AAH expression and directional motility. Molecular and biochemical approaches were used to characterize GSK-3beta regulation of AAH and neuronal motility. Ethanol reduced IGF-1 stimulated AAH protein expression and directional motility without inhibiting AAH's mRNA. Further analysis revealed that: (1) AAH protein could be phosphorylated by GSK-3beta; (2) high levels of GSK-3beta activity decreased AAH protein; (3) inhibition of GSK-3beta and/or global Caspases increased AAH protein; (4) AAH protein was relatively more phosphorylated in ethanol-treated compared with control cells; and (5) chemical inhibition of GSK-3beta and/or global Caspases partially rescued ethanol-impaired AAH protein expression and motility. Ethanol-impaired neuronal migration is associated with reduced IGF-I stimulated AAH protein expression. This effect may be mediated by increased GSK-3beta phosphorylation and Caspase degradation of AAH. Therapeutic strategies to rectify CNS developmental abnormalities in FASD should target factors underlying the ethanol-associated increases in GSK-3beta and Caspase activation, e.g. IGF resistance and increased oxidative stress. PMID:18478238

  16. Altered tryptophan hydroxylase 2 expression in enteric serotonergic nerves in Hirschsprung’s-associated enterocolitis

    PubMed Central

    Coyle, David; Murphy, Justin M; Doyle, Brian; O’Donnell, Anne Marie; Gillick, John; Puri, Prem

    2016-01-01

    AIM: To determine if expression of colonic tryptophan hydroxylase-2 (TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung’s-associated enterocolitis. METHODS: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung’s disease (HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung’s-associated enterocolitis (HAEC). Controls were collected at colostomy closure in children with anorectal malformation (n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers. RESULTS: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα+ cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zone bowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC. CONCLUSION: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC. PMID:27217698

  17. Serotonin synthesis rate and the tryptophan hydroxylase-2: G-703T polymorphism in social anxiety disorder.

    PubMed

    Furmark, Tomas; Marteinsdottir, Ina; Frick, Andreas; Heurling, Kerstin; Tillfors, Maria; Appel, Lieuwe; Antoni, Gunnar; Hartvig, Per; Fischer, Håkan; Långström, Bengt; Eriksson, Elias; Fredrikson, Mats

    2016-10-01

    It is disputed whether anxiety disorders, like social anxiety disorder, are characterized by serotonin over- or underactivity. Here, we evaluated whether our recent finding of elevated neural serotonin synthesis rate in patients with social anxiety disorder could be reproduced in a separate cohort, and whether allelic variation in the tryptophan hydroxylase-2 (TPH2) G-703T polymorphism relates to differences in serotonin synthesis assessed with positron emission tomography. Eighteen social anxiety disorder patients and six healthy controls were scanned during 60 minutes in a resting state using positron emission tomography and 5-hydroxy-L-[β -(11)C]tryptophan, [(11)C]5-HTP, a substrate of the second enzymatic step in serotonin synthesis. Parametric images were generated, using the reference Patlak method, and analysed using Statistical Parametric Mapping (SPM8). Blood samples for genotyping of the TPH2 G-703T polymorphism were obtained from 16 social anxiety disorder patients (T carriers: n=5, GG carriers: n=11). A significantly elevated [(11)C]5-HTP accumulation rate, indicative of enhanced decarboxylase activity and thereby serotonin synthesis capacity, was detected in social anxiety disorder patients compared with controls in the hippocampus and basal ganglia nuclei and, at a more lenient (uncorrected) statistical threshold, in the amygdala and anterior cingulate cortex. In patients, the serotonin synthesis rate in the amygdala and anterior cingulate cortex was significantly elevated in TPH2 T carriers in comparison with GG homozygotes. Our results support that social anxiety disorder entails an overactive presynaptic serotonergic system that, in turn, seems functionally influenced by the TPH2 G-703T polymorphism in emotionally relevant brain regions.

  18. Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma

    PubMed Central

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P.; Martin, William; De Groot, Anne S.; Gregory, Stephen H.; Wands, Jack R.

    2015-01-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA Class I and II peptides to generate antigen specific CD4+ and CD8+ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4+ and CD8+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity. PMID:25629522

  19. 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.

