Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Suicidal inactivation by acetylenic fatty acids.

PubMed

Shak, S; Reich, N O; Goldstein, I M; Ortiz de Montellano, P R

1985-10-25

Human polymorphonuclear leukocytes (PMN) not only generate and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation (probably due to the action of a cytochrome P-450 enzyme). To develop pharmacologically useful inhibitors of the LTB4 omega-hydroxylase in human PMN, we devised a general scheme for synthesizing terminal acetylenic fatty acids based on the "acetylenic zipper" reaction. We found that the LTB4 omega-hydroxylase in intact PMN and in PMN sonicates is inactivated in a concentration-dependent fashion by terminal acetylenic analogues of lauric, palmitic, and stearic acids (i.e. 11-dodecynoic, 15-hexadecynoic, and 17-octadecynoic acids). Consistent with a suicidal process, inactivation of the LTB4 omega-hydroxylase requires molecular oxygen and NADPH, is time-dependent, and follows pseudo-first-order kinetics. Inactivation of the omega-hydroxylase by acetylenic fatty acids also is dependent on the terminal acetylenic moiety and the carbon chain length. Saturated fatty acids lacking a terminal acetylenic moiety do not inactivate the omega-hydroxylase. In addition, the two long-chain (C16, C18) acetylenic fatty acids inactivate the omega-hydroxylase at much lower concentrations (less than 5.0 microM) than those required for inactivation by the short-chain (C12) terminal acetylenic fatty acid (100 microM). Potent suicidal inhibitors of the LTB4 omega-hydroxylase in human PMN will help elucidate the roles played by LTB4 and its omega-oxidation products in regulating PMN function and in mediating inflammation.

Molecular evolution of flavonoid dioxygenases in the family Apiaceae.

PubMed

Gebhardt, Yvonne; Witte, Simone; Forkmann, Gert; Lukacin, Richard; Matern, Ulrich; Martens, Stefan

2005-06-01

Plant species of the family Apiaceae are known to accumulate flavonoids mainly in the form of flavones and flavonols. Three 2-oxoglutarate-dependent dioxygenases, flavone synthase or flavanone 3 beta-hydroxylase and flavonol synthase are involved in the biosynthesis of these secondary metabolites. The corresponding genes were cloned recently from parsley (Petroselinum crispum) leaves. Flavone synthase I appears to be confined to the Apiaceae, and the unique occurrence as well as its high sequence similarity to flavanone 3beta-hydroxylase laid the basis for evolutionary studies. In order to examine the relationship of these two enzymes throughout the Apiaceae, RT-PCR based cloning and functional identification of flavone synthases I or flavanone 3beta-hydroxylases were accomplished from Ammi majus, Anethum graveolens, Apium graveolens, Pimpinella anisum, Conium maculatum and Daucus carota, yielding three additional synthase and three additional hydroxylase cDNAs. Molecular and phylogenetic analyses of these sequences were compatible with the phylogeny based on morphological characteristics and suggested that flavone synthase I most likely resulted from gene duplication of flavanone 3beta-hydroxylase, and functional diversification at some point during the development of the apiaceae subfamilies. Furthermore, the genomic sequences from Petroselinum crispum and Daucus carota revealed two introns in each of the synthases and a lack of introns in the hydroxylases. These results might be explained by intron losses from the hydroxylases occurring at a later stage of evolution.

Tyrosine hydroxylase expression and activity in the rat brain: differential regulation after long-term intermittent or sustained hypoxia.

PubMed

Gozal, Evelyne; Shah, Zahoor A; Pequignot, Jean-Marc; Pequignot, Jacqueline; Sachleben, Leroy R; Czyzyk-Krzeska, Maria F; Li, Richard C; Guo, Shang-Z; Gozal, David

2005-08-01

Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O(2)), intermittent hypoxia (alternating room air and 10% O(2)), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser(40) tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.

[Prolyl hydroxylase activity in liver specimens in chronic liver diseases (author's transl)].

PubMed

Langness, U; Clausnitzer, H; Verspohl, M; Grasedyck, K

1978-08-25

100 patients were laparoscopied, liver tissue specimens taken from atypically altered areas. Prolyl hydroxylase was determined in the specimen, in parallel tissue was examined by light microscope. 8 groups of patients could be differentiated: Patients 1. with active, 2, with inactive cirrhosis, 3. with fatty infiltrations, 4. with fatty infiltration and mesenchymal reaction, 5. with aggressive, 6. with persistent, 7. with reactive hepatitis, 8. patients without histological changes. In the case of connective tissue increase in the liver prolyl hydroxylase activities were statistically significant above normal. In addition, there was a statistically significant difference between the enzyme activities of each group. A correlation could be found between prolyl hydroxylase activity and morphologically estimated connective tissue formation, but not the serum enzyme activities usually determined in liver diseases. Therefore, could be concluded that prolyl hydroxylase activity is an index of actual collagen biosynthesis in chronic liver diseases.

Abscinazole-F1, a conformationally restricted analogue of the plant growth retardant uniconazole and an inhibitor of ABA 8'-hydroxylase CYP707A with no growth-retardant effect.

PubMed

Todoroki, Yasushi; Kobayashi, Kyotaro; Shirakura, Minaho; Aoyama, Hikaru; Takatori, Kokichi; Nimitkeatkai, Hataitip; Jin, Mei-Hong; Hiramatsu, Saori; Ueno, Kotomi; Kondo, Satoru; Mizutani, Masaharu; Hirai, Nobuhiro

2009-09-15

To develop a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, seed dormancy, and other various physiological events, we designed and synthesized conformationally restricted analogues of uniconazole (UNI), a well-known plant growth retardant, which inhibits a biosynthetic enzyme (ent-kaurene oxidase) of gibberellin as well as ABA 8'-hydroxylase. Although most of these analogues were less effective than UNI in inhibition of ABA 8'-hydroxylase and rice seedling growth, we found that a lactol-bridged analogue with an imidazole is a potent inhibitor of ABA 8'-hydroxylase but not of plant growth. This compound, abscinazole-F1, induced drought tolerance in apple seedlings upon spray treatment with a 10 microM solution.

Long-Term Behavioral Recovery in Parkinsonian Rats by an HSV Vector Expressing Tyrosine Hydroxylase

PubMed Central

Naegele, Janice R.; O’Malley, Karen L.; Geller, Alfred I.

2006-01-01

One therapeutic approach to treating Parkinson’s disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (l-dopa)–producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine–lesioned rats, used as a model of Parkinson’s disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity. PMID:7669103

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency.

PubMed

Khattab, Ahmed; Haider, Shozeb; Kumar, Ameet; Dhawan, Samarth; Alam, Dauood; Romero, Raquel; Burns, James; Li, Di; Estatico, Jessica; Rahi, Simran; Fatima, Saleel; Alzahrani, Ali; Hafez, Mona; Musa, Noha; Razzghy Azar, Maryam; Khaloul, Najoua; Gribaa, Moez; Saad, Ali; Charfeddine, Ilhem Ben; Bilharinho de Mendonça, Berenice; Belgorosky, Alicia; Dumic, Katja; Dumic, Miroslav; Aisenberg, Javier; Kandemir, Nurgun; Alikasifoglu, Ayfer; Ozon, Alev; Gonc, Nazli; Cheng, Tina; Kuhnle-Krahl, Ursula; Cappa, Marco; Holterhus, Paul-Martin; Nour, Munier A; Pacaud, Daniele; Holtzman, Assaf; Li, Sun; Zaidi, Mone; Yuen, Tony; New, Maria I

2017-03-07

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1 , a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

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

USDA-ARS?s Scientific Manuscript database

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...

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.

Detection of tyrosine hydroxylase in dopaminergic neuron cell using gold nanoparticles-based barcode DNA.

PubMed

An, Jeung Hee; Oh, Byung-Keun; Choi, Jeong Woo

2013-04-01

Tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosysthesis, is predominantly expressed in several cell groups within the brain, including the dopaminergic neurons of the substantia nigra and ventral tegmental area. We evaluated the efficacy of this protein-detection method in detecting tyrosine hydroxylase in normal and oxidative stress damaged dopaminergic cells. In this study, a coupling of DNA barcode and bead-based immnunoassay for detecting tyrosine hydroxylaser with PCR-like sensitivity is reported. The method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes were identified by PCR analysis. The concentration of tyrosine hydroxylase in dopaminergic cell can be easily and rapidly detected using bio-barcode assay. The bio-barcode assay is a rapid and high-throughput screening tool to detect of neurotransmitter such as dopamine.

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.

Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

PubMed

Fernández-Espejo, Emilio; Bis-Humbert, Cristian

2018-06-06

3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in cellular, enteric and brain approaches of Parkinsonism in mice. Copyright © 2018 Elsevier B.V. All rights reserved.

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; ...

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.

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)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanderson, J.T.; Giesy, J.P.; Janz, D.M.

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},more » 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.« less

Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

PubMed Central

Hlouchova, Klara; Rudolph, Johannes; Pietari, Jaana M.H.; Behlen, Linda S.; Copley, Shelley D.

2014-01-01

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The kcat for PCP (0.024 s−1) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure/activity studies reveal that substrate binding and activity are enhanced by a low pKa for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 – 100% with different substrates. Uncoupling is increased by the presence of bulky substituents in the 3-, 4-, or 5-position, and lessened by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with TCHQ, a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ. PMID:22482720

Tyrosine hydroxylase in the ventral tegmental area of rams with high or low libido-A role for dopamine.

PubMed

Kramer, A C; Mirto, A J; Austin, K J; Roselli, C E; Alexander, B M

2017-12-01

Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D 2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (P<0.05) neurons were positive for tyrosine hydroxylase in the VTA. Sexually inactive rams had fewer (P<0.05) FRA and tyrosine hydroxylase positive neurons in the VTA than sexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine. Copyright © 2017 Elsevier B.V. All rights reserved.

[Response of vasopressin and tyrosine hydroxylase expressing neurons of the rat supraoptic nucleus to chronic osmotic stimulation].

PubMed

Abramova, M A; Calas, A; Maiily, P; Thibault, J; Ugriumov, M V

1999-06-01

This study has evaluated the dynamic of intracellular vasopressin and tyrosine hydroxylase contents in the neuron cell bodies in the supraoptic nucleus and in the axons of the posterior lobe in rats drinking 2% NaCl for 1, 2, and 3 weeks. The number of vasopressin-immunoreactive neurons increased by the end of the second week of osmotic stimulation that might be explained by the onset of vasopressin synthesis in the neurons which do not synthesize this neurohormone under normal physiological conditions. The concentration of vasopressin fell down continuously during the first two weeks of salt-loading, apparently, due to predominance of the vasopressin release over its synthesis. Over the third week of salt-loading, the intracellular concentration of vasopressin was not changed significantly suggesting the establishment of the dynamic equilibrium between the vasopressin synthesis and release. The number of tyrosine hydroxylase-immunoreactive neurons and the amount of tyrosine hydroxylase in cell bodies and the large axonal swellings, Herring bodies, increased gradually showing that the rate of tyrosine hydroxylase synthesis prevailed over that of its enzymatic degradation. Thus, the chronic stimulation of vasopressin neurons is accompanied by a number of the adaptive reactions; the most important is related to the onset of vasopressin and tyrosine hydroxylase synthesis in the neurons which do not synthetize both of them under normal conditions.

[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. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Regulation of embryonic neurotransmitter and tyrosine hydroxylase protein levels by ascorbic acid

PubMed Central

Meredith, M. Elizabeth; May, James M.

2013-01-01

Scope: Ascorbic acid (ascorbate) is required to recycle tetrahydrobiopterin, which is necessary for neurotransmitter synthesis by the rate-limiting enzymes tyrosine and tryptophan hydroxylases. We sought to determine whether ascorbate might regulate embryonic brain cortex monoamine synthesis utilizing transgenic mouse models with varying intracellular ascorbate levels. Methods and Results: In embryos lacking the sodium-dependent vitamin C transporter 2 (SVCT2), very low levels of brain ascorbate decreased cortex levels of norepinephrine and dopamine by approximately 33%, but had no effect on cortex serotonin or its metabolite, 5-hydroxyindole acetic acid. This decrease in ascorbate also led to a decrease in protein levels of tyrosine hydroxylase, but not of tryptophan hydroxylase. Increased cortex ascorbate in embryos carrying extra copies of the SVCT2 resulted in increased levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as serotonin and 5-hydroxyindole acetic acid. Conclusion: The dependence of embryonic brain cortex neurotransmitter synthesis and tyrosine hydroxylase expression on intracellular ascorbate emphasizes the importance of receiving adequate ascorbate during development. PMID:24095796

Prolyl hydroxylase activity in serum and rectal mucosa in inflammatory bowel disease.

PubMed Central

Farthing, M F; Dick, A P; Heslop, G; Levene, C I

1978-01-01

Prolyl hydroxylase activity in rectal mucosa was found to be significantly greater in 11 patients with Crohn's disease than in 11 control subjects with the irritable bowel syndrome and 16 patients with ulcerative colitis (P less than 0.005). Seven of the patients with Crohn's disease had a histologically normal rectum. This abnormality in apparently normal mucosa supports the concept that Crohn's disease is a 'continuous' disease of the gastrointestinal tract. Although there was no significant difference in prolyl hydroxylase activity between control subjects and patients with ulcerative colitis, those patients with quiescent disease tended to have lower values than those with active mucosal inflammation. Prolyl hydroxylase activity could not, however, be detected in the sera of either healthy control subjects or patients with inflammatory bowel disease. PMID:210089

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats.

PubMed

Murray, M

1988-05-01

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.

Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

PubMed

Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

1991-02-01

A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

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

24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs.

PubMed

Tryfonidou, M A; Oosterlaken-Dijksterhuis, M A; Mol, J A; van den Ingh, T S G A M; van den Brom, W E; Hazewinkel, H A W

2003-03-01

A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D(3) toxicosis.

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation.

PubMed

Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V

2018-06-01

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2007-06-21

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

Recommendations for the nutrition management of phenylalanine hydroxylase deficiency

PubMed Central

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.

2014-01-01

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. Genet Med 16 2, 121–131. PMID:24385075

Genetics Home Reference: dopamine beta-hydroxylase deficiency

MedlinePlus

... deficiency Sources for This Page Cubells JF, Zabetian CP. Human genetics of plasma dopamine beta-hydroxylase activity: ... 16. Review. Citation on PubMed Kim CH, Zabetian CP, Cubells JF, Cho S, Biaggioni I, Cohen BM, Robertson ...

Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia.

PubMed

Yu, Bingwu; Ruppert, Martin; Stöckigt, Joachim

2002-08-01

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria.

PubMed

Danecka, Marta K; Woidy, Mathias; Zschocke, Johannes; Feillet, François; Muntau, Ania C; Gersting, Søren W

2015-03-01

In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.

PubMed

Ma, Gang; Zhang, Lancui; Yungyuen, Witchulada; Tsukamoto, Issei; Iijima, Natsumi; Oikawa, Michiru; Yamawaki, Kazuki; Yahata, Masaki; Kato, Masaya

2016-06-29

Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein. CitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

PubMed

Jia, Longgang; Dong, Jianzhang; Wang, Ruijie; Mao, Shuhong; Lu, Fuping; Singh, Suren; Wang, Zhengxiang; Liu, Xiaoguang

2017-08-01

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

[Role of vitamin E in regulation of cholecalciferol hydroxylation in hypovitaminosis D and hypervitaminosis D].