  20. Association of Dopamine Beta-Hydroxylase (DBH) Polymorphisms with Susceptibility to Parkinson's Disease.

    PubMed

    Shao, Peng; Yu, Yun-Xia; Bao, Jing-Xi

    2016-01-01

    BACKGROUND The purpose of this study was to explore the association between 2 single-nucleotide polymorphisms (SNPs) in the dopamine β-hydroxylase (DBH) gene (rs1611115 and rs732833) and the susceptibility to Parkinson's disease (PD). MATERIAL AND METHODS Polymerase chain reaction direct sequencing (PCR-DS) was used to test the genotypes of DBH polymorphisms in 95 PD patients and 100 healthy examinees frequency-matched with the former by age and sex. The genotype and allele distribution differences between the case and control groups were analyzed by chi-square test, and the relative risk of PD in southern Chinese populations was expressed by odds ratio (OR) and 95% confidence interval (CI). Hardy-Weinberg equilibrium (HWE) was also checked by chi-square test. RESULTS The genotype and allele distribution frequencies in rs1611115 were obviously different between PD patients and the healthy control group (P<0.05). The TT genotype may lead to a 2.95 times higher risk of PD occurrence compared with the common genotype CC (OR=2.95, 95%CI=1.02-8.51), and the C allele increased risk of onset of PD (OR=1.81, 95%CI=1.17-2.82). Cognition of the PD patients was different between CC and CT+TT genotypes of rs1611115 (P=0.047). CONCLUSIONS DBH rs1611115 polymorphism was likely to be associated with the susceptibility to PD, but we did not find that rs732833 is a susceptibility marker for PD. PMID:27177268

  1. Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat.

    PubMed

    Loureiro, Ana I; Bonifácio, Maria João; Fernandes-Lopes, Carlos; Igreja, Bruno; Wright, Lyndon C; Soares-da-Silva, Patrício

    2014-10-01

    Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions. PMID:25058908

  2. 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.

  3. Mapping of tyrosine hydroxylase in the alpaca (Lama pacos) brainstem and colocalization with CGRP.

    PubMed

    Marcos, P; Arroyo-Jimenez, M M; Lozano, G; Aguilar, L A; Coveñas, R

    2011-03-01

    The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics.

  4. 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.

  5. Hypnotic Hypersensitivity to Volatile Anesthetics and Dexmedetomidine in Dopamine β-Hydroxylase Knockout Mice

    PubMed Central

    Hu, Frances Y.; Hanna, George M.; Han, Wei; Mardini, Feras; Thomas, Steven A.; Wyner, Abraham J.; Kelz, Max B.

    2012-01-01

    BACKGROUND Multiple lines of evidence suggest that the adrenergic system can modulate sensitivity to anesthetic-induced immobility and anesthetic-induced hypnosis as well. However, several considerations prevent the conclusion that the endogenous adrenergic ligands norepinephrine and epinephrine alter anesthetic sensitivity. METHODS Using dopamine β-hydroxylase (Dbh−/−) mice genetically engineered to lack the adrenergic ligands and their siblings with normal adrenergic levels, we test the contribution of the adrenergic ligands upon volatile anesthetic induction and emergence. Moreover, we investigate the effects of intravenous dexmedetomidine in adrenergic-deficient mice and their siblings using both righting reflex and processed electroencephalographic measures of anesthetic hypnosis. RESULTS We demonstrate that the loss of norepinephrine and epinephrine and not other neuromodulators copackaged in adrenergic neurons is sufficient to cause hypersensitivity to induction of volatile anesthesia. However, the most profound effect of adrenergic deficiency is retarding emergence from anesthesia, which takes two to three times as long in Dbh−/− mice for sevoflurane, isoflurane, and halothane. Having shown that Dbh−/− mice are hypersensitive to volatile anesthetics, we further demonstrate that their hypnotic hypersensitivity persists at multiple doses of dexmedetomidine. Dbh−/− mice exhibit up to 67% shorter latencies to loss of righting reflex and up to 545% longer durations of dexmedetomidine-induced general anesthesia. Central rescue of adrenergic signaling restores control-like dexmedetomidine sensitivity. A novel continuous electroencephalographic analysis illustrates that the longer duration of dexmedetomidine-induced hypnosis is not due to a motor confound, but occurs because of impaired anesthetic emergence. CONCLUSIONS Adrenergic signaling is essential for normal emergence from general anesthesia. Dexmedetomidine-induced general anesthesia does

  6. Cortical injury increases cholesterol 24S hydroxylase (Cyp46) levels in the rat brain.

    PubMed

    Cartagena, Casandra M; Ahmed, Farid; Burns, Mark P; Pajoohesh-Ganji, Ahdeah; Pak, Daniel T; Faden, Alan I; Rebeck, G William

    2008-09-01

    In traumatic brain injury (TBI), cellular loss from initial impact as well as secondary neurodegeneration leads to increased cholesterol and lipid debris at the site of injury. Cholesterol accumulation in the periphery can trigger inflammatory mechanisms while cholesterol clearance may be anti-inflammatory. Here we investigated whether TBI altered the regulation of cholesterol 24S-hydroxylase (Cyp46), an enzyme that converts cholesterol to the more hydrophilic 24S-hydroxycholesterol. We examined by Western blot and immunohistochemistry changes in Cyp46 expression following fluid percussion injury. Under normal conditions, most Cyp46 was present in neurons, with very little measurable in glia. Cyp46 levels were significantly increased at 7 days post-injury, and cell type specific analysis at 3 days post-injury showed a significant increase in levels of Cyp46 (84%) in microglia. Since 24-hydroxycholesterol induces activation of genes through the liver X receptor (LXR), we examined protein levels of ATP-binding cassette transporter A1 and apolipoprotein E, two LXR regulated cholesterol homeostasis proteins. Apolipoprotein E and ATP-binding cassette transporter A1 were increased at 7 days post-injury, indicating that increased LXR activity coincided with increased Cyp46 levels. We found that activation of primary rat microglia by LPS in vitro caused increased Cyp46 levels. These data suggest that increased microglial Cyp46 activity is part of a system for removal of damaged cell membranes post-injury, by conversion of cholesterol to 24-hydroxycholesterol and by activation of LXR-regulated gene transcription.

  7. [Health status of adults with congenital adrenal hyperplasia due to 21-hydroxylase deficiency].

    PubMed

    Bachelot, Anne; Touraine, Philippe

    2014-04-01

    Congenital adrenal hyperplasia (CAH) is the commonest genetic endocrine disorder. Mutations in the 21-hydroxylase gene account for 95 % of cases. CAH is classified according to symptoms and signs and to age of presentation. The clinical phenotype is typically classified as classic, the severe form, or nonclassic (NCF), the mild or late-onset form. Classic CAH is a life-long chronic disorder. In childhood, treatment focuses on genital surgery and optimization of growth and pubertal development. Priorities change with increasing age, typically focusing on fertility in early adult life and prevention of metabolic syndrome and osteoporosis in middle and older age. Recent studies highlight the importance of long-term follow-up of these patients and of transitional care between childhoods to adult life. In nonclassic CAH women, subfertility is mild compared with the classic form and seems to be mainly due to hormonal imbalance. Menstrual cycle or ovulation disorders observed in these women who consulted for infertility are in most cases corrected by hydrocortisone treatment, which led to simultaneous lowering of plasma androgen levels and rapid occurrence of pregnancy. Hydrocortisone also reduces the incidence of miscarriages. Several studies have reported that near 60 % of nonclassic CAH patients are carriers of a severe mutation. These patients may therefore give birth to a child with the classical form of CAH if their partner is also carrying a severe mutation. Due to the high frequency of CYP21A2 mutations in the general population, it is essential to genotype the partner of NC-CAH patients with one severe mutation to offer genetic counselling.

  8. 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

  9. [Health status of adults with congenital adrenal hyperplasia due to 21-hydroxylase deficiency].

    PubMed

    Bachelot, Anne; Touraine, Philippe

    2014-04-01

    Congenital adrenal hyperplasia (CAH) is the commonest genetic endocrine disorder. Mutations in the 21-hydroxylase gene account for 95 % of cases. CAH is classified according to symptoms and signs and to age of presentation. The clinical phenotype is typically classified as classic, the severe form, or nonclassic (NCF), the mild or late-onset form. Classic CAH is a life-long chronic disorder. In childhood, treatment focuses on genital surgery and optimization of growth and pubertal development. Priorities change with increasing age, typically focusing on fertility in early adult life and prevention of metabolic syndrome and osteoporosis in middle and older age. Recent studies highlight the importance of long-term follow-up of these patients and of transitional care between childhoods to adult life. In nonclassic CAH women, subfertility is mild compared with the classic form and seems to be mainly due to hormonal imbalance. Menstrual cycle or ovulation disorders observed in these women who consulted for infertility are in most cases corrected by hydrocortisone treatment, which led to simultaneous lowering of plasma androgen levels and rapid occurrence of pregnancy. Hydrocortisone also reduces the incidence of miscarriages. Several studies have reported that near 60 % of nonclassic CAH patients are carriers of a severe mutation. These patients may therefore give birth to a child with the classical form of CAH if their partner is also carrying a severe mutation. Due to the high frequency of CYP21A2 mutations in the general population, it is essential to genotype the partner of NC-CAH patients with one severe mutation to offer genetic counselling. PMID:24630263

  10. Dopamine- and Tyrosine Hydroxylase-Immunoreactive Neurons in the Brain of the American Cockroach, Periplaneta americana.