PubMed

Apukhovs'ka, L I; Velykyĭ, M M; Lotots'ka, O Iu; Khomenko, A V

2009-01-01

It is established, that dose-dependent influence of vitamin E on vitamin D3 metabolism, is conditioned by degree of cholecalciferol sufficiency. Under a condition of D-hypovitaminosis, contents of 25OHD3 in blood serum is 2-fold reduced and vitamin D3 25-hydroxylase enzymes activity increased in rat hepatocytes. Vitamin E (0.726-7.26 IU) significantly stimulated the effect of vitamin D3 (40 IU) in animals with D-hypovitaminosis and led to further increase of 25OHD3 content in the blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. In D-hypervitaminosis the contents of 25OHD3 in blood serum was more than 3-fold increased and vitamin D3 25-hydroxylase enzymes activity was inhibited. Vitamin E (0,726-7,26 IU) lowered the vitamin D toxicity, decreased contents of 25OHD3 in blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. High doses of vitamin E (36.3 IU) under these conditions demonstrated negative effect on vitamin D3 metabolism. The mechanism of vitaminE participation in the vitamin D3 metabolism under D-hypovitaminosis and D-hypervitaminosis may be its influence on the activity of different vitamin D3 25-hydroxylase systems of hepatocytes.

Genetics Home Reference: 17 alpha-hydroxylase/17,20-lyase deficiency

MedlinePlus

... Center Frequency 17α-hydroxylase/17,20-lyase deficiency accounts for about 1 percent of congenital adrenal hyperplasia cases. It is estimated to occur in 1 in 1 million people worldwide. Related Information What information about a genetic condition can statistics ...

Identification of a Novel Allosteric Inhibitory Site on Tryptophan Hydroxylase 1 Enabling Unprecedented Selectivity Over all Related Hydroxylases

PubMed Central

Petrassi, Mike; Barber, Rob; Be, Celine; Beach, Sarah; Cox, Brian; D’Souza, Anne-Marie; Duggan, Nick; Hussey, Martin; Fox, Roy; Hunt, Peter; Jarai, Gabor; Kosaka, Takatoshi; Oakley, Paul; Patel, Viral; Press, Neil; Rowlands, David; Scheufler, Clemens; Schmidt, Oliver; Srinivas, Honnappa; Turner, Mary; Turner, Rob; Westwick, John; Wolfreys, Alison; Pathan, Nuzhat; Watson, Simon; Thomas, Matthew

2017-01-01

Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT1B receptor) and complex lesions (SERT, 5-HT1B, 5-HT2B receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT2B receptor). Selective inhibitors of individual signaling elements – SERT, 5-HT2A, 5HT2B, and combined 5-HT2A/B receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine in vivo system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH. PMID:28529483

[Tyrosine hydroxylase in telencephalon and diencephalon of Rhodeus sericeus (Cyprinidae)].

PubMed

Pushchina, E V

2009-01-01

Immunohistochemical labeling of tyrosine hydroxylase was used to demonstrate catecholaminergic neuronal populations in the telencephalon and diencephalonof adult cypryniform fish Rhodeus sericeus. Various immunoreactive cell populations have been found in the telencephalon (ventral, central and lateral nuclei of ventral telencephalic area). Immunoreactive cells and fibers were discovered in dorsal nucleus of ventral telencephalic area and supracomissural nucleus in the caudal part of the telencephalon. In the diencephalon, periventricular nuclei (preoptic, periventricular nucleus of posterior tuberculum and periventricular organ) contained considerable TH-ergic cells. High activity of tyrosine hydroxylase was revealed in the pretectal, ventro-medial, ventro-lateral and suprachiasmatic nuclei. Periventricular hypothalamic nuclei also displayed high activity of tyrosine hydroxylase. Pseudounipolar neurons prevailed in all TH-immunereactive structures of the telencephalon and diencephalon: numerous bipolar liquor-contacting cells were discovered in the periventricular nuclei. Large pear-shaped cells and bipolar TH-ergic cells were found in posterior tuberculum. These cells may be functionally related to the dopamine-acquiring system.

Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

PubMed

Sadiku, Pranvera; Willson, Joseph A; Dickinson, Rebecca S; Murphy, Fiona; Harris, Alison J; Lewis, Amy; Sammut, David; Mirchandani, Ananda S; Ryan, Eilise; Watts, Emily R; Thompson, A A Roger; Marriott, Helen M; Dockrell, David H; Taylor, Cormac T; Schneider, Martin; Maxwell, Patrick H; Chilvers, Edwin R; Mazzone, Massimilliano; Moral, Veronica; Pugh, Chris W; Ratcliffe, Peter J; Schofield, Christopher J; Ghesquiere, Bart; Carmeliet, Peter; Whyte, Moira Kb; Walmsley, Sarah R

2017-09-01

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

PubMed Central

Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

2016-01-01

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

PubMed

Jaiswal, A K; Nebert, D W; Eisen, H W

1985-08-01

The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

An Unusual Case: The Comorbidity of Mood Disorder and 17-α-Hydroxylase Deficiency

PubMed Central

TUNÇ, Serhat; YİĞİTER, Sera; ALTINBAŞ, Kürşat; KURT, Erhan; ORAL, Timuçin

2013-01-01

17-α-hydroxylase enzyme has a crucial role in the steroid biosynthesis and, deficiency of this enzyme is an autosomal recessive monogenic disorder which is one of the two hypertensive form of congenital adrenal hyperplasia. It is characterized with the deficiency in glucocorticoid, adrenal androgen, and sex steroid synthesis with concomitant mineralocorticoid excess due to genetic defect in steroid biosynthesis. The relationship of hormone system physiology with psychiatric signs and syndromes are complex. Any problem in the hypothalamo-pituitary axis may cause psychiatric syndromes. On the other hand, many psychiatric disorders, such as mood-anxiety symptoms, depression, mania, psychosis, and delirium can be seen secondary to the treatment of hormone deficiency. We present the case of a male patient with pseudohermaphroditism who has been followed and treated in Raşit Tahsin Mood Clinic with the diagnosis of mood disorder not otherwise specified and was diagnosed with 46, XY karyotype and 17-α-hydroxylase deficiency after referring to a hospital with delayed puberty. Considering the medical literature, 17-α-hydroxylase deficiency has been evaluated from the aspects of gender-related behavioral disorders, psychological developmental and anxiety disorders. To the best of our knowledge, in the medical literature, this is the first case of 17-α-hydroxylase deficiency associated with mood disorder. Here, the relationship between mood disorders and hypothalamo-pituitary axis is discussed in the light of the literature PMID:28360556

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 acetylenesmore » 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.« less

CINNAMIC ACID HYDROXYLASE IN SPINACH,

DTIC Science & Technology

An acetone precipitate from an extract of spinach leaves catalysed the hydroxylation of trans- cinnamic acid to p-coumaric acid . The enzyme was...and addition of L-phenylalanine inhibited cinnamic acid hydroxylase activity. (Author)...Tetrahydrofolic acid and a reduced pyridine nucleotide coenzyme were necessary for maximum activity. Aminopterin was a potent inhibitor of the hydroxylating

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

USDA-ARS?s Scientific Manuscript database

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 ...

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

[Features of cholecalciferol hydroxylation in the liver of rats in conditions of D-hypervitaminosis and activity of alpha-tocopherol].

PubMed

Velykyĭ, M M; Apukhovs'ka, L I; Vasylevs'ka, V M; Lotots'ka, O Iu; Besusiak, A I; Khomenko, A V

2010-01-01

It is shown, that hepatocytes contain two (microsomal and mitochondrial) vitamin D3 25-hydroxylase enzymes, which differ as to their activity and function with maximal activity at different concentrations to substrate, namely at 15 microM and 100 microM of vitamin D3, accordingly. Activity of vitamin D3 25-hydroxylase enzymes of hepatocytes is regulated by cholecalciferol and alpha-tocopherol. The general and microsomal vitamin D3 25-hydroxylase enzymes activity of hepatocytes is lowered, but mitochondrial isoform is increased under D-hypervitaminosis conditions. Vitamin E increases microsomal vitamin D3 25-hydroxylase activity and decreases mitochondrial isoform activity of rats hepatocytes under D-hypervitaminosis conditions. It is established that D-hypervitaminosis is accompanied by expressed hypercalcemia and hyperphosphatemia, by decreased contents of mineral components in the bone tissue and high activity of alkaline phosphatase in the blood serum. The physiological doses of vitamin E under these conditions normalized the mineral metabolism, contents of calcium, phosphates and activity of alkaline phosphatase isoform in the blood serum.

Autoantibodies against aromatic amino acid hydroxylases in patients with autoimmune polyendocrine syndrome type 1 target multiple antigenic determinants and reveal regulatory regions crucial for enzymatic activity.

PubMed

Bratland, Eirik; Magitta, Ng'weina Francis; Bøe Wolff, Anette Susanne; Ekern, Trude; Knappskog, Per Morten; Kämpe, Olle; Haavik, Jan; Husebye, Eystein Sverre

2013-06-01

Patients with autoimmune polyendocrine syndrome type 1 (APS-1) frequently have autoantibodies directed against the aromatic amino acid hydroxylases tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). We aimed to characterize these autoantibodies with regard to their antigenic determinants, their influence on enzymatic activity and their clinical associations. In particular, we wanted to compare autoantibodies against the two different isoforms of TPH, which display different tissue distribution. Using sera from 48 Scandinavian APS-1 patients we identified 36 patients (75%) with antibodies against one or more of these three enzymes. Antibodies against TPH1, but not TPH2, were associated with malabsorption in the whole Scandinavian cohort, while TH antibodies were associated with dental enamel hypoplasia in Norwegian patients. Subsequent experiments with selected patient sera indicated that while the C-terminal domain was the immunodominant part of TPH1, the epitopes of TPH2 and TH were mainly located in the N-terminal regulatory domains. We also identified a TPH1 specific epitope involved in antibody mediated inhibition of enzyme activity, a finding that provides new insight into the enzymatic mechanisms of the aromatic amino acid hydroxylases and knowledge about structural determinants of enzyme autoantigens. In conclusion, TPH1, TPH2 and TH all have unique antigenic properties in spite of their structural similarity. Copyright © 2012 Elsevier GmbH. All rights reserved.

Programming of Dopaminergic Neurons by Neonatal Sex Hormone Exposure: Effects on Dopamine Content and Tyrosine Hydroxylase Expression in Adult Male Rats

PubMed Central

Espinosa, Pedro; Silva, Roxana A.; Sanguinetti, Nicole K.; Venegas, Francisca C.; Riquelme, Raul; González, Luis F.; Cruz, Gonzalo; Renard, Georgina M.; Moya, Pablo R.; Sotomayor-Zárate, Ramón

2016-01-01

We sought to determine the long-term changes produced by neonatal sex hormone administration on the functioning of midbrain dopaminergic neurons in adult male rats. Sprague-Dawley rats were injected subcutaneously at postnatal day 1 and were assigned to the following experimental groups: TP (testosterone propionate of 1.0 mg/50 μL); DHT (dihydrotestosterone of 1.0 mg/50 μL); EV (estradiol valerate of 0.1 mg/50 μL); and control (sesame oil of 50 μL). At postnatal day 60, neurochemical studies were performed to determine dopamine content in substantia nigra-ventral tegmental area and dopamine release in nucleus accumbens. Molecular (mRNA expression of tyrosine hydroxylase) and cellular (tyrosine hydroxylase immunoreactivity) studies were also performed. We found increased dopamine content in substantia nigra-ventral tegmental area of TP and EV rats, in addition to increased dopamine release in nucleus accumbens. However, neonatal exposure to DHT, a nonaromatizable androgen, did not affect midbrain dopaminergic neurons. Correspondingly, compared to control rats, levels of tyrosine hydroxylase mRNA and protein were significantly increased in TP and EV rats but not in DHT rats, as determined by qPCR and immunohistochemistry, respectively. Our results suggest an estrogenic mechanism involving increased tyrosine hydroxylase expression, either by direct estrogenic action or by aromatization of testosterone to estradiol in substantia nigra-ventral tegmental area. PMID:26904299

Programming of Dopaminergic Neurons by Neonatal Sex Hormone Exposure: Effects on Dopamine Content and Tyrosine Hydroxylase Expression in Adult Male Rats.

PubMed

Espinosa, Pedro; Silva, Roxana A; Sanguinetti, Nicole K; Venegas, Francisca C; Riquelme, Raul; González, Luis F; Cruz, Gonzalo; Renard, Georgina M; Moya, Pablo R; Sotomayor-Zárate, Ramón

2016-01-01

We sought to determine the long-term changes produced by neonatal sex hormone administration on the functioning of midbrain dopaminergic neurons in adult male rats. Sprague-Dawley rats were injected subcutaneously at postnatal day 1 and were assigned to the following experimental groups: TP (testosterone propionate of 1.0 mg/50 μL); DHT (dihydrotestosterone of 1.0 mg/50 μL); EV (estradiol valerate of 0.1 mg/50 μL); and control (sesame oil of 50 μL). At postnatal day 60, neurochemical studies were performed to determine dopamine content in substantia nigra-ventral tegmental area and dopamine release in nucleus accumbens. Molecular (mRNA expression of tyrosine hydroxylase) and cellular (tyrosine hydroxylase immunoreactivity) studies were also performed. We found increased dopamine content in substantia nigra-ventral tegmental area of TP and EV rats, in addition to increased dopamine release in nucleus accumbens. However, neonatal exposure to DHT, a nonaromatizable androgen, did not affect midbrain dopaminergic neurons. Correspondingly, compared to control rats, levels of tyrosine hydroxylase mRNA and protein were significantly increased in TP and EV rats but not in DHT rats, as determined by qPCR and immunohistochemistry, respectively. Our results suggest an estrogenic mechanism involving increased tyrosine hydroxylase expression, either by direct estrogenic action or by aromatization of testosterone to estradiol in substantia nigra-ventral tegmental area.

An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.

PubMed

Meesapyodsuk, Dauenpen; Qiu, Xiao

2008-07-01

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

PubMed

Shak, S; Goldstein, I M

1985-09-01

Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism

NASA Technical Reports Server (NTRS)

Anterola, Aldwin M.; Jeon, Jae-Heung; Davin, Laurence B.; Lewis, Norman G.

2002-01-01

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.

PubMed

Anterola, Aldwin M; Jeon, Jae-Heung; Davin, Laurence B; Lewis, Norman G

2002-05-24

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

Functional characterization of a p-coumaroyl quinate/shikimate 3’-hydroxylase from potato (Solanum tuberosum)

USDA-ARS?s Scientific Manuscript database

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3’-hydroxylase (C3’H) for CGA biosynthesis has not been isolated or characterized i...

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…

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

USDA-ARS?s Scientific Manuscript database

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2, which encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rod...

Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH)2D3-26,23-lactone

PubMed Central

Prosser, David E.; Kaufmann, Martin; O'Leary, Brendan; Byford, Valarie; Jones, Glenville

2007-01-01

Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1α,25-(OH)2D3, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1α,25-(OH)2D3-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. PMID:17646648

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

Aging and a long-term diabetes mellitus increase expression of 1 α-hydroxylase and vitamin D receptors in the rat liver.

PubMed

Vuica, Ana; Ferhatović Hamzić, Lejla; Vukojević, Katarina; Jerić, Milka; Puljak, Livia; Grković, Ivica; Filipović, Natalija

2015-12-01

Diabetes mellitus (DM) is a metabolic disorder associated with serious liver complications. As a metabolic chronic disease, DM is very common in the elderly. Recent studies suggest ameliorating effects of vitamin D on metabolic and oxidative stress in the liver tissue in an experimental model of DM. The aim of this study was to investigate the expression of vitamin D receptors (VDRs) and 1α-hydroxylase, the key enzyme for the production of active vitamin D form (calcitriol) in the liver during long-term diabetes mellitus type 1 (DM1) in aging rats. We performed immunohistochemical analysis of liver expression of 1α-hydroxylase and VDRs during aging in long-term streptozotocin-induced DM1. 1α-Hydroxylase was identified in the monocyte/macrophage system of the liver. In addition to the nuclear expression, we also observed the expression of VDR in membranes of lipid droplets within hepatocytes. Aging and long-term DM1 resulted in significant increases in the number of 1α-hydroxylase immunoreactive cells, as well as the percentage of strongly positive VDR hepatocytes. In conclusion, the liver has the capacity for active vitamin D synthesis in its monocyte/macrophage system that is substantially increased in aging and long-term diabetes mellitus. These conditions are also characterized by significant increases in vitamin D receptor expression in hepatocytes. The present study suggests that VDR signaling system could be a potential target in prevention of liver complications caused by diabetes and aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior

USDA-ARS?s Scientific Manuscript database

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...