    PubMed

    Hamanaka, Yoshitaka; Minoura, Run; Nishino, Hiroshi; Miura, Toru; Mizunami, Makoto

    2016-01-01

    The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine's actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of "dopaminergic" neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory formation

  11. Preliminary assessment of the C13-side chain 2'-hydroxylase involved in Taxol biosynthesis

    SciTech Connect

    Long, Robert M.; Croteau, Rodney . E-mail: croteau@wsu.edu

    2005-12-09

    The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts {alpha}-phenylalanine to {beta}-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield {beta}-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both {beta}-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of {beta}-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of {beta}-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.

  12. Biochemical characterization of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients.

    PubMed

    Dobrowolski, S F; Pey, A L; Koch, R; Levy, H; Ellingson, C C; Naylor, E W; Martinez, A

    2009-02-01

    The biochemical properties of mutant phenylalanine hydroxylase (PAH) enzymes and clinical characteristics of hyperphenylalaninaemic patients who bear these mutant enzymes were investigated. Biochemical characterization of mutant PAH enzymes p.D143G, p.R155H, p.L348V, p.R408W and p.P416Q included determination of specific activity, substrate activation, V(max), K(m) for (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), K (d) for BH(4), and protein stabilization by BH(4). Clinical data from 22 patients either homozygous, functionally hemizygous, or compound heterozygous for the mutant enzymes of interest were correlated with biochemical parameters of the mutant enzymes. The p.L348V and p.P416Q enzymes retain significant catalytic activity yet were observed in classic and moderate PKU patients. Biochemical studies demonstrated that BH(4) rectified the stability defects in p.L348V and p.P416Q; additionally, patients with these variants responded to BH(4) therapy. The p.R155H mutant displayed low PAH activity and decreased apparent affinity for L-Phe yet was observed in mild hyperphenylalaninaemia. The p.R155H mutant does not display kinetic instability, as it is stabilized by BH(4) similarly to wild-type PAH; thus the residual activity is available under physiological conditions. The p.R408W enzyme is dysfunctional in nearly all biochemical parameters, as evidenced by disease severity in homozygous and hemizygous patients. Biochemical assessment of mutant PAH proteins, especially parameters involving interaction with BH(4) that impact protein folding, appear useful in clinical correlation. As additional patients and mutant proteins are assessed, the utility of this approach will become apparent.

  13. 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

  14. Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris.

    PubMed

    McKinney, Jeffrey; Knappskog, Per M; Pereira, Jacinto; Ekern, Trude; Toska, Karen; Kuitert, Baukje B; Levine, David; Gronenborn, Angela M; Martinez, Aurora; Haavik, Jan

    2004-02-01

    Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.

  15. A Rare Combination: Congenital Adrenal Hyperplasia Due To 21 Hydroxylase Deficiency and Turner Syndrome

    PubMed Central

    Peltek Kendirci, Havva Nur; Aycan, Zehra; Çetinkaya, Semra; Baş, Veysel Nijat; Ağladıoğlu, Sebahat Yılmaz; Önder, Aşan

    2012-01-01

    A combination of Turner syndrome (TS) and classical congenital adrenal hyperplasia (CAH) is rare. A one-day-old newborn was referred to our hospital with ambiguous genitalia. The parents were third-degree relatives. The infant’s weight was 3350g (50-75p), and the head circumference was 34.5cm (50p). The gonads were nonpalpable. Presence of a 3 cm phallus, one urogenital opening into the perineum, and incomplete labial fusion were identified. Laboratory tests revealed a classical type of CAH due to 21-hydroxylase deficiency. Karyotyping revealed a 45X0(35)/46XX(22) pattern with negative sex-determining region Y (SRY) on gene analysis. At the most recent follow-up visit, the patient appeared to be in good health - her height was 70.4 cm [-1.5 standard deviation (SD)] and her weight was 9.8 kg (0.3 SD). She was receiving hydrocortisone in a dose of 10 mg/m2/day, fludrocortisone acetate in a dose of 0.075 mg/day, and oral salt of 1 g/day. System examinations were normal. The patient’s electrolyte levels were found to be normal and she was in good metabolic control. The findings of this patient demonstrate that routine karyotyping during investigation of patients with sexual differentiation disorders can reveal TS. Additionally, signs of virilism should always be investigated at diagnosis or during physical examinations for follow-up of TS cases. [i][/i]SRY analysis should be performed primarily when signs of virilism are observed. CAH should also be considered in patients with negative [i]SRY[/i]. Conflict of interest:None declared. PMID:23261864

  16. 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.