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

USDA-ARS?s Scientific Manuscript database

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...

Only two mutations detected in 15 Tunisian patients with 11β-hydroxylase deficiency: the p.Q356X and the novel p.G379V.

PubMed

Kharrat, M; Trabelsi, S; Chaabouni, M; Maazoul, F; Kraoua, L; Ben Jemaa, L; Gandoura, N; Barsaoui, S; Morel, Y; M'rad, R; Chaabouni, H

2010-10-01

Steroid 11β-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β-hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β-hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population. © 2010 John Wiley & Sons A/S.

Identification and expression analysis of the genes involved in serotonin biosynthesis and transduction in the field cricket Gryllus bimaculatus.

PubMed

Watanabe, T; Sadamoto, Hitoshi; Aonuma, H

2011-10-01

Serotonin (5-HT) modulates various aspects of behaviours such as aggressive behaviour and circadian behaviour in the cricket. To elucidate the molecular basis of the cricket 5-HT system, we identified 5-HT-related genes in the field cricket Gryllus bimaculatus DeGeer. Complementary DNA of tryptophan hydroxylase and phenylalanine-tryptophan hydroxylase, which convert tryptophan into 5-hydroxy-L-tryptophan (5-HTP), and that of aromatic L-amino acid decarboxylase, which converts 5-HTP into 5-HT, were isolated from a cricket brain cDNA library. In addition, four 5-HT receptor genes (5-HT(1A) , 5-HT(1B) , 5-HT(2α) , and 5-HT(7) ) were identified. Expression analysis of the tryptophan hydroxylase gene TRH and phenylalanine-tryptophan hydroxylase gene TPH, which are selectively involved in neuronal and peripheral 5-HT synthesis in Drosophila, suggested that two 5-HT synthesis pathways co-exist in the cricket neuronal tissues. The four 5-HT receptor genes were expressed in various tissues at differential expression levels, suggesting that the 5-HT system is widely distributed in the cricket. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 andmore » 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.« less

Effects of 25-hydroxyvitamin D3 on cathelicidin production and antibacterial function of human oral keratinocytes.

PubMed

Wang, Qi; Zhang, Wu; Li, Hao; Aprecio, Raydolf; Wu, Wan; Lin, Yiqiao; Li, Yiming

2013-01-01

Vitamin D and its metabolites have been recognized as key determinants in innate immune modulation. In this study, we investigated the regulation of antibacterial functions of oral keratinocyte cells by 25-hydroxyvitamin D3 (25VD3). OKF6/TERT2 cells, an immortalized human oral keratinocyte cell line, were transfected with or without 24-hydroxylase small interfering RNA (siRNA) and incubated with different amounts of 25VD3. These epithelial cells expressed high levels of inactivating 24-hydroxylase (CYP24A1) and relatively low levels of activating 1α-hydroxylase (CYP27B1) in the presence of 25VD3. 25VD3 influenced the expression of vitamin D-driven genes and cathelicidin in a dose-related manner. SiRNA specific to 24-hydroxylase augmented the cathelicidin production and subseqently influenced the antibacterial activity on multispecies of oral pathogens. These observations suggest that 25VD3 is capable of stimulating cathelicidin production and modulating antibacterial function upon CYP24A1 knochdown in oral epithelial cells, and indicate novel mechanisms that 25VD3 may enhance antibacterial ability in oral keratinocytes. Copyright © 2013 Elsevier Inc. All rights reserved.

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

PubMed

Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang

2014-11-03

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fardella, C.E.; Hum, D.W.; Miller, W.L.

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-1more » 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.« less

Defense Health: Coordinating Authority Needed for Psychological Health and Traumatic Brain Injury Activities

DTIC Science & Technology

2012-01-01

Placebo-Controlled Trial of the Dopamine Beta Hydroxylase (DBH) Inhibitor, Nepicastat, for the Treatment of PTSD in Operation Iraqi Freedom (OIF...Operation Enduring Freedom (OEF) Veterans 1 A Randomized, Placebo-Controlled Trial of the Dopamine -?-Hydroxylase (DBH) Inhibitor, Nepicastat for the...Reduction: Predeployment Stress Inoculation Training 1 Combat, Sexual Assault, and Post-Traumatic Stress in OIF/OEF Military Women 1 Comparing

Effects of coumarate 3-hydroxylase down-regulation on lignin structure

Treesearch

John Ralph; Takuya Akiyama; Hoon Kim; Fachuang Lu; Paul F. Schatz; Jane M. Marita; Sally A. Ralph; M.S. Srinivasa Reddy; Fang Chen; Richard A. Dixon

2006-01-01

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to thenormally dominant guaiacyl (G) and syringyl (S) units Stem levels of up to ~65% P (from wild-type levels of ~1%) resulting from down-regulation of C3H were measured by traditional degradative...

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

ERIC Educational Resources Information Center

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

2011-01-01

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

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

USDA-ARS?s Scientific Manuscript database

The effects of azole-type P450 inhibitors and two metabolism-resistant ABA analogs 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 expr...

Prolyl Hydroxylase EGLN3 Regulates Skeletal Myoblast Differentiation through an NF-κB-dependent Pathway

PubMed Central

Fu, Jian; Taubman, Mark B.

2010-01-01

The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the α subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1α by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-κB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IκBα. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-κB activation induced by overexpressed TRAF2 or IκB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-κB. These data suggest that EGLN3 is a negative regulator of NF-κB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-κB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent. PMID:20089853

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 bemore » 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.« less

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.

Renal cytochrome P450 omega-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride.

PubMed

Oyekan, A O; Youseff, T; Fulton, D; Quilley, J; McGiff, J C

1999-10-01

Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.

Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

PubMed

Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni

2005-10-01

We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

PubMed

Ishikawa, Yoshihiro; Bächinger, Hans Peter

2013-11-01

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

A search for association between schizophrenia and dopamine-related alleles.

PubMed

Jönsson, E; Brené, S; Geijer, T; Terenius, L; Tylec, A; Persson, M L; Sedvall, G

1996-01-01

Dopamine receptor dysfunction and altered tyrosine hydroxylase activity have both been implicated in the pathophysiology of schizophrenia. Schizophrenic patients and control subjects were examined for allele frequencies in the tyrosine hydroxylase and dopamine D2 and D4 receptor genes. No significant differences of allele or genotype frequencies were found between the two groups after adjustment for multiple comparisons. Neither were any significant relationships observed between allele frequencies and a number of clinical variables within the schizophrenic subsample. When no adjustment was made for multiple testing a few significant tendencies were obtained which warrant further research in extended patient and control materials. The results are compatible with the view that the tyrosine hydroxylase, dopamine receptor D2 and D4 gene polymorphisms examined are not of major importance in the aetiology or pathophysiology of schizophrenia.

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.

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 themore » 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.« less

Mechanism-based inactivation of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity by acetylenic and olefinic polycyclic arylhydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gan, L.S.

A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxygenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene (EP), 3-ethynylperylene (EPL), cis- and trans-1-(2-bromo-vinyl)pyrene (c-BVP and t-BVP), and 1-allylpyrene (AP) serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene (BP) hydroxylase, while 1-vinyl-pyrene (VP) and phenyl 1-pyrenyl acetylene (PPA) do not cause a detectable suicide inhibition of the BP hydroxylase. The mechanism-based loss of BP hydroxylase activity caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes. In themore » presence of NADPH, /sup 3/H-labeled EP covalently attached to P-450 isozymes with a measured stoichiometry of one mole of EP per mole of the P-450 heme. The results of the effects of these aryl derivatives in the mammalian cell-mediated mutagenesis assay and toxicity assay show that none of the compounds examined nor any of the their metabolites produced in the incubation system are cytotoxic to V79 cells.« less

Functional analysis of Antirrhinum kelloggii flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes; critical role in flower color and evolution in the genus Antirrhinum.

PubMed

Ishiguro, Kanako; Taniguchi, Masumi; Tanaka, Yoshikazu

2012-05-01

The enzymes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3'5'H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3'H and two F3'5'H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3'Hs and F3'5'Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3'H and F3'5'H, respectively, and that the ancestor of A. majus lost F3'5'H activity after its speciation from the ancestor of A. kelloggii.

[Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

PubMed

Sorokina, E A; Sibiriak, S V; Sergeeva, S A

2002-01-01

Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity.

Lignification in transgenics deficient in 4-coumarate 3-hydroxylase (C3H)or the associated hydroxycinnamoyl transferase (HCT)

Treesearch

John Ralph; Takuya Akiyama; Hoon Kim; Fachuang Lu; Sally A. Ralph; Clint Chapple; Ramesh B. Nair; Armin Wagner; Fang Chen; M.S. Srinivasa Reddy; Richard A Dixon; Heather D. Coleman; Shawn D. Mansfield

2006-01-01

Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in angiosperms massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant syringyl (S) and guaiacyl (G) units. Alfalfa stem levels of up to ~65% P (from wild-type (WT) levels of ~1%) resulting from down-regulation of C3H were measured by...

Characterization of genes encoding ABA 8'-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.).

PubMed

Yang, Seung-Hwan; Choi, Dongsu

2006-11-24

Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daniels, J.; Williams, J.; Asherson, P.

1995-02-27

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

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

DOE PAGES

Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; ...

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 (C 2’H) from sweet potato. By comparing these twomore » structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

Effect of mCOUP-TF1 deficiency on the glossopharyngeal and vagal sensory ganglia.

PubMed

Ichikawa, H; Lin, S-C; Tsai, S Y; Tsai, M-J; Sugimoto, T

2004-07-16

Immunohistochemistry for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase and calbindin D-28k was performed on the glossopharyngeal and vagal ganglia in mCOUP-TFI knockout mice to know the effect of its deficiency on different types of primary sensory neurons. In wild type and heterozygous mice, the glossopharyngeal and vagal ganglia contained abundant CGRP-, tyrosine hydroxylase- and calbindin D-28k-immunoreactive (IR) neurons. In the ganglia of mCOUP-TFI knockout mice, a 38% decrease of CGRP-IR neurons was detected. However, the number of tyrosine hydroxylase- or calbindin D-28k-neurons was not altered by the mCOUP-TFI deficiency. In the tongue of knockout mice, the number of CGRP-IR nerve fibers decreased compared to wild-type and heterozygous mice. The development of CGRP-IR petrosal neurons, which supply innervation of the tongue, may depend on mCOUP-TFI.

Regulation of Phenylalanine Hydroxylase: Conformational Changes Upon Phenylalanine Binding Detected by H/D Exchange and Mass Spectrometry†

PubMed Central

Li, Jun; Dangott, Lawrence J.; Fitzpatrick, Paul F.

2010-01-01

Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains. PMID:20307070

Effects of biogenic aldehydes and aldehyde dehydrogenase inhibitors on rat brain tryptophan hydroxylase activity in vitro.

PubMed

Nilsson, G E; Tottmar, O

1987-04-21

The effect of indole-3-acetaldehyde, 5-hydroxyindole-3-acetaldehyde, disulfiram, diethyldithiocarbamate, coprine, and 1-amino-cyclopropanol on tryptophan hydroxylase activity was studied in vitro using high performance liquid chromatography with electro-chemical detection. With the analytical method developed, 5-hydroxytryptophan, serotonin, and 5-hydroxyindole-3-acetic acid could be measured simultaneously. Indole-3-acetaldehyde (12-1200 microM) was found to cause a 6-33% inhibition of the enzyme. Dependent upon the nature of the sulfhydryl- or reducing-agent (dithiotreitol, glutathione, or ascorbate) present in the incubates, the degree of inhibition by disulfiram varied, probably due to the formation of various mixed disulfides. Also the presence of diethyldithiocarbamate (160-1600 microM) was found to inhibit tryptophan hydroxylase (28-91%), while 5-hydroxyindole-3-acetaldehyde, coprine, or 1-aminocyclopropanol appeared to have no effect on the enzyme activity.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani

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 (C 2’H) from sweet potato. By comparing these twomore » structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

Exome sequencing and SNP analysis detect novel compound heterozygosity in fatty acid hydroxylase-associated neurodegeneration

PubMed Central

Pierson, Tyler Mark; Simeonov, Dimitre R; Sincan, Murat; Adams, David A; Markello, Thomas; Golas, Gretchen; Fuentes-Fajardo, Karin; Hansen, Nancy F; Cherukuri, Praveen F; Cruz, Pedro; Blackstone, Craig; Tifft, Cynthia; Boerkoel, Cornelius F; Gahl, William A

2012-01-01

Fatty acid hydroxylase-associated neurodegeneration due to fatty acid 2-hydroxylase deficiency presents with a wide range of phenotypes including spastic paraplegia, leukodystrophy, and/or brain iron deposition. All previously described families with this disorder were consanguineous, with homozygous mutations in the probands. We describe a 10-year-old male, from a non-consanguineous family, with progressive spastic paraplegia, dystonia, ataxia, and cognitive decline associated with a sural axonal neuropathy. The use of high-throughput sequencing techniques combined with SNP array analyses revealed a novel paternally derived missense mutation and an overlapping novel maternally derived ∼28-kb genomic deletion in FA2H. This patient provides further insight into the consistent features of this disorder and expands our understanding of its phenotypic presentation. The presence of a sural nerve axonal neuropathy had not been previously associated with this disorder and so may extend the phenotype. PMID:22146942

Tumoral vitamin D synthesis by CYP27B1 1-alpha-hydroxylase delays mammary tumor progression in the PyMT-MMTV mouse model and its action involves NF-kappaB modulation

USDA-ARS?s Scientific Manuscript database

Biologically-active vitamin D (1,25(OH)2D) is synthetized from inactive prohormone 25(OH)D by the enzyme CYP27B1 1-a-hydroxylase in kidney and several extra-renal tissues including breast. While the development of breast cancer has been linked to inadequate vitamin D status, the importance of bioac...

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

PubMed Central

Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.

2009-01-01

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397

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.

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

PubMed Central

Topp, E; Xun, L Y; Orser, C S

1992-01-01

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative. PMID:1610174

Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa-Responsive Dystonia

PubMed Central

Fossbakk, Agnete; Kleppe, Rune; Knappskog, Per M; Martinez, Aurora; Haavik, Jan

2014-01-01

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients. PMID:24753243

Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens

PubMed Central

Perez, Dale M.; Richards, Mark P.; Parker, Robert S.; Berres, Mark E.; Wright, Aaron T.; Sifri, Mamduh; Sadler, Natalie C; Tatiyaborworntham, Nantawat; Li, Na

2016-01-01

Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens. PMID:26653675

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka

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 completelymore » 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.« less

The Mechanisms and Effects of the Plant Activation of Chemicals in the Environment

DTIC Science & Technology

1991-12-02

amino- Uilich et al., 1973; Raz- fluorene-N-hydroxylase. zouk et al., 1980 * Did not inhibit the TXI cell activation of m-PDA, howev- Wagner et al...P450 in mammals and Goujon et al., 1972, Car- yeast. Inhibited 2-AF hydroxylase. ratore et al., 1986; Raz- zouk et al., 1980 i Did not inhibit the TXI...Activation of 2-aminofluorene by cultured plant cells. Science 219:1427-1429 Poulsen LL, R.M. Hyslop , D.M. Ziegler. 1974. S-oxidation of

Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

PubMed

Carlson, G P; Dziezak, J D; Johnson, K M

1979-07-01

1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

[Medullary layer activity of the rat adrenals after a flight on the Kosmos-1129 biosatellite].

PubMed

Kvetnanský, R; Blazicek, P; Tigranian, R A

1982-01-01

After a 18.5-day space flight on Cosmos-1129 rat adrenals were investigated for the concentration of catecholamines and activity of enzymes involved in their synthesis, i.e. tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenyl ethanol amine-N-methyl transferase. It was found that inflight the sympatho-adreno-medullary system of rats was not exposed to a prolonged or strong stressogenic effect. Postflight the rats showed an increased reactivity to the immobilization stress.

Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

PubMed

Wang, Weixue; Liang, Alexandria D; Lippard, Stephen J

2015-09-15

A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of "Fe2O2" intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.

On the role of brain serotonin in expression of genetic predisposition to catalepsy in animal models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popova, N.K.; Kulikov, A.V.

1995-06-19

The activity of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, in the striatum but not in the hippocampus and midbrain of rats bred for predisposition to catalepsy was higher than in nonselected rats. Mice of the highly susceptible to catalepsy CBA strain also differed from other noncataleptic mouse strains by the highest tryptophan hydroxylase activity in the striatum. Inhibition of tryptophan hydroxylase with p-chlorophenylalanine and p-chloromethamphetamine drastically decreased immobility time in hereditary predisposed to catalepsy animals. A decrease in the {sup 3}H-ketanserin specific binding in the striatum of cataleptic rats and CBA mice was found. It was suggested thatmore » this decrease in 5-HT2A serotonin receptor density represented a down regulation of the receptors due to an activation of serotonergic transmission in striatum. It is suggested that hereditary catalepsy may be resulted from genetic changes in the regulation of serotonin metabolism in striatum. 32 refs., 6 figs.« less

A Connective Tissue Disorder Caused by Mutations of the Lysyl Hydroxylase 3 Gene

PubMed Central

Salo, Antti M.; Cox, Helen; Farndon, Peter; Moss, Celia; Grindulis, Helen; Risteli, Maija; Robins, Simon P.; Myllylä, Raili

2008-01-01

Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders. PMID:18834968

Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil.

PubMed

Mahmoud, Soheil S; Williams, Matthew; Croteau, Rodney

2004-03-01

cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.

[Class I HLA antigens in children from families with congenital adrenal hyperplasia].

PubMed

Turowska-Heydel, D; Pietrzyk, J J; Turowski, G

1995-02-01

Congenital adrenal hyperplasia (CAH) is a syndrome of adrenal steroid metabolism errors with an autosomal inheritance model. The most common metabolic defect is 21-hydroxylase deficiency. It has been demonstrated that 21-hydroxylase genes are in close association with HLA antigens. I HLA antigens were typed in a group of 32 families of children with CAH-type 21-hydroxylase deficiency with salt loss. The antigen frequencies were determined and compared to those of the control population. The studies revealed that two HLA antigens determined by the B Locus, i.e. HLA-B47 and HLA-B61, showed a highly significant frequency (chi 2 corresponding to 404,5259 and 23,7808, respectively). The calculated relative risk and etiologic fraction values were extremely high, distinguishing the population of patients and their parents. The RR value among patients was 427.1 for HLA-B47 and 7.8 for HLA-B61 antigen. Studies on the correlation between HLA and CAH indicate an association with HLA-B47 and HLA-B61 antigens.

Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

PubMed Central

Suidan, Georgette L.; 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

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.

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: description of a new mutation, R384X.

PubMed

Matallana-Rhoades, Audrey Mary; Corredor-Castro, Juan David; Bonilla-Escobar, Francisco Javier; Mecias-Cruz, Bony Valentina; Mejia de Beldjena, Liliana

2016-09-30

It is presented the phenotype of a new compound heterozygous mutation of the genes R384X and Q356X encoding the enzyme of 11-beta-hydroxylase. Severe virilization, peripheral hypertension, and early puberty. Managed with hormone replacement therapy (corticosteroid) and antihypertensive therapy (beta-blocker), resulting in the control of physical changes and levels of arterial tension. According to the phenotypic characteristics of the patient, it is inferred that the R384X mutation carries an additional burden on the Q356X mutation, with the latter previously described as a cause of 11-beta-hydroxylase deficiency. The description of a new genotype, as in this case, expands the understanding of the hereditary burden and deciphers the various factors that lead to this pathology as well as the other forms of congenital adrenal hyperplasia (CAH), presenting with a broad spectrum of clinical presentations. This study highlights the importance of a complete description of the patient's CAH genetic profile as well as their parents' genetic profile.

Differential regulation of catecholamine synthesis and transport in rat adrenal medulla by fluoxetine treatment.

PubMed

Spasojevic, Natasa; Jovanovic, Predrag; Dronjak, Sladjana

2015-03-01

We have recently shown that chronic fluoxetine treatment acted significantly increasing plasma norepinephrine and epinephrine concentrations both in control and chronically stressed adult male rats. However, possible effects of fluoxetine on catecholamine synthesis and re-uptake in adrenal medulla have been largely unknown. In the present study the effects of chronic fluoxetine treatment on tyrosine hydroxylase, a rate-limiting enzyme in catecholamine synthesis, as well as a norepinephrine transporter and vesicular monoamine transporter 2 gene expressions in adrenal medulla of animals exposed to chronic unpredictable mild stress (CUMS) for 4 weeks, were investigated. Gene expression analyses were performed using a real-time quantitative reverse transcription-PCR. Chronically stressed animals had increased tyrosine hydroxylase mRNA levels and decreased expression of both transporters. Fluoxetine increased tyrosine hydroxylase and decreased norepinephrine transporter gene expression in both unstressed and CUMS rats. These findings suggest that chronic fluoxetine treatment increased plasma catecholamine levels by affecting opposing changes in catecholamine synthesis and uptake.

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

Effect of coexposure to asbestos and kerosene soot on pulmonary drug-metabolizing enzyme system.

PubMed Central

Arif, J M; Khan, S G; Mahmood, N; Aslam, M; Rahman, Q

1994-01-01

This article reports the effect of coexposure to Indian chrysotile asbestos (5 mg/rat) and kerosene soot (5 mg/rat) on the pulmonary phase I and phase II drug-metabolizing enzymes 1, 4, 8, 16, 30, 90, and 150 days after a single intratracheal inoculation. Exposure to soot resulted in a significant induction of the pulmonary microsomal cytochrome P450 and the activity of dependent monooxygenase, benzo(a)pyrene (B[a]P) hydroxylase, and epoxide hydrase at all time intervals. On the other hand, the cytosolic glutathione S-transferase (GST) activity was induced at days 1, 4, 8, 16, and 30 after exposure, followed by inhibition in the enzyme activity. In contrast, chrysotile exposure depleted cytochrome P450, B[a]P hydroxylase, epoxide hydrase, and GST at initial stages, while all these parameters except GST were induced at later stages. However, coexposure to chrysotile and soot led to a significant inhibition in the cytochrome P450 levels, activities of B[a]P hydroxylase, epoxide hydrase, and GST at initial stages of exposure. At advanced stages, however, an additional increase in cytochrome P450, B[a]P hydroxylase, and epoxide hydrase but a decrease in GST was observed. These results clearly show that the intratracheal coexposure to high levels of asbestos and kerosene soot alters the metabolic activity of the lung, which is turn may retain toxins in the system for a longer period, resulting in adverse pathological disorders. PMID:7882926

Electronic structure description of the cis-MoOS unit in models for molybdenum hydroxylases.

PubMed

Doonan, Christian J; Rubie, Nick D; Peariso, Katrina; Harris, Hugh H; Knottenbelt, Sushilla Z; George, Graham N; Young, Charles G; Kirk, Martin L

2008-01-09

The molybdenum hydroxylases catalyze the oxidation of numerous aromatic heterocycles and simple organics and, unlike other hydroxylases, utilize water as the source of oxygen incorporated into the product. The electronic structures of the cis-MoOS units in CoCp2[TpiPrMoVOS(OPh)] and TpiPrMoVIOS(OPh) (TpiPr = hydrotris(3-isopropylpyrazol-1-yl)borate), new models for molybdenum hydroxylases, have been studied in detail using S K-edge X-ray absorption spectroscopy, vibrational spectroscopy, and detailed bonding calculations. The results show a highly delocalized Mo=S pi* LUMO redox orbital that is formally Mo(dxy) with approximately 35% sulfido ligand character. Vibrational spectroscopy has been used to quantitate Mo-Ssulfido bond order changes in the cis-MoOS units as a function of redox state. Results support a redox active molecular orbital that has a profound influence on MoOS bonding through changes to the relative electro/nucleophilicity of the terminal sulfido ligand accompanying oxidation state changes. The bonding description for these model cis-MoOS systems supports enzyme mechanisms that are under orbital control and dominantly influenced by the unique electronic structure of the cis-MoOS site. The electronic structure of the oxidized enzyme site is postulated to play a role in polarizing a substrate carbon center for nucleophilic attack by metal activated water and acting as an electron sink in the two-electron oxidation of substrates.

Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

PubMed

Sakowski, Stacey A; Geddes, Timothy J; Thomas, David M; Levi, Edi; Hatfield, James S; Kuhn, Donald M

2006-04-26

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase.

PubMed

McCoy, G D

1980-03-01

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

25-Hydroxyvitamin D3-1 alpha-hydroxylase in porcine hepatic tissue: subcellular localization to both mitochondria and microsomes.

PubMed Central

Hollis, B W

1990-01-01

In vitro studies were performed to assess the ability of hepatic homogenates, mitochondria, and microsomes to 1 alpha-hydroxylate 25-hydroxyvitamin D3 [25(OH)D3]. Addition of 25(OH)D3 to either hepatic mitochondria or microsomes caused a concentration-dependent increase in the production of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Hepatic homogenates also produced purported 1,25(OH)2D3, although at a much reduced efficiency as compared with hepatic mitochondria or microsomes. Purported 1,25(OH)2D3 synthesized by hepatic mitochondria or microsomes was identified by its mobility on several high-performance liquid chromatographic systems and, ultimately, by its ability to interact with the bovine thymus 1,25(OH)2D3 receptor protein. Production of 1,25(OH)2D3 by hepatic mitochondria and microsomes was dependent on time of incubation, protein content, and pH of incubation medium, and it required an adequate source of reducing equivalents. Generation of 1,25(OH)2D3 by these organelles could be totally blocked by the cytochrome P-450 inhibitor ketoconazole. The microsomal 1 alpha-hydroxylase could not be saturated even at the highest concentration (240 microM) of 25(OH)D3 used. The mitochondrial 1 alpha-hydroxylase, however, displayed saturation at approximately 40 microM 25(OH)D3. Eadie-Hofstee reciprocal plot analysis of the hepatic mitochondrial 1 alpha-hydroxylase gave a Km of 17 microM 25(OH)D3 and a Vmax of 481 pg of 1,25(OH)2D3 per min per mg of protein. Because of its inability to achieve substrate saturation, meaningful kinetic parameters could not be calculated for the hepatic microsomal 1 alpha-hydroxylase. These data demonstrate the liver to be an even more dynamic organ than was previously believed with respect to vitamin D metabolism in that the liver has the potential to produce 1,25(OH)2D3 in situ by at least two separate mechanisms. PMID:2385581

Superiority of dietary safflower oil over olive oil in lowering serum cholesterol and increasing hepatic mRnas for the LDL receptor and cholesterol 7alpha-hydroxylase in exogenously hypercholesterolemic (exHC) rats.

PubMed

Sato, M; Yoshida, S; Nagao, K; Imaizumi, K

2000-06-01

The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of serum cholesterol.

Carotenoid Biosynthesis in the Primitive Red Alga Cyanidioschyzon merolae▿

PubMed Central

Cunningham, Francis X.; Lee, Hansel; Gantt, Elisabeth

2007-01-01

Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and β-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, β-carotene, and zeaxanthin. Carotenoids with ɛ-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only β-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene β-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related ɛ-ring cyclases from several plant species were found to be less specific, yielding products with β-rings as well as ɛ-rings. C. merolae lacks orthologs of genes that encode the two types of β-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of β-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae β-carotene hydroxylase therefore remains uncertain. PMID:17085635

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.

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

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

PubMed

Hansdottir, Sif; Monick, Martha M; Hinde, Sara L; Lovan, Nina; Look, Dwight C; Hunninghake, Gary W

2008-11-15

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Characterization and two-dimensional crystallization of membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1.

PubMed

Alonso, Hernan; Roujeinikova, Anna

2012-11-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

PubMed

Meng, C X; Wei, W; Su, Z- L; Qin, S

2006-10-01

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.

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

A Cell surface β-Hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma

PubMed Central

Aihara, Arihiro; Huang, Chiung-Kuei; Olsen, Mark J.; Lin, Qiushi; Chung, Waihong; Tang, Qi; Dong, Xiaoqun; Wands, Jack R.

2014-01-01

Hepatocellular carcinoma (HCC) has a poor prognosis due to widespread intrahepatic and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(Asparaginyl)-β-hydroxylase (ASPH) is a cell surface enzyme that generates enhanced cell motility, migration, invasion and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer assisted drug design. Candidate compounds were tested for inhibition of β-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biologic effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor MO-I-1100 was selected since it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion and anchorage independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. Conclusions These studies suggest that the enzymatic activity of ASPH was important for hepatic oncogenesis. Reduced β-hydroxylase activity generated by the SMI MO-I-1100 led to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease. PMID:24954865

The effects of Urtica dioica L. leaf extract on aniline 4-hydroxylase in mice.

PubMed

Ozen, Tevfik; Korkmaz, Halil

2009-01-01

The effects of hydroalcoholic (80% ethanol-20% water) extract of Urtica dioica L. on microsomal aniline 4-hydroxylase (A4H) were investigated in the liver of Swiss albino mice (8- 10-weeks-old) treated with two doses (50 and 100 mg/kg body weight, given orally for 14 days ). The activities of A4H showed a significant increase in the liver at both dose levels of extract treatment. The hydroalcoholic extract of Urtica dioica induced the activities of A4H that had been increased by treatment of metal ions (Mg2+ and Ca2+) and the mixture of cofactors (NADH and NADPH). At saturated concentration of cofactor, microsomal A4H exhibited significantly even higher activities in the presence of the mixture of cofactors than NADPH and NADH. Mg2+ and Ca2+ ions acted as stimulants in vitro. The present results suggest that the hydroalcoholic extract of Urtica dioica may have modalatory effect on aniline hydroxylase at least in part and enhance the activity of A4H adding metals ions and cofactors.

Le pronostic statural après une pseudo-puberté précoce par déficit en 11ß hydroxylase chez une fille de 7 ans: à propos d'un cas avec revue de la literature

PubMed Central

Latrech, Hanane; Gaouzi, Ahmed

2015-01-01

L'hyperplasie congénitale des surrénales par déficit en 11ß hydroxylase se voit dans 5 à 10% des cas et est diagnostiquée, habituellement, devant des signes de virilisation d'un nouveau né ou d'un fœtus 46 XX et plus tard devant une HTA avec une hypokaliémie. Le pronostic de taille chez ces patients est très souvent compromis. Nous rapportons le cas d'un déficit en 11ß hydroxylase chez une enfant âgée de 7 ans révélé par une pseudo- puberté précoce traitée par hydrocortisone. La survenue d'une puberté précoce centrale complique encore plus la situation par la mise en jeu du pronostic de taille finale et nécessitant une prise en charge thérapeutique particulière. PMID:26113905

Induction of hepatic aryl hydrocarbon hydroxylase and epoxide hydrase in Wistar rats pretreated with oral methadone hydrochloride.