  17. Serotonin synthesis rate and the tryptophan hydroxylase-2: G-703T polymorphism in social anxiety disorder.

    PubMed

    Furmark, Tomas; Marteinsdottir, Ina; Frick, Andreas; Heurling, Kerstin; Tillfors, Maria; Appel, Lieuwe; Antoni, Gunnar; Hartvig, Per; Fischer, Håkan; Långström, Bengt; Eriksson, Elias; Fredrikson, Mats

    2016-10-01

    It is disputed whether anxiety disorders, like social anxiety disorder, are characterized by serotonin over- or underactivity. Here, we evaluated whether our recent finding of elevated neural serotonin synthesis rate in patients with social anxiety disorder could be reproduced in a separate cohort, and whether allelic variation in the tryptophan hydroxylase-2 (TPH2) G-703T polymorphism relates to differences in serotonin synthesis assessed with positron emission tomography. Eighteen social anxiety disorder patients and six healthy controls were scanned during 60 minutes in a resting state using positron emission tomography and 5-hydroxy-L-[β -(11)C]tryptophan, [(11)C]5-HTP, a substrate of the second enzymatic step in serotonin synthesis. Parametric images were generated, using the reference Patlak method, and analysed using Statistical Parametric Mapping (SPM8). Blood samples for genotyping of the TPH2 G-703T polymorphism were obtained from 16 social anxiety disorder patients (T carriers: n=5, GG carriers: n=11). A significantly elevated [(11)C]5-HTP accumulation rate, indicative of enhanced decarboxylase activity and thereby serotonin synthesis capacity, was detected in social anxiety disorder patients compared with controls in the hippocampus and basal ganglia nuclei and, at a more lenient (uncorrected) statistical threshold, in the amygdala and anterior cingulate cortex. In patients, the serotonin synthesis rate in the amygdala and anterior cingulate cortex was significantly elevated in TPH2 T carriers in comparison with GG homozygotes. Our results support that social anxiety disorder entails an overactive presynaptic serotonergic system that, in turn, seems functionally influenced by the TPH2 G-703T polymorphism in emotionally relevant brain regions. PMID:27189957

  18. Reduction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

    PubMed

    Sucharitakul, Jeerus; Wongnate, Thanyaporn; Montersino, Stefania; van Berkel, Willem J H; Chaiyen, Pimchai

    2012-05-29

    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M(-1) s(-1). In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s(-1). At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ~51 s(-1), whereas without 3-HB, the rate constant is 0.43 s(-1) at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme-product (2,5-DHB) complex, with a rate constant of 45 ± 2 s(-1). The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em 0 for the enzyme-substrate complex is -179 ± 1 mV compared to an E(m)(0) of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme-ligand complex to a fully activated form before flavin reduction takes place. PMID:22559817

  19. 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

  20. 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.

  1. Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation.

    PubMed

    Lukacin, R; Gröning, I; Schiltz, E; Britsch, L; Matern, U

    2000-03-15

    Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.

  2. Adrenal-derived 11-Oxygenated 19-Carbon Steroids are the Dominant Androgens in Classic 21-Hydroxylase Deficiency

    PubMed Central

    Turcu, Adina F.; Nanba, Aya T.; Chomic, Robert; Upadhyay, Sunil K.; Giordano, Thomas J.; Shields, James J.; Merke, Deborah P.; Rainey, William E.; Auchus, Richard J.

    2016-01-01

    Objective To comprehensively characterize androgens and androgen precursors in classic 21-hydroxylase deficiency (21OHD) and to gain insight to the mechanisms of their formation. Design Serum samples were obtained from 38 patients (19 men) with classic 21OHD, age 3-59, and 38 sex- and age-matched controls; 3 patients with 11β-hydroxylase deficiency; 4 patients with adrenal insufficiency; and 16 patients (8 men) undergoing adrenal vein sampling. Paraffin-embedded normal (n=5) and 21OHD adrenal tissue (n=3) was used for immunohistochemical studies. Methods We measured 11 steroids in all sera using liquid chromatography-tandem mass spectrometry. Immunofluroescence localized 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) within the normal and 21OHD adrenals. Results Four 11-oxygenated 19-carbon (11oxC19) steroids were significantly higher in male and female 21OHD patients than in controls: 11β-hydroxyandrostenedione, 11-ketoandrostenedione 11β-hydroxytestosterone, and 11-ketotestosterone (3-4-fold, p< 0.0001). For 21OHD patients, testosterone and 11-ketotestosterone were positively correlated in females, but inversely correlated in males. All 11oxC19 steroids were higher in adrenal vein than in inferior vena cava samples from men and women and rose with cosyntropin stimulation. Only trace amounts of 11oxC19 steroids were found in sera from patients with 11β-hydroxylase deficiency and adrenal insufficiency, confirming their adrenal origin. HSD3B2 and CYB5A immunoreactivities were sharply segregated in the normal adrenal glands, whereas areas of overlapping expression were identified in the 21OHD adrenals. Conclusions All four 11oxC19 steroids are elevated in both men and women with classic 21OHD. Our data suggest that 11oxC19 steroids are specific biomarkers of adrenal-derived androgen excess. PMID:26865584

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Primary infertility in 45-year-old man with untreated 21-hydroxylase deficiency: successful outcome with glucocorticoid therapy.

    PubMed

    Tiitinen, Aila; Välimäki, Matti

    2002-06-01

    We describe a 45-yr-old man, who presented with primary infertility of 2 yr. He had small testicles with severe oligoasthenozoospermia and low serum gonadotropins, but normal serum T. The suppression of gonadotropin secretion by increased adrenal steroids due to untreated 21-hydroxylase deficiency appeared to underlie the failure in spermatogenesis. Hydrocortisone treatment was started and was modified later to include prednisolone to get optimal suppression of the secretion of ACTH and adrenal steroids. Within a few months, the gonadotropin levels became normal, and spermatogenesis was improved. A normal pregnancy was achieved.

  8. Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

    PubMed Central

    Teutsch, H G; Hasenfratz, M P; Lesot, A; Stoltz, C; Garnier, J M; Jeltsch, J M; Durst, F; Werck-Reichhart, D

    1993-01-01

    Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a lambda gt11 cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a lambda gt10 library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (M(r) = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family. Images Fig. 4 PMID:8097885

  9. 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

  10. 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.

  11. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism

    PubMed Central

    2014-01-01

    Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain. PMID:25401074

  12. Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain

    PubMed Central

    Park, Sangkyu; Choi, Min Ji; Lee, Jong Yeol; Kim, Jae Kwang; Ha, Sun-Hwa; Lim, Sun-Hyung

    2016-01-01

    Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3′,4′-dihydroxylated leucocyanidin. 3′-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3′H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3′H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice. PMID:27649148

  13. Association between the transcriptional levels of Htr-1a and tryptophan hydroxylase-1 in the hippocampus and the antifatigue effects of leucine on rats with postoperative fatigue

    PubMed Central

    WU, TIANTIAN; CHEN, JING; ZHU, JIANG; YU, ZHEN

    2014-01-01

    Leucine (Leu), a branched-chain amino acid (BCAA), is widely used in clinical practice following severe burns, gastrointestinal surgery, trauma and sepsis. In the present study, the antifatigue effects of BCAAs on a postoperative fatigue (POF) rat model, induced by 70% intestinal resection, were investigated. Leu (16.5 g/l) was administered intraperitoneally at a dose of 18 ml/kg/day. The fatigue level and antifatigue effects of Leu were evaluated by open-field testing on day 1, 3, 5 and 7 after surgery. In addition, mRNA specimens were extracted and measured using a quantitative polymerase chain reaction method. The open-field test results indicated that Leu exhibited a significant antifatigue effect. The total distance travelled and the number of times the rats passed from the outermost grids of an open-top case were greatly improved in the Leu treatment group when compared with the POF model group. With the exception of the normal group, the mRNA expression levels of Htr-1a exhibited a similar trend in all other groups, reaching a climax on day 3 and 5, while being restored to a normal level on day 7. With regard to the Leu intervention group, the mRNA expression level of Htr-1a decreased significantly on day 3 and 5 following surgery. The mRNA expression levels of tryptophan hydroxylase-1 were unchanged in this short time period; however, the levels were increased gradually in the Leu treatment group. Therefore, Leu exhibited an apparent antifatigue effect on various 5-hydroxytryptamine-associated genes. PMID:25289072

  14. Effects of embryonic and adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic microsomal testosterone hydroxylase activities in great blue herons (Ardea herodias)

    SciTech Connect

    Sanderson, J.T.; Giesy, J.P.; Janz, D.M.; Bellward, G.D.