PubMed

Bellward, G D; Gontovnick, L S; Otten, M

1977-01-01

Methadone-HCl added to the drinking water of adult female Wistar rats for 4 weeks produced an increase in the aryl hydrocarbon hydroxylase activity of the hepatic microsomal fraction to 222% of control levels. No change was seen in epoxide hydrase activity. In contrast, when male rats were treated similarly, there was an increase in epoxide hydrase activity to 212% of controls with no change in aryl hydrocarbon hydroxylase activity. No such changes were observed when the subcutaneous route of administration or chronic, low-dose, intraperitoneal injections were used. There were no differences in hepatic cytochrome P-450 or protein concentrations in treated animals as compared to their respective control groups. Control studies were carried out with quinine sulfate in the drinking water to decrease water intake to the level of the methadone-treated group. No elevation in either enzyme activity occurred in this control group. Similarly, paired-feeding studies showed the elevation of enzyme activity to be due to the methadone, not food deprivation. The effects of concurrent therapy of methadone with phenobarbital sodium or 3-methylcholanthrene were compared.

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

PubMed

Zhang, Nan; Han, Zhentai; Sun, Guiling; Hoffman, Angela; Wilson, Iain W; Yang, Yanfang; Gao, Qian; Wu, Jianqiang; Xie, Dan; Dai, Jungui; Qiu, Deyou

2014-01-17

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp. Copyright © 2013 Elsevier Inc. All rights reserved.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Therapeutic inhibition of prolyl hydroxylase domain-containing enzymes in surgery: putative applications and challenges

PubMed Central

Harnoss, Jonathan Michael; Strowitzki, Moritz Johannes; Radhakrishnan, Praveen; Platzer, Lisa Katharina; Harnoss, Julian Camill; Hank, Thomas; Cai, Jun; Ulrich, Alexis; Schneider, Martin

2015-01-01

Oxygen is essential for metazoans to generate energy. Upon oxygen deprivation adaptive and protective pathways are induced, mediated by hypoxia-inducible factors (HIFs) and prolyl hydroxylase domain-containing enzymes (PHDs). Both play a pivotal role in various conditions associated with prolonged ischemia and inflammation, and are promising targets for therapeutic intervention. This review focuses on aspects of therapeutic PHD modulation in surgically relevant disease conditions such as hepatic and intestinal disorders, wound healing, innate immune responses, and tumorigenesis, and discusses the therapeutic potential and challenges of PHD inhibition in surgical patients. PMID:27774478

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

[Noradrenaline and the enzymes of its synthesis and breakdown in the rat hypothalamus after a flight on the Kosmos-936 biosatellite].

PubMed

Torda, T; Kvetnansky, R; Tigranian, R A; Chulman, J; Genin, A M

1981-01-01

In the hypothalamus of the weightless and centrifuged rats flown for 18.5 days on board the biosatellite Cosmos-936 the noradrenaline concentration and activity of the enzymes involved in the catecholamine synthesis and degradation were measured. It was found that under the space flight influence the noradrenaline concentration and tyrosine hydroxylase, dopamine-beta-hydroxylase and monoamine oxidase activities remained unaltered. These findings indicate that a prolonged exposure to weightlessness was not a stressogenic agent that could activate the adrenergic system in the rat hypothalamus.

The contribution of hepatic inactivation of testosterone to the lowering of serum testosterone levels by ketoconazole.

PubMed

Wilson, V S; LeBlanc, G A

2000-03-01

Hepatic biotransformation processes can be modulated by chemical exposure and these alterations can impact the biotransformation of endogenous substrates. Furthermore, chemically mediated alterations in the biotransformation of endogenous steroid hormones have been implicated as a mechanism by which steroid hormone homeostasis can be disrupted. The fungicide ketoconazole has been shown to lower serum testosterone levels and alter both gonadal synthesis and hepatic inactivation of testosterone. The present study examined whether the effects of ketoconazole on the hepatic biotransformation of testosterone contribute to its lowering of serum testosterone levels. Results also were used to validate further the use of the androgen-regulated hepatic testosterone 6alpha/15alpha-hydroxylase ratio as an indicator of androgen status. Male CD-1 mice were fed from 0 to 160 mg/kg ketoconazole in honey. Four h after the initial treatment, serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylase activity decreased, and the hepatic testosterone 6alpha/15alpha-hydroxylase ratio increased in a dose-dependent manner. Immunoblot analysis indicated that the transient decline in hepatic biotransformation was not due to reduced P450 protein levels. Rather, hepatic testosterone biotransformation activities were found to be differentially susceptible to direct inhibition by ketoconazole. Differential inhibition was also responsible for the increase seen in the 6alpha/15alpha-hydroxylase ratio. The changes in serum testosterone levels could be explained by decreased gonadal synthesis of testosterone and were not impacted by decreased hepatic biotransformation of testosterone. These results demonstrate that changes in the hepatic hydroxylation of testosterone by ketoconazole, and perhaps other chemicals, have little or no influence serum testosterone levels.

Relative Expression of Vitamin D Hydroxylases, CYP27B1 and CYP24A1, and of Cyclooxygenase-2 and Heterogeneity of Human Colorectal Cancer in Relation to Age, Gender, Tumor Location, and Malignancy: Results from Factor and Cluster Analysis.

PubMed

Brozek, Wolfgang; Manhardt, Teresa; Kállay, Enikö; Peterlik, Meinrad; Cross, Heide S

2012-07-26

Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1a-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression.

Relative Expression of Vitamin D Hydroxylases, CYP27B1 and CYP24A1, and of Cyclooxygenase-2 and Heterogeneity of Human Colorectal Cancer in Relation to Age, Gender, Tumor Location, and Malignancy: Results from Factor and Cluster Analysis

PubMed Central

Brozek, Wolfgang; Manhardt, Teresa; Kállay, Enikö; Peterlik, Meinrad; Cross, Heide S.

2012-01-01

Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1α-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression. PMID:24213465

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

PubMed

Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils

2017-12-01

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

PubMed

Flachowsky, Henryk; Halbwirth, Heidi; Treutter, Dieter; Richter, Klaus; Hanke, Magda-Viola; Szankowski, Iris; Gosch, Christian; Stich, Karl; Fischer, Thilo C

2012-02-01

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

PubMed Central

Nakano, Masashi; Kino, Kuniki

2015-01-01

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA. PMID:25795668

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

PubMed Central

Alonso, Hernan

2012-01-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C12E8]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism. PMID:22941083

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

PubMed

Hutabarat, Olly Sanny; Flachowsky, Henryk; Regos, Ionela; Miosic, Silvija; Kaufmann, Christine; Faramarzi, Shadab; Alam, Mohammed Zobayer; Gosch, Christian; Peil, Andreas; Richter, Klaus; Hanke, Magda-Viola; Treutter, Dieter; Stich, Karl; Halbwirth, Heidi

2016-05-01

Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3'-hydroxylase (F3'H). Using recombinant F3'H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3'H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.

RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

PubMed

Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

2017-05-01

Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

Endocrine and molecular investigations in a cohort of 25 adolescent males with prominent/persistent pubertal gynecomastia.

PubMed

Paris, F; Gaspari, L; Mbou, F; Philibert, P; Audran, F; Morel, Y; Biason-Lauber, A; Sultan, C

2016-03-01

Pubertal gynecomastia is a common condition observed in up to 65% of adolescent males. It is usually idiopathic and tends to regress within 1-2 years. In this descriptive cross-sectional study, we investigated 25 adolescent males with prominent (>B3) and/or persistent (>2 years) pubertal gynecomastia (P/PPG) to determine whether a hormonal/genetic defect might underline this condition. Endocrine investigation revealed the absence of hormonal disturbance for 18 boys (72%). Three patients presented Klinefelter syndrome and three a partial androgen insensitivity syndrome (PAIS) as a result of p.Ala646Asp and p.Ala45Gly mutations of the androgen receptor gene. The last patient showed a 17α-hydroxylase/17,20-lyase deficiency as a result of a compound heterozygous mutation of the CYP17A1 gene leading to p.Pro35Thr(P35T) and p.Arg239Stop(R239X) in the P450c17 protein. Enzymatic activity was analyzed: the mutant protein bearing the premature stop codon R239X showed a complete loss of 17α-hydroxylase and 17,20-lyase activity. The mutant P35T seemed to retain 15-20% of 17α-hydroxylase and about 8-10% of 17,20-lyase activity. This work demonstrates that P/PPG had an endocrine/genetic cause in 28% of our cases. PAIS may be expressed only by isolated gynecomastia as well as by 17α-hydroxylase/17,20-lyase deficiency. Isolated P/PPG is not always a 'physiological' condition and should thus be investigated through adequate endocrine and genetic investigations, even though larger studies are needed to better determine the real prevalence of genetic defects in such patients. © 2016 American Society of Andrology and European Academy of Andrology.

Quantitative RT-PCR and immunoblot analyses reveal acclimated A2 noradrenergic neuron substrate fuel transporter, glucokinase, phospho-AMPK, and dopamine-β-hydroxylase responses to hypoglycemia.

PubMed

Cherian, Ajeesh Koshy; Briski, Karen P

2011-07-01

Cellular metabolic stasis is monitored in discrete brain sites, including the dorsal vagal complex (DVC), where A2 noradrenergic neurons perform this sensory function. Single-cell qPCR and high-sensitivity immunoblotting were used to determine if A2 neurons adapt to chronic hypoglycemia by increasing substrate fuel transporter expression, and whether such adjustments coincide with decreased cellular energy instability during this systemic metabolic stress. Tyrosine hydroxylase-immunolabeled neurons were laser-microdissected from the caudal DVC 2 hr after single or serial neutral protamine Hagedorn insulin (NPH) dosing. Preceding hypoglycemia suppressed basal A2 MCT2, GLUT3, and GLUT4 profiles and diminished MCT2, GLUT4, and glucokinase responses to recurring hypoglycemia. Acute NPH caused a robust increase in A2 phospho-AMPK protein levels; baseline phospho-AMPK expression was elevated after 3 days of insulin treatment but only slight augmented after a fourth NPH injection. Transcripts encoding the catecholamine biosynthetic enzyme dopamine-β-hydroxylase were unaffected by acute NPH but were diminished by serial insulin dosing. This evidence for diminished basal A2 glucose and lactate uptake and attenuated phospho-AMPK-mediated detection of hypoglycemia-associated energy deficits suggests that these cells acclimate to chronic hypoglycemia by adopting a new metabolic steady state characterized by energy paucity and reduced sensitivity to hypoglycemia. Because dopamine-β-hydroxylase mRNA was reduced after serial, but not single NPH dosing, A2 neurotransmitter biosynthesis may be impervious to acute hypoglycemia but inhibited when posthypoglycemic metabolic deficiency is exacerbated by recurring hypoglycemia. This research suggests that chronic hypoglycemia-associated adjustments in A2-sensory neurotransmission may reflect cellular energetic debilitation rather than adaptive attenuation of cellular metabolic imbalance. Copyright © 2011 Wiley-Liss, Inc.

Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph Methylosulfonomonas methylovora

PubMed Central

de Marco, Paolo; Moradas-Ferreira, Pedro; Higgins, Timothy P.; McDonald, Ian; Kenna, Elizabeth M.; Murrell, J. Colin

1999-01-01

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria. PMID:10094704

The α-Amylase Induction in Endosperm during Rice Seed Germination Is Caused by Gibberellin Synthesized in Epithelium1

PubMed Central

Kaneko, Miyuki; Itoh, Hironori; Ueguchi-Tanaka, Miyako; Ashikari, Motoyuki; Matsuoka, Makoto

2002-01-01

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3β-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909–8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3β-hydroxylase genes and also an α-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and α-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3β-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3β-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for α-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for α-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced α-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not. PMID:11950975

Maternal drug abuse and human term placental xenobiotic and steroid metabolizing enzymes in vitro.

PubMed

Paakki, P; Stockmann, H; Kantola, M; Wagner, P; Lauper, U; Huch, R; Elovaara, E; Kirkinen, P; Pasanen, M

2000-02-01

We evaluated the impact of maternal drug abuse at term on human placental cytochrome P450 (CYP)-mediated (Phase I) xenobiotic and steroid-metabolizing activities [aromatase, 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), pyrene 1-hydroxylase (P1OH), and testosterone hydroxylase], and androstenedione-forming isomerase, NADPH quinone oxidoreductase (Phase II), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) activities in vitro. Overall, the formation of androstenedione, P1OH, and testosterone hydroxylase was statistically significant between control and drug-abusing subjects; we observed no significant differences in any other of the phase I and II activities. In placentas from drug-abusing mothers, we found significant correlations between ECOD and P1OH activities (p < 0. 001), but not between ECOD and aromatase or P1OH and EROD activities; we also found significant correlations between blood cotinine and UGT activities (p < 0.01). In contrast, in controls (mothers who did not abuse drugs but did smoke cigarettes), the P1OH activity correlated with ECOD, EROD (p < 0.001), and testosterone hydroxylase (p < 0.001) activities. Our results (wider variation in ECOD activity among tissue from drug-abusing mothers and the significant correlation between P1OH and ECOD activities, but not with aromatase or EROD activities) indicate that maternal drug abuse results in an additive effect in enhancing placental xenobiotic metabolizing enzymes when the mother also smokes cigarettes; this may be due to enhancing a "silent" CYP form, or a new placental CYP form may be activated. The change in the steroid metabolism profile in vitro suggests that maternal drug abuse may alter normal hormonal homeostasis during pregnancy.

Tryptophan Hydroxylase 2 Gene Polymorphism in Anxiety and Depressive Disorder in Kashmiri Population

PubMed Central

Shoib, Sheikh; Shah, Tabindah; Mushtaq, Sahil

2014-01-01

Background: The gene of tryptophan hydroxylase is widely recognized as a major candidate gene in many psychiatric disorders. However, no study has been done which investigates tryptophan hydroxylase 2 gene polymorphism in anxiety and depressive disorders in Kashmiri population (India). Objectives: To study tryptophan hydroxylase 2 (TPH2) C 11993 A gene polymorphism in anxiety and depressive disorders. Method: Sixty patients of depression disorder, 60 patients of anxiety disorder and 40 unrelated healthy volunteers (control) were studied in a case control design. Polymorphism was determined using polymerase chain reaction (PCR) and agarose gel electrophoresis after digestion with HAP II enzyme. Genotypes and allele frequencies were compared using Chi-square tests, Fischer’s exact test, odds ratio, 95% confidence interval (C.I) and p-value of <0.05 was considered to be statistical significant. Results: The mean age ± SD of anxiety, depression and control group was 32.73±10.99, 32.20±10 and 29.75±10.12 respectively and the difference was found to be statistically non significant (p=0.349).The mean HAM-A (Hamilton rating scale for anxiety) score and HAM-D (Hamilton rating scale for depression) score was high in both groups (anxiety and depression) and found to be statistically significant (p=0.001).Depression group had AA genotype (55.2%) than control (37.5%) and was found to be statistically non significant (p=0.890).Comparison of allelic frequency revealed no association of A allele in anxiety group (76.67%) compared with control (75.5%) and was found to be statistically non significant (p= 0.866), OR 1.09 (0.56-2.11). Conclusion: TPH2C 11993 A gene was not found to be associated with major depressive disorder (MDD) and anxiety disorder in Kashmiri population. PMID:25121048

In vitro differentiation of quail neural crest cells into sensory-like neuroblasts

NASA Technical Reports Server (NTRS)

Sieber-Blum, Maya; Kumar, Sanjiv R.; Riley, Danny A.

1988-01-01

Data are presented that demonstrate the ability of quail neural-crest embrionic cells grown as primary culture to differentiate in vitro into sensorylike neuroblasts. After 7-14 days of growth as primary culture, many of the putative sensory neuroblasts displayed substance P (SP)-like immunoreactivity and some exhibited histochemical carbonic anhydrase activity. Double staining experiments showed that the SP-like immunoreactive neuroblasts did not contain detectable levels of tyrosine hydroxylase or dopamine-beta-hydroxylase. The neuronal nature of the cultured sensorylike neuroblasts was further documented by double labeling for antibodies against the 68 kDa neurofilament polypeptide and substance P.

Immunology of Addison's disease and premature ovarian failure.

PubMed

Husebye, Eystein S; Løvås, Kristian

2009-06-01

Autoimmune Addison's disease and autoimmune ovarian insufficiency are caused by selective targeting by T and B lymphocytes to the steroidogenic apparatus in these organs. Autoantibodies toward 21-hydroxylase are a clinically useful marker for autoimmune Addison's disease. Autoantibodies to 21-hydroxylase are found in premature ovarian insufficiency, but others also can be present, notably antibodies against side-chain cleavage enzyme. The autoimmune response primarily targets the theca cells, yielding elevated concentrations of inhibin, which is emerging as a useful diagnostic marker for autoimmune etiology of ovarian insufficiency. Little is known about its immunogenetics, but in contrast to Addison's disease, several experimental models of autoimmune premature ovarian insufficiency are available for study.

The effects of estrus cycle on drug metabolism in the rat.

PubMed

Brandstetter, Y; Kaplanski, J; Leibson, V; Ben-Zvi, Z

1986-01-01

The effect of the female rat estral cycle on microsomal drug metabolism in-vivo and in-vitro has been studied. Two microsomal enzymes, aminopyrine-N-demethylase and aniline hydroxylase showed a greater specific activity (p less than 0.01) in the diestrus phase of the estral cycle while the oxidative enzyme aryl hydrocarbon hydroxylase and the conjugative enzyme, glucuronyl transferase, were not affected. In vivo studies which included theophylline and antipyrine metabolism, and hexobarbital sleeping times showed no difference between the different phases of the estral cycle. Conflicting evidence about the effect of steroid sex hormones on hepatic drug metabolism is discussed.

XAFS of human tyrosine hydroxylase

NASA Astrophysics Data System (ADS)

Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.

1995-02-01

Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

PubMed

Li, Jun; Long, Deng-Kai; Zhou, Tao; Ding, Ling; Zheng, Wei; Jiang, Wei-Ke

2016-07-01

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress. Copyright© by the Chinese Pharmaceutical Association.

Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility.

PubMed

Astuti, Dewi; Ricketts, Christopher J; Chowdhury, Rasheduzzaman; McDonough, Michael A; Gentle, Dean; Kirby, Gail; Schlisio, Susanne; Kenchappa, Rajappa S; Carter, Bruce D; Kaelin, William G; Ratcliffe, Peter J; Schofield, Christopher J; Latif, Farida; Maher, Eamonn R

2011-02-01

Germline mutations in the von Hippel-Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.

Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO.

PubMed

Michiel, Magalie; Perchat, Nadia; Perret, Alain; Tricot, Sabine; Papeil, Aude; Besnard, Marielle; de Berardinis, Véronique; Salanoubat, Marcel; Fischer, Cécile

2012-12-01

In aerobic cells, urate is oxidized to 5-hydroxyisourate by two distinct enzymes: a coenzyme-independent urate oxidase (EC 1.7.3.3) found in eukaryotes and bacteria like Bacillus subtilis and a prokaryotic flavoprotein urate hydroxylase (HpxO) originally found in some Klebsiella species. More cases of analogous or non-homologous isofunctional enzymes (NISE) for urate catabolism have been hypothesized by inspecting bacterial genomes. Here, we used a functional complementation approach in which a candidate gene for urate oxidation is integrated by homologous recombination in the Acinetobacter baylyi ADP1 genome at the locus of its original hpxO gene. Catabolism of urate was restored in A. baylyi ADP1 expressing a FAD-dependent protein from Xanthomonas campestris, representing a new urate hydroxylase family that we called HpyO. This enzyme was kinetically characterized and compared with other HpxO enzymes. In contrast to the latter, HpyO is a typical Michaelian enzyme. This work provides the first experimental evidences for the function of HpyO in bacterial urate catabolism and establishes it as a NISE of HpxO. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1α-hydroxylase

PubMed Central

Ohnishi, Mutsuko; Nakatani, Teruyo; Lanske, Beate; Razzaque, M. Shawkat

2011-01-01

Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice. PMID:19225558

A novel homozygous mutation IVS6+5G>T in CYP11B1 gene in a Vietnamese patient with 11β-hydroxylase deficiency.

PubMed

Nguyen, Thi Phuong Mai; Nguyen, Thu Hien; Ngo, Diem Ngoc; Vu, Chi Dung; Nguyen, Thi Kim Lien; Nong, Van Hai; Nguyen, Huy Hoang

2015-07-10

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is characterized by a deficiency of one of the enzymes involved in the synthesis of cortisol from cholesterol by the adrenal cortex. CAH cases arising from impaired 11β-hydroxylase are the second most common form. Mutations in the CYP11B1 gene are the cause of 11β-hydroxylase deficiency. This study was performed on a patient with congenital adrenal hyperplasia and with premature development such as enlarged penis, muscle development, high blood pressure, and bone age equivalent of 5 years old at 2 years of chronological age. Biochemical tests for steroids confirmed the diagnosis of CAH. We used PCR and sequencing to screen for mutations in CYP11B1 gene. Results showed that the patient has a novel homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5G>T). The analysis of this mutation by MaxEntScan boundary software indicated that this mutant could affect the gene splicing during transcription. Copyright © 2015 Elsevier B.V. All rights reserved.

Combination growth hormone and gonadotropin releasing hormone analog therapy in 11beta-hydroxylase deficiency.

PubMed

Bajpai, Anurag; Kabra, Madhulika; Menon, P S N

2006-06-01

Diagnosis of 11beta-hydroxylase deficiency was made in a boy at the age of 2 1/2 years on the basis of peripheral precocious puberty, growth acceleration (height standard deviation score +4.4) with advanced skeletal maturation (bone age 8.4 years) and elevated deoxycortisol levels. Glucocorticoid supplementation led to normalization of blood pressure but was associated with progression to central precocious puberty and increase in bone age resulting in decrease in predicted adult height to 133.7 cm (target height 163 cm). The child was started on GnRH analog (triptorelin 3.75 mg every 28 days), which led to improvement in predicted adult height by 3.1 cm over 15 months. Addition of growth hormone (0.1 IU/kg/day) resulted in improvement in predicted adult height (151 cm) and height deficit (12 cm) over the next 3.6 years. Final height (151 cm) exceeded predicted height at the initiation of GnRH analog treatment by 17.3 cm. This report suggests that combination GH and GnRH analog treatment may be useful in improving height outcome in children with 11beta-hydroxylase deficiency and compromised final height.

Duration-dependence of the effect of treadmill exercise on hyperactivity in attention deficit hyperactivity disorder rats.

PubMed

Ji, Eun-Sang; Kim, Chang-Ju; Park, Jun Heon; Bahn, Geon Ho

2014-04-01

Attention-deficit hyperactivity disorder (ADHD) is a common neurobehavioral disorder, and its symptoms are hyperactivity and deficits in learning and memory. Physical exercise increases dopamine synthesis and neuronal activity in various brain regions. In the present study, we investigate the duration-dependence of the treadmill exercise on hyperactivity in relation with dopamine expression in ADHD. Spontaneously hypertensive rats were used for the ADHD rats and Wistar-Kyoto rats were used for the control rats. The rats in the exercise groups were forced to run on a treadmill for 10 min, 30 min, and 60 min once daily for 28 consecutive days. For this experiment, open field test and immunohistochemistry for tyrosine hydroxylase were conducted. The present results revealed that ADHD rats showed hyperactivity, and tyrosine hydroxylase expression in the striatum and substantia nigra were decreased in ADHD rats. Treadmill exercise alleviated hyperactivity and also increased TH expression in ADHD rats. Treadmill exercise for 30 min per day showed most potent suppressing effect on hyperactivity, and this dose of treadmill exercise also most potently inhibited tyrosine hydroxylase expression. The present study suggests that treadmill exercise for 30 min once a day is the most effective therapeutic intervention for ADHD patients.

Vitamin D-Dependent Rickets Type 1B (25-Hydroxylase Deficiency): A Rare Condition or a Misdiagnosed Condition?

PubMed

Molin, Arnaud; Wiedemann, Arnaud; Demers, Nick; Kaufmann, Martin; Do Cao, Jérémy; Mainard, Laurent; Dousset, Brigitte; Journeau, Pierre; Abeguile, Geneviève; Coudray, Nadia; Mittre, Hervé; Richard, Nicolas; Weryha, Georges; Sorlin, Arthur; Jones, Glenville; Kottler, Marie-Laure; Feillet, Francois

2017-09-01

Vitamin D requires a two-step activation by hydroxylation: The first step is catalyzed by hepatic 25-hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α-hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25-(OH) 2 D. Mutations of CYP2R1 have been associated with vitamin D-dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss-of-function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss-of-function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25-OH-D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25-(OH) 2 D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa-hydroxyvitamin D 3 or alfacalcidol (1α-OH-D 3 ) treatment, and we observed a dramatic increase in the 1,25-(OH) 2 D serum concentration, which indicated the role of accessory 25-hydroxylase enzymes. Lastly, in patients who received calcifediol (25-OH-D 3 ), we documented normal 24-hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25-OH-D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

Insights into the Catalytic Mechanisms of Phenylalanine and Tryptophan Hydroxylase from Kinetic Isotope Effects on Aromatic Hydroxylation†

PubMed Central

Pavon, Jorge Alex; Fitzpatrick, Paul F.

2006-01-01

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine as substrates. All Dkcat values are normal with Δ117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12–1.41. In contrast, for Δ117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the Dkcat value with [4-2H]-phenylalanine is 0.92 but is normal with [3,5-2H2]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Δ117PheH V379D shows a similar inverse isotope effect with [4-2H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-2H]-and [3,52H2]-phenylalanine, are identical for Δ117PheH and Δ117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the Dkcat value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight (∼34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With 2H5-indole-tryptophan as a substrate for Δ117PheH, the Dkcat value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the Dkcat values are within error of those for Δ117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine. PMID:16953590

Vascular endothelial overexpression of human CYP2J2 (Tie2-CYP2J2 Tr) modulates cardiac oxylipin profiles and enhances coronary reactive hyperemia in mice

PubMed Central

Hanif, Ahmad; Edin, Matthew L.; Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

2017-01-01

Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05). Similarly, inhibition of ω-hydroxylases increased repayment volume and repayment duration, in Tie2-CYP2J2 Tr compared to WT mice (P < 0.05). Endothelial overexpression of CYP2J2 significantly changed oxylipin profiles, including increased EETs (P < 0.05), increased EpOMEs (P < 0.05), and decreased 8-iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid-chain HETEs (5-, 11-, 12-, 15-HETEs P < 0.05) and HODEs (P < 0.05). These data demonstrate that vascular endothelial overexpression of CYP2J2, through changing the oxylipin profiles, enhances CRH. Inhibition of CYP epoxygenases decreases CRH, whereas inhibition of ω-hydroxylases enhances CRH. PMID:28328948

Estradiol or fluoxetine alters depressive behavior and tryptophan hydroxylase in rat raphe.

PubMed

Yang, Fu-Zhong; Wu, Yan; Zhang, Wei-Guo; Cai, Yi-Yun; Shi, Shen-Xun

2010-03-10

The effects of 17beta-estradiol and fluoxetine on behavior of ovariectomized rats subjected to the forced swimming test and the expression of tryptophan hydroxylase (TPH) in dorsal and median raphe were investigated, respectively through time sampling technique of behavior scoring and immunohistochemistry. Both estradiol and fluoxetine increased swimming and decreased immobility in the forced swimming test. The forced swimming stress decreased integrated optical density of TPH-positive regions in dorsal and median raphe. Both estradiol and fluoxetine administration prevented integrated optical density of TPH-positive regions from being decreased by forced swimming stress. These observations suggest that both estradiol and fluoxetine have protective bearing on ovariectomized rats enduring forced swimming stress.

Mucor hiemalis mediated 14α-hydroxylation on steroids: in vivo and in vitro investigations of 14α-hydroxylase activity.

PubMed

Kolet, Swati P; Haldar, Saikat; Niloferjahan, Siddiqui; Thulasiram, Hirekodathakallu V

2014-07-01

Transformation of testosterone and progesterone into synthetically challenging 14α-hydroxy derivatives was achieved by using fungal strain Mucor hiemalis. Prolonged incubation led to the formation of corresponding 6β/7α,14α-dihydroxy metabolites. The position and stereochemistry of newly introduced hydroxyl group was determined by detailed spectroscopic analyses. The time course experiment indicated that fungal strain initiated transformation by hydroxylation at 14α-position followed by at 6β- or 7α-positions. Studies using cell-free extracts suggest that the 14α-hydroxylase activity is NADPH dependent and belongs to the cytochrome P450 family. Copyright © 2014 Elsevier Inc. All rights reserved.

Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.

PubMed

Selak, Mary A; Armour, Sean M; MacKenzie, Elaine D; Boulahbel, Houda; Watson, David G; Mansfield, Kyle D; Pan, Yi; Simon, M Celeste; Thompson, Craig B; Gottlieb, Eyal

2005-01-01

Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1alpha. These results suggest a mechanistic link between SDH mutations and HIF-1alpha induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.

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.

Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: Indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17

PubMed Central

Bank, Ruud A.; Robins, Simon P.; Wijmenga, Cisca; Breslau-Siderius, Liesbeth J.; Bardoel, Alfons F. J.; Van der Sluijs, Hans A.; Pruijs, Hans E. H.; TeKoppele, Johan M.

1999-01-01

Bruck syndrome is characterized by the presence of osteoporosis, joint contractures, fragile bones, and short stature. We report that lysine residues within the telopeptides of collagen type I in bone are underhydroxylated, leading to aberrant crosslinking, but that the lysine residues in the triple helix are normally modified. In contrast to bone, cartilage and ligament show unaltered telopeptide hydroxylation as evidenced by normal patterns of crosslinking. The results provide compelling evidence that collagen crosslinking is regulated primarily by tissue-specific enzymes that hydroxylate only telopeptide lysine residues and not those destined for the helical portion of the molecule. This new family of enzymes appears to provide the primary regulation for controlling the different pathways of collagen crosslinking and explains why crosslink patterns are tissue specific and not related to a genetic collagen type. A genome screen identified only a single region on chromosome 17p12 where all affected sibs shared a cluster of haplotypes identical by descent; this might be the BS (Bruck syndrome) locus and consequently the region where bone telopeptidyl lysyl hydroxylase is located. Further knowledge of this enzyme has important implications for conditions where aberrant expression of telopeptide lysyl hydroxylase occurs, such as fibrosis and scar formation. PMID:9927692

Roux-en-Y gastric bypass in the treatment of non-classic congenital adrenal hyperplasia due to 11-hydroxylase deficiency.