    1997-06-01

    In a continuing effort to evaluate biomarkers of exposure of great blue herons (Ardea herodias) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons, the authors examined the effect of TCDD on hepatic microsomal testosterone hydroxylase activities. Heron embryos were exposed in ovo to 2 {micro}g TCDD/kg egg (or corn oil vehicle) and sacrificed at hatch or 7 d posthatch. Adult herons were exposed intraperitoneally to 20 {micro}g TCDD/kg and sacrificed 2 weeks later. The sex of the birds was known for the adults only. Hepatic microsomes of herons of each age group were able to hydroxylate testosterone at the 2{beta}, 6{beta}, 15{alpha}, 16{alpha}, or 16{beta} positions. In 7-d-old chicks, an additional unidentified compound was formed. The age of the untreated herons had a strong influence on the activities of the five hydroxylases, with changes of up to 17-fold. The TCDD significantly induced 2{beta}-, 6{beta}, and 15{alpha}-testosterone hydroxylase activities in the adult females, 15{alpha} in the adult males, and 6{beta}-testosterone hydroxylase activity in the hatchlings. In the 7-d-old chicks, induction was no longer apparent. A significant correlation existed between hepatic microsomal ethoxyresorufin O-deethylase (EROD) and 6{beta}-testosterone hydroxylase activity in hatchlings and adult female herons. The TCDD-induced changes in testosterone hydroxylase activities occurred at doses that resulted in tissue concentrations and levels of EROD induction that were environmentally relevant, but did not result in overt toxicities.

  15. Dopamine-β-Hydroxylase Activity and Levels of Its Cofactors and Other Biochemical Parameters in the Serum of Arsenicosis Patients of Bangladesh

    PubMed Central

    Rahman, M. Khalilur; Choudhary, M. Iqbal; Arif, M.; Morshed, M. Monzur

    2014-01-01

    Dopamine-β-hydroxylase (DBH) is a neurotransmitter (catecholamine)-mediating enzyme, which catalyzes the formation of norepinephrine from dopamine. The levels of DBH activity, its coenzyme (ascorbic acid) and cofactor (Cu++) and other biochemical parameters were measured in the serum of 32 arsenicosis patients of Bangladesh at three different age groups, namely, group 1 (10–18 years, 9 patients), group 2 (19–40 years, 14 patients) and group 3 (41–70 years, 9 patients) of the locality of Stadium Para of Meherpur district of Bangladesh. The values were compared with the same number of age-matched normal healthy individuals of the respective group. DBH activity was markedly decreased in the patients of group 1 as compared to that of the normal healthy people. The activities of DBH were decreased to lesser extents for the other two age groups. The total protein contents in the serum of arsenicosis patients were also significantly low as compared to that in the age-matched control groups. The levels of ascorbic acid and copper were found to be decreased in the serum of arsenicosis patients. The serum glucose levels were elevated in arsenicosis patients, as compared to that of the respective healthy controls. Other parameters, such as zinc and vitamin A levels were also decreased in the serum of arsenicosis patients. It was evident from the results of drinking of the arsenic contaminated water of shallow tube wells that the levels of DBH activity decreased significantly as compared to the control healthy persons. The levels of proteins, ascorbic acid, copper, zinc and vitamin A were decreased in the serum of people drinking the arsenic contaminated tube wells water as compared to that in the control healthy people with the exception that the levels of glucose were elevated in the serum of these patients. The pathophysiological significance of the results could be correlated with the decreased in proteins and that in DBH activities as DBH deficiency is

  16. OXYGEN-SENSITIVE RESET OF INDUCIBLE HIF TRANSACTIVATION RESPONSE: PROLYL HYDROXYLASES TUNE THE BIOLOGICAL NORMOXIC SETPOINT