PubMed

Kalani, Amir; Thomas, Nithin; Sacerdote, Alan; Bahtiyar, Gül

2013-03-18

Non-classic adrenal hyperplasia (NCAH) has been associated with insulin resistance (IR). Therapies such as metformin, thiazolidinediones and lifestyle alterations improve IR and also ameliorate the biochemical and clinical abnormalities of NCAH, much as they do in polycystic ovarian syndrome (PCOS). More recently, bariatric surgery, such as Roux-en-Y gastric bypass (RYGBP), has also been associated with improvement in IR and amelioration of PCOS and may, therefore, be beneficial in NCAH. We report a case of a 39-year-old, deaf-mute, obese woman with NCAH due to 11-hydroxylase deficiency who underwent RYGBP followed by improvement of NCAH manifestations. She was initially treated with metformin and pioglitazone, which lowered serum 11-deoxycortisol from 198 ng/dl (<51) to 26 ng/dl. Five weeks after undergoing RYGBP her body mass index fell from 44.18 kg/m(2) to 39.54 kg/m(2) and, despite not taking metformin or pioglitazone, serum 11-deoxycortisol remained normal at <40 ng/dl. Concurrently and subsequently, her NCAH symptoms, for example, alopecia, hirsutism and irregular menses normalised as well. We conclude that RYGBP, like other interventions that reduce IR, may be another way of treating non-classic 11-hydroxylase deficiency in selected patients.

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase.

PubMed

Rashidi, Mohammad-Reza; Beedham, Christine; Smith, John S; Davaran, Soodabeh

2007-08-01

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.

A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia.

PubMed

Kasajima, Ichiro; Sasaki, Katsutomo

2016-05-03

The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3'-hydroxylase (F3'H) and Flavonoid 3'5'-hydroxylase (F3'5'H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis.

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

Design, synthesis, and evaluation of (2S,4R)-Ketoconazole sulfonamide analogs as potential treatments for Metabolic Syndrome.

PubMed

Blass, Benjamin E; Iyer, Pravin; Abou-Gharbia, Magid; Childers, Wayne E; Gordon, John C; Ramanjulu, Mercy; Morton, George; Arumugam, Premkumar; Boruwa, Joshodeep; Ellingboe, John; Mitra, Sayan; Nimmareddy, Rajashekar Reddy; Paliwal, Shalini; Rajasekhar, Jamallamudi; Shivakumar, Savithiri; Srivastava, Pratima; Tangirala, Raghuram S; Venkataramanaiah, Konda; Yanamandra, Mahesh

2016-12-01

Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11β-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Genetics Home Reference: 21-hydroxylase deficiency

MedlinePlus

... Urinary Tract Defects Orphanet: Congenital adrenal hyperplasia Screening, Technology, and Research in Genetics (PDF) Vanderbilt Childrens Hospital: Congenital Adrenal Hyperplasia (PDF) Virginia Department of ...

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

PubMed

Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons that is independent of the MAP kinase pathway.

CYP86B1 Is Required for Very Long Chain ω-Hydroxyacid and α,ω-Dicarboxylic Acid Synthesis in Root and Seed Suberin Polyester1[W][OA

PubMed Central

Compagnon, Vincent; Diehl, Patrik; Benveniste, Irène; Meyer, Denise; Schaller, Hubert; Schreiber, Lukas; Franke, Rochus; Pinot, Franck

2009-01-01

Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid ω-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven β-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22- and C24-hydroxyacids and α,ω-dicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development. PMID:19525321

Atheroprotective Effects of Methotrexate on Reverse Cholesterol Transport Proteins and Foam Cell Transformation in THP-1 Human Monocytes/Macrophages

PubMed Central

Reiss, Allison B.; Carsons, Steven E.; Anwar, Kamran; Rao, Soumya; Edelman, Sari D.; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N.; Chan, Edwin S.L.

2008-01-01

OBJECTIVE: To determine whether MTX can overcome the atherogenic effect of COX-2 inhibitors and IFN-γ, both of which suppress cholesterol efflux protein levels and promote foam cell transformation in THP-1 human monocytes/macrophages. METHODS: Message and protein level of the reverse cholesterol transport (RCT) proteins cholesterol 27-hydroxylase and ABCA1 in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFN-γ with/without MTX. Foam cell transformation of lipid-loaded THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS: MTX increased 27-hydroxylase message and completely blocked NS398-induced downregulation of 27-hydroxylase (112.8±13.1% for NS398+MTX versus 71.1±4.3% for NS398 alone, with untreated as 100%, n=3, p<0.01). MTX also negated COX-2 inhibitor-mediated downregulation of ABCA1. Reversal of inhibitory effects on 27-hydroxylase and ABCA1 in the presence of MTX were blocked by the adenosine A2A receptor-specific antagonist ZM-241385. MTX also prevented NS398 and IFN-γ from increasing transformation of lipid-loaded THP-1 macrophages into foam cells. CONCLUSIONS: This study provides evidence supporting the atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes RCT and limits foam cell formation in THP-1 macrophages. This is the first evidence that any commonly used medication can increase expression of anti-atherogenic RCT proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular mortality in RA patients is through facilitation of cholesterol outflow from cells of the artery wall. PMID:19035488

Adrenal-derived 11-oxygenated 19-carbon steroids are the dominant androgens in classic 21-hydroxylase deficiency.

PubMed

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

2016-05-01

To comprehensively characterize androgens and androgen precursors in classic 21-hydroxylase deficiency (21OHD) and to gain insights into the mechanisms of their formation. Serum samples were obtained from 38 patients (19 men) with classic 21OHD, aged 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 tissues (n = 3) were used for immunohistochemical studies. We measured 11 steroids in all sera by liquid chromatography-tandem mass spectrometry. Immunofluroescence localized 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) within the normal and 21OHD adrenals. 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 the adrenal vein than in the inferior vena cava samples from men and women and rose with cosyntropin stimulation. Only trace amounts of 11oxC19 steroids were found in the sera of 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. 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. © 2016 European Society of Endocrinology.

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig.

PubMed

Bogus-Nowakowska, Krystyna; Równiak, Maciej; Hermanowicz-Sobieraj, Beata; Wasilewska, Barbara; Najdzion, Janusz; Robak, Anna

2016-12-01

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA. Copyright © 2016 Elsevier B.V. All rights reserved.

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-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst. Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure. PMID:24714029

Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocyte/macrophages.

PubMed

Reiss, Allison B; Carsons, Steven E; Anwar, Kamran; Rao, Soumya; Edelman, Sari D; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N; Chan, Edwin S L

2008-12-01

To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation.

PubMed

Pavon, Jorge Alex; Fitzpatrick, Paul F

2006-09-12

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Peat, Judy; Garg, Uttam

2016-01-01

Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. Increased phenylalanine and decreased tyrosine in blood are used in the diagnosis and follow-up of patients with PKU. LC/MS/MS method is described for the quantification of phenylalanine and tyrosine.

Expression, Purification, Crystallization And Preliminary X-Ray Studies of a Prolyl-4-Hydroxylase Protein From Bacillus Anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, M.A.; Scott, E.E.; Limburg, J.

2009-05-26

Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 {angstrom} resolution.

Delta 9 -tetrahydrocannabinol and ethanol: differential effects on sympathetic activity in differing environmental setting.

PubMed

Ng, L K; Lamprecht, F; Williams, R B; Kopin, I J

1973-06-29

Serum dopamine beta-hydroxylase activity, a useful biochemical index of peripheral sympathetic nervous activity, was measured in rats treated with Delta(9)-tetrahydrocannabinol or ethanol or both substances. After 7 days of treatment with either substance, serum dopamine beta-hydroxylase activity decreased significantly. Combined treatment with both agents enhanced the effects of each given alone. In rats subjected to immobilization stress, treatment with Delta(9)- tetrahydrocannabinol appeared to potentiate the stress-induced increase in serum enzyme activity. Treatment with ethanol, with or without Delta(9)-tetrahydrocannabinol, effectively blocked this increase in enzyme activity. These results show that both substances have significant effects on the sympathetic nervous system which are critically influenced by environmental setting.

p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase.

PubMed

Teodoro, Jose G; Parker, Albert E; Zhu, Xiaochun; Green, Michael R

2006-08-18

Recent evidence suggests that antiangiogenic therapy is sensitive to p53 status in tumors, implicating a role for p53 in the regulation of angiogenesis. Here we show that p53 transcriptionally activates the alpha(II) collagen prolyl-4-hydroxylase [alpha(II)PH] gene, resulting in the extracellular release of antiangiogenic fragments of collagen type 4 and 18. Conditioned media from cells ectopically expressing either p53 or alpha(II)PH selectively inhibited growth of primary human endothelial cells. When expressed intracellularly or exogenously delivered, alpha(II)PH significantly inhibited tumor growth in mice. Our results reveal a genetic and biochemical linkage between the p53 tumor suppressor pathway and the synthesis of antiangiogenic collagen fragments.

Differential dose- and time-dependent effects of molindone on dopamine neurons of rat brain: mediation by irreversible inhibition of monoamine oxidase.

PubMed

Meller, E; Friedman, E

1982-03-01

The effects of molindone (2.5, 10 and 40 mg/kg) on striatal tyrosine hydroxylase activity and dopamine (DA), 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were measured as a function of time (0-72 hr). Whereas a dose of 2.5 mg/kg produced effects typical of DA receptor blockade (activation of synaptosomal tyrosine hydroxylase, increased DA metabolite levels and unchanged DA levels), a dose of 40 mg/kg produced opposite effects (decreased tyrosine hydroxylase activity and metabolite concentrations and elevated DA levels). A dose of 10 mg/kg elicited intermediate effects. The atypical effects of both higher doses were long-lasting (less than 72 hr). Molindone at doses of 10 or 40 mg/kg, but nor 2.5 mg/kg, selectively, irreversibly and dose-dependently inhibited type A monoamine oxidase. This inhibition appeared to be due to a metabolite, inasmuch as the drug itself inhibited monoamine oxidase (reversibly) only at high concentrations (less than or equal to 10(-4) M). The heretofore unsuspected inhibition of monoamine oxidase by molindone provided a consistent mechanistic interpretation of the differential dose- and time-dependent effects of the drug on dopaminergic neuronal activity. This mechanism may also serve to explain the reported efficacy of molindone in animal tests for antidepressant activity as well as its inability to produce increased DA receptor binding after chronic treatment.

Conditional Depletion of Hippocampal Brain-Derived Neurotrophic Factor Exacerbates Neuropathology in a Mouse Model of Alzheimer's Disease.

PubMed

Braun, David J; Kalinin, Sergey; Feinstein, Douglas L

2017-01-01

Damage occurring to noradrenergic neurons in the locus coeruleus (LC) contributes to the evolution of neuroinflammation and neurodegeneration in a variety of conditions and diseases. One cause of LC damage may be loss of neurotrophic support from LC target regions. We tested this hypothesis by conditional unilateral knockout of brain-derived neurotrophic factor (BDNF) in adult mice. To evaluate the consequences of BDNF loss in the context of neurodegeneration, the mice harbored familial mutations for human amyloid precursor protein and presenilin-1. In these mice, BDNF depletion reduced tyrosine hydroxylase staining, a marker of noradrenergic neurons, in the rostral LC. BDNF depletion also reduced noradrenergic innervation in the hippocampus, the frontal cortex, and molecular layer of the cerebellum, assessed by staining for dopamine beta hydroxylase. BDNF depletion led to an increase in cortical amyloid plaque numbers and size but was without effect on plaque numbers in the striatum, a site with minimal innervation from the LC. Interestingly, cortical Iba1 staining for microglia was reduced by BDNF depletion and was correlated with reduced dopamine beta hydroxylase staining. These data demonstrate that reduction of BDNF levels in an LC target region can cause retrograde damage to LC neurons, leading to exacerbation of neuropathology in distinct LC target areas. Methods to reduce BDNF loss or supplement BDNF levels may be of value to reduce neurodegenerative processes normally limited by LC noradrenergic activities.

Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1

PubMed Central

Li, Xinliang; Zhang, Xuwang; Zhou, Jiti

2012-01-01

Background Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically. Findings A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PHw1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis. Conclusions/Significance We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production. PMID:23028517

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450.

PubMed

Iwasaki, M; Juvonen, R; Lindberg, R; Negishi, M

1991-02-25

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library.

PubMed

Kim, B S; Kim, S Y; Park, J; Park, W; Hwang, K Y; Yoon, Y J; Oh, W K; Kim, B Y; Ahn, J S

2007-05-01

Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.

MANAGEMENT OF ENDOCRINE DISEASE: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: update on the management of adult patients and prenatal treatment.

PubMed

Bachelot, Anne; Grouthier, Virginie; Courtillot, Carine; Dulon, Jérôme; Touraine, Philippe

2017-04-01

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is characterized by cortisol and in some cases aldosterone deficiency associated with androgen excess. Goals of treatment are to replace deficient hormones and control androgen excess, while avoiding the adverse effects of exogenous glucocorticoid. Over the last 5 years, cohorts of adults with CAH due to 21-hydroxylase deficiency from Europe and the United States have been described, allowing us to have a better knowledge of long-term complications of the disease and its treatment. Patients with CAH have increased mortality, morbidity and risk for infertility and metabolic disorders. These comorbidities are due in part to the drawbacks of the currently available glucocorticoid therapy. Consequently, novel therapies are being developed and studied in an attempt to improve patient outcomes. New management strategies in the care of pregnancies at risk for congenital adrenal hyperplasia using fetal sex determination and dexamethasone have also been described, but remain a subject of debate. We focused the present overview on the data published in the last 5 years, concentrating on studies dealing with cardiovascular risk, fertility, treatment and prenatal management in adults with classic CAH to provide the reader with an updated review on this rapidly evolving field of knowledge. © 2017 European Society of Endocrinology.

Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

PubMed Central

Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

2014-01-01

ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

Alpha-Hydroxylation of lignoceric and nervonic acids in the brain. Effects of altered thyroid function on postnatal development of the hydroxylase activity.

PubMed

Murad, S; Strycharz, G D; Kishimoto, Y

1976-09-10

Rat brain postnuclear preparations catalyzed the alpha-hydroxylation of nervonic acid with an apparent Km of 3 muM. Evidence has been presented which suggests that nervonic acid in the brain is hydroxylated by the same enzyme system which hydroxylates lignoceric acid. The hydroxylase activity in brains of normal (euthyroid) rats increased rapidly from a low in the period immediately following birth to a maximum at the 23rd day and then declined to a low level characteristic of the mature brain. Neonatal hypothyroidism retarded the development of the activity and shifted its peak to the 39th day after birth. Conversely, neonatal hyperthyroidism accelerated the entire developmental pattern and shifted the peak to the 16th day after birth. The hydroxylase activity in mouse brain was also increased by thyroid hormone administration from the 13th through the 18th day after birth. Unlike normal mice, the low activity in jimpy mice was not affected by this treatment. It is concluded that thyroid hormones play an important role in the control of brain fatty acid alpha-hydroxylation. The stimulation of alpha-hydroxy fatty acid synthesis in response to hyperthyroidism during the early postnatal period may be one of the major effects of thyroid hormones in accelerating myelination of the central nervous system.

Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis.

PubMed

Carrera, P; Barbieri, A M; Ferrari, M; Righetti, P G; Perego, M; Gelfi, C

1997-11-01

A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming because of the high number of hybridizations with radioactive probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.

Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

PubMed

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-06-26

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

Biochemical Characterization of the Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B Complex*

PubMed Central

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-01-01

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969

Overexpression of parkin in the rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity.

PubMed

Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-09-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Overexpression of parkin in rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity

PubMed Central

Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-01-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. PMID:23313192

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

2016-05-25

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.

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

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

PubMed

Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita

2006-06-25

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts[W

PubMed Central

Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.

2010-01-01

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559

Origins of serotonin innervation of forebrain structures

NASA Technical Reports Server (NTRS)

Kellar, K. J.; Brown, P. A.; Madrid, J.; Bernstein, M.; Vernikos-Danellis, J.; Mehler, W. R.