    PubMed Central

    Khanna, Savita; Roy, Sashwati; Maurer, Mariah; Ratan, Rajiv R; Sen, Chandan K

    2006-01-01

    Cellular O2 sensing enables physiological adjustments to variations in tissue pO2. Under basal conditions, cells are adjusted to an O2 environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O2-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological read-out of hypoxia. In the presence of sufficient O2, HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase isoenzymes PHD1, 2 and 3. Using HT22 neurons stably transfected with a HIF reporter construct, we tested a novel hypothesis postulating that biological cells are capable of resetting their normoxic set-point by O2-sensitive changes in PHD expression. Results of this study show that the pO2 of the mouse brain cortex was 35 mm Hg or 5% O2. Exposure of HT22, adjusted to growing in 20% O2, to 5% O2 resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O2 did not report hypoxia. Cells adjusted to growing in 30% O2 reported hypoxia when acutely exposed to room air culture conditions. When grown under high O2 conditions, cells reset their normoxic set-point upwards by down-regulating the expression of PHD1–3. When grown under low O2 conditions, cells reset their normoxic set-point downwards by inducing the expression of PHD1–3. Exposure of mice in vivo to a hypoxic 10% O2 environment lowered blood as well as brain pO2. Such hypoxic exposure induced PHD1–3. Exposure of mice to a hyperoxic 50% O2 ambience repressed the expression of PHD1–3 indicating that O2-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knock-down of PHD expression revealed that O2-sensitive regulation of PHD may contribute to tuning the normoxic set-point in biological cells. PMID:16785028

  17. 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.

  18. ‘Roid rage in rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase

    PubMed Central

    Wood, Ruth I.; Armstrong, Abigail; Fridkin, Vlad; Shah, Vivek; Najafi, Allison; Jakowec, Michael

    2013-01-01

    In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight. Alternatively, AAS may increase impulsive behavior, consistent with the popular image of ‘roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5 mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to respond on a nose-poke on a 10 min fixed-interval schedule for the opportunity to fight in their home cage with an unfamiliar rat. Although testosterone increased aggression (6.3±1.3 fights/5 min vs 2.4±0.8 for controls, p<0.05), there was no difference in operant responding (28.4±1.6 nose-pokes/ 10 min for testosterone, 32.4±7.0 for vehicle). This suggests that testosterone does not enhance motivation for aggression. To test for impulsivity, rats were trained to respond for food in a delay-discounting procedure. In an operant chamber, one lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0, 15, 30 or 45 s). In testosterone- and vehicle-treated rats, body weights and food intake did not differ. However, testosterone-treated rats chose the larger, delayed reward more often (4.5±0.7 times in 10 trials with 45 s delay) than vehicle controls (2.5±0.5 times, p<0.05), consistent with a reduction in impulsive choice. Thus, although chronic high-dose testosterone enhances aggression, this does not include an increase in impulsive behavior or motivation to fight. This is further supported by measurement of tyrosine hydroxylase (TH) by Western immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and executive function (medial prefrontal cortex, PFC). There were no differences in TH between testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly reduced TH (to 76.9±3.1% of controls, p<0.05) in the caudate-putamen, a

  19. Tyrosine Hydroxylase Phosphorylation in Catecholaminergic Brain Regions: A Marker of Activation following Acute Hypotension and Glucoprivation

    PubMed Central

    Damanhuri, Hanafi A.; Burke, Peter G. R.; Ong, Lin K.; Bobrovskaya, Larisa; Dickson, Phillip W.; Dunkley, Peter R.; Goodchild, Ann K.

    2012-01-01

    The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased (prefrontal cortex) or in conjunction with pSer40 (caudal C1). Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the

  20. ZBP-89 Regulates Expression of Tryptophan Hydroxylase I and Mucosal Defense Against Salmonella Typhimurium in Mice

    PubMed Central

    Essien, Bryan; Grasberger, Helmut; Romain, Rachael D.; Law, David J.; Veniaminova, Natalia A.; Saqui-Salces, Milena; El-Zaatari, Mohamad; Tessier, Arthur; Hayes, Michael M.; Yang, Alexander C.; Merchant, Juanita L.

    2013-01-01

    Background & Aims ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium. Methods We generated mice with a conditional null allele of Zfp148 (ZBP-89FL/FL), using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89FL/FL), and then breeding the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89FL/FL and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S typhimurium after administration of streptomycin. Results Microarray analysis revealed that the colonic mucosa of ZBP-89FL/FL mice had reduced levels of tryptophan hydroxylase 1 (Tph1) mRNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5HT) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89FL/FL mice did not increase mucosal levels of 5HT in response to S typhimurium infection and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89FL/FL mice, correlating with fecal levels of butyrate. Conclusions ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis following infection with S typhimurium, partly due to reduced

  1. 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, structur