1977-01-01

The tryptophan hydroxylase activity and high-affinity uptake of (3H) serotonin ((3H)5-HT) were measured in five discrete brain regions of rats following lesions of the dorsal or median raphe nuclei. Dorsal raphe lesions reduced enzyme and uptake activity in the striatum only. Median raphe lesions reduced activities in the hippocampus, septal area, frontal cortex, and, to a lesser extent, in the hypothalamus. These data are consistent with the suggestion that the dorsal and median raphe nuclei are the origins of two separate ascending serotonergic systems - one innervating striatal structures and the other mesolimbic structures, predominantly. In addition, the data suggest that measurements of high-affinity uptake of (3H)5-HT may be a more reliable index of innervation than either 5-HT content or tryptophan hydroxylase activity.

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.

Chronic stress effects on working memory: association with prefrontal cortical tyrosine hydroxylase.

PubMed

Lee, Young-A; Goto, Yukiori

2015-06-01

Chronic stress causes deficits in cognitive function including working memory, for which transmission of such catecholamines as dopamine and noradrenaline transmission in the prefrontal cortex (PFC) are crucial. Since catecholamine synthesis depends on the rate-limiting enzyme, tyrosine hydroxylase (TH), TH is thought to play an important role in PFC function. In this study, we found that two distinct population existed in Sprague-Dawley rats in terms of working memory capacity, one with higher working memory capacity, and the other with low capacity. This distinction of working memory capacity became apparent after rats were exposed to chronic stress. In addition, such working memory capacity and alterations of working memory function by chronic stress were associated with TH expression in the PFC. Copyright © 2015 Elsevier B.V. All rights reserved.

Photobiogenesis and metabolism of vitamin D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holick, M.F.; Clark, M.B.

1978-10-01

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitarymore » hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined.« less

[Recommendations for the diagnosis and treatment of classic forms of 21-hydroxylase-deficient congenital adrenal hyperplasia].

PubMed

Rodríguez, Amparo; Ezquieta, Begoña; Labarta, José Igancio; Clemente, María; Espino, Rafael; Rodriguez, Amaia; Escribano, Aranzazu

2017-08-01

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations in the CYP21A2 gene. Cortisol and aldosterone synthesis are impaired in the classic forms (adrenal insufficiency and salt-wasting crisis). Females affected are virilised at birth, and are at risk for genital ambiguity. In this article we give recommendations for an early as possible diagnosis and an appropriate and individualised treatment. A patient and family genetic study is essential for the diagnosis of the patient, and allows genetic counselling, as well as a prenatal diagnosis and treatment for future pregnancy. Copyright © 2016 Asociación Española de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

PubMed Central

Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

2014-01-01

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

Pleiotrophin over-expression provides trophic support to dopaminergic neurons in parkinsonian rats.

PubMed

Taravini, Irene Re; Chertoff, Mariela; Cafferata, Eduardo G; Courty, José; Murer, Mario G; Pitossi, Fernando J; Gershanik, Oscar S

2011-06-07

Pleiotrophin is known to promote the survival and differentiation of dopaminergic neurons in vitro and is up-regulated in the substantia nigra of Parkinson's disease patients. To establish whether pleiotrophin has a trophic effect on nigrostriatal dopaminergic neurons in vivo, we injected a recombinant adenovirus expressing pleiotrophin in the substantia nigra of 6-hydroxydopamine lesioned rats. The viral vector induced pleiotrophin over-expression by astrocytes in the substantia nigra pars compacta, without modifying endogenous neuronal expression. The percentage of tyrosine hydroxylase-immunoreactive cells as well as the area of their projections in the lesioned striatum was higher in pleiotrophin-treated animals than in controls. These results indicate that pleiotrophin over-expression partially rescues tyrosine hydroxylase-immunoreactive cell bodies and terminals of dopaminergic neurons undergoing 6-hydroxydopamine-induced degeneration.

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 transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools. PMID:16433923

Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptamine in rat brain.

PubMed

Rastogi, R B; Singhal, R L

1976-09-01

In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats. Whereas administration of l-triiodothyronine (10 mug/100 g/day) for 30 days to 120-day-old rats increased the levels of tyrosine by 23% and of tryptophan by 43%, no appreciable change was noted in tryptophan hydroxylase activity. In contrast to neonatal hyperthyroidism, excess of thyroid hormone in adult rats failed to produce any change in motor activity and tended to decrease striatal tyrosine hydroxylase activity only slightly. The concentration of dopamine remained unchanged in all regions of the brain except in midbrain where it rose by 19%. Whereas norepinephrine concentration was altered in hypothalamus, pons-medulla and midbrain, the levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, were significantly decreased in striatum and cerebellum. Since dopaminergic and noradrenergic neurons are the critical components of the motor system, the possibility exists that elevated behavioral activity in young L-triiodothyronine-treated animals might be associated with increased turnover of catecholamines in neuronal tissue.

Cryptic Production of trans-3-Hydroxyproline in Echinocandin B Biosynthesis.

PubMed

Mattay, Johanna; Houwaart, Stefanie; Hüttel, Wolfgang

2018-04-01

Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: trans -4-hydroxy-l-proline and, in most echinocandin structures, ( trans -2,3)-3-hydroxy-( trans -2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by Glarea lozoyensis , both amino acids are found in pneumocandin A 0 , while in pneumocandin B 0 the latter residue is replaced by trans -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by Aspergillus species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Aspergillus pachycristatus Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Cryptosporiopsis IMPORTANCE Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A 0 and B 0 are produced by the fungus Glarea lozoyensis They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A 0 and B 0 , three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans -3-hydroxyproline. However, echinocandin B biosynthesis does not require trans -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase. Copyright © 2018 American Society for Microbiology.

17-Hydroxyprogesterone Test

MedlinePlus

... or complete lack of the enzyme 21-hydroxylase, accounting for about 90% of cases. Because a low ... a CAH diagnosis in people with symptoms. Diagnosis Measurement of 17-OHP in the blood may be ...

Functional Consequences of Seven Novel Mutations in the CYP11B1 Gene: Four Mutations Associated with Nonclassic and Three Mutations Causing Classic 11β-Hydroxylase Deficiency

PubMed Central

Parajes, Silvia; Loidi, Lourdes; Reisch, Nicole; Dhir, Vivek; Rose, Ian T.; Hampel, Rainer; Quinkler, Marcus; Conway, Gerard S.; Castro-Feijóo, Lidia; Araujo-Vilar, David; Pombo, Manuel; Dominguez, Fernando; Williams, Emma L.; Cole, Trevor R.; Kirk, Jeremy M.; Kaminsky, Elke; Rumsby, Gill; Arlt, Wiebke; Krone, Nils

2010-01-01

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD. PMID:20089618

Non-classic 21-hydroxylase deficiency in an 18-year-old female athlete. A case report

PubMed

Silverman; Coupey; Muram

2000-05-01

Background: In non-classic 21-hydroxylase deficiency, age at presentation and genital findings are variable. Late diagnosis with dramatic signs of virilization precludes early treatment and thus prevention of anatomic and psychosocial consequences. The following case illustrates the complexity of late diagnosis.Case: An 18-year-old West Indian female was seen for evaluation of clitoromegaly and hirsutism discovered in the emergency department when she presented after sexual assault. She had allegedly been drugged and raped in her dorm room. She was a college student with an athletic scholarship and had a striking masculinized, broad-shouldered appearance. She denied any use of anabolic steroids or other drugs. Menarche was at age 16 with infrequent menses. She was sexually active with 4 life-time partners, all male. On physical exam, her height was 152 cm, weight 50 kg, blood pressure 110/70 mm Hg. Breasts were hypoplastic with hyperpigmented nipples. She was hirsute with a Ferriman-Gallwey score of 14. Genitalia were abnormal with clitoris measuring 5.5 x 1.5 cm and posterior labial fusion. Initial non-fasting serum 17-hydroxyprogesterone level was 2890 ng/dL, testosterone was 274 ng/dL, and cortisol was 9 &mgr;g/dL. Chromosome analysis was 46, XX and ACTH stimulation test confirmed the diagnosis of 21-hydroxylase deficiency. The patient was initially reluctant to begin glucocorticoid treatment because of concern that it would decrease her muscle mass and negatively impact on her athletic performance and scholarship support. One year after diagnosis and 10 months after beginning treatment, she elected surgical correction of her clitoromegaly because of extreme embarrassment over having erections during sex. She underwent excision of most of the corpus cavernosum with repositioning of the glans. The neurovascular elements were preserved. The patient is pleased with the cosmetic result and reports no change in achieving orgasm. She has not notices any change in muscle mass or athletic performance since beginning glucocorticoid therapy.Conclusion: This case illustrates the somatic and genital abnormalities as well as the psychosocial impact of a delayed diagnosis of 21-hydroxylase deficiency in this young female athlete.

STUDIES ON THE ORIGIN OF CIRCULATING 18-HYDROXYCORTISOL AND 18-OXOCORTISOL IN NORMAL HUMAN SUBJECTS.

PubMed Central

Freel, E Marie; Shakerdi, Loai A; Friel, Elaine C; Wallace, A Michael; Davies, Eleanor; Fraser, Robert; Connell, John MC

2005-01-01

18-hydroxycortisol (18-OHF) and 18-oxocortisol (18oxo-F) are derivatives of cortisol found in Primary Aldosteronism but whose origin and regulation in normal subjects is uncertain. 18-OHF can be synthesised by zona fasciculata 11-β hydroxylase; 18-oxoF can only be produced by zona glomerulosa aldosterone synthase (AS). Stably transfected cell lines expressing either CYP11B1 (11β-hydroxylase) or CYP11B2 (AS) were incubated with cortisol and other substrates over a range of concentrations. Both enzymes could synthesise 18-OHF from cortisol but only AS could synthesise 18-oxoF. AS was more efficient than 11β-hydroxylase at 18-hydroxylation. The apparent Km of AS for cortisol was estimated to be 2.6μM. In 5 patients with adrenal insufficiency maintained on hydrocortisone, urinary free cortisol and cortisone levels were high; 18-oxoF was detectable in all patients and 18-hydroxycortisol in 3. It is likely that the 18-oxygenated steroids were synthesised from circulating cortisol, either in the zona glomerulosa or at extra-adrenal sites. In 8 male volunteers, dexamethasone treatment decreased urinary excretion rates of free cortisol, cortisone, 18-OHF and 18-oxoFl, confirming dependence of 18-oxygenated steroid levels on cortisol availability. In both groups, hydrocortisone administration resulted in detectable levels of 18-OHF and raised levels of 18-oxoF. There was close correlation between 18-oxoF and cortisol excretion during hydrocortisone administration in normal subjects (r=0.86, p<0.001). These data show, for the first time, that 18-OHF and 18oxoF can be synthesised from circulating cortisol. The close correlation between 18-oxoF and cortisol suggests that 18-oxoF is normally produced by the action of aldosterone synthase utilising circulating cortisol as a substrate. Although 18OHF can be synthesized using circulating cortisol as substrate, our data suggest this is normally produced in the zona fasciculata by 11β-hydroxylase from locally available cortisol. PMID:15356073

Types of Leukodystrophies

MedlinePlus

... Leukoencephalopathy (RNASET2 related) Elongation of Very Long-Chain Fatty Acids-4 (ELOVL4; Pseudo-Sjogren-Larsson) Extensive Cerebral White ... with Adult Onset Dementia and Abnormal Glycolipid Storage Fatty Acid 2-Hydroxylase Deficiency Fucosidosis Fukuyama Congential Muscular Dystrophy ...

Tyrosine hydroxylase and dopamine D4 receptor allelic distribution in Scandinavian chronic alcoholics.

PubMed

Geijer, T; Jönsson, E; Neiman, J; Persson, M L; Brené, S; Gyllander, A; Sedvall, G; Rydberg, U; Wasserman, D; Terenius, L

1997-02-01

Associations of polymorphic genetic markers at the tyrosine hydroxylase (TH) and dopamine D4 receptor (DRD4) loci were examined in Scandinavian chronic alcoholics (n = 72) and control subjects (n = 67). Patients were divided into subgroups with regard to the presence of parental alcoholism and age of onset. Neither the TH nor the DRD4 allele distributions were significantly different when alcoholic samples were compared with control subjects. However, a tendency to high prevalence for 1 of the 5 TH alleles assayed (TH-K3) was observed in a subsample of 44 alcoholics characterized by late onset when compared with control subjects (27.3% vs. 10.6%, p = 0.041). Results suggest that no major influence on alcoholism is exerted through genes associated with the DRD4 or TH allelic markers examined.

Pseudovitamin D deficiency rickets—a report from the Indian subcontinent

PubMed Central

Zargar, A. H.; Mithal, A.; Wani, A. I.; Laway, B. A.; Masoodi, S. R.; Bashir, M. I.; Ganie, M. A.

2000-01-01

Pseudovitamin D deficiency rickets (also called vitamin D dependent rickets type I) is one of the types of inherited rickets and is caused by a deficit in renal 25-hydroxyvitamin D 1α-hydroxylase. This form of rickets has not been reported from the Indian subcontinent. Three patients with this disorder are presented. These patients were all females aged 3-20 years and presented with growth failure and skeletal deformities. All had florid clinical and radiological rickets. The biochemical abnormalities seen included hypocalcaemia, hypophosphataemia, and hyperphosphatasia. All patients had grossly raised 25-hydroxyvitamin D concentrations and markedly low to undetectable concentrations of 1,25-dihydroxyvitamin D. A disturbing feature of this study was the late referral of the patients.


Keywords: 1α-hydroxylase; calcitriol; inherited rickets; vitamin D dependent rickets PMID:10824056

Rapid carrier screening using short tandem repeats in the phenylalanine hydroxylase gene.

PubMed

Shawky, R M; el-Aleem, K A; Rifaat, M M; el-Naggar, R L; Marzouk, G M

2002-01-01

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by defects in the phenylalanine hydroxylase (PAH) system. Our work aimed to screen the PAH locus for the presence of potentially useful short tandem repeats (STR) as markers for carrier detection in PKU families in Egypt, and to determine the level of PAH heterozygosity within the Egyptian population. The system contains at least eight independent alleles in the Egyptian population, transmitted in a Mendelian fashion. Variations in the number of STR in the 16 families studied gave rise to polymorphisms that proved to be suitable markers for PKU carrier detection and prenatal diagnosis. The most frequent allelic fragment size in PKU patients was 246 bp (35.7%), which together with a fragment of 254 bp accounted for 60.7% of the mutant chromosomes.

Zebrafish have an ethanol-inducible hepatic 4-nitrophenol hydroxylase that is not CYP2E1-like.

PubMed

Hartman, Jessica H; Kozal, Jordan S; Di Giulio, Richard T; Meyer, Joel N

2017-09-01

Zebrafish are an attractive model organism for toxicology; however, an important consideration in translating between species is xenobiotic metabolism/bioactivation. CYP2E1 metabolizes small hydrophobic molecules, e.g. ethanol, cigarette smoke, and diesel exhaust components. CYP2E1 is thought to only be conserved in mammals, but recent reports identified homologous zebrafish cytochrome P450s. Herein, ex vivo biochemical measurements show that unlike mammals, zebrafish possess a low-affinity 4-nitrophenol hydroxylase (K m ∼0.6 mM) in hepatic microsomes and mitochondria that is inducible only 1.5- to 2-fold by ethanol and is insensitive to 4-methylpyrazole inhibition. In closing, we suggest creating improved models to study CYP2E1 in zebrafish. Copyright © 2017 Elsevier B.V. All rights reserved.

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

NASA Technical Reports Server (NTRS)

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

2004-01-01

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

Pleiotrophin over-expression provides trophic support to dopaminergic neurons in parkinsonian rats

PubMed Central

2011-01-01

Background Pleiotrophin is known to promote the survival and differentiation of dopaminergic neurons in vitro and is up-regulated in the substantia nigra of Parkinson's disease patients. To establish whether pleiotrophin has a trophic effect on nigrostriatal dopaminergic neurons in vivo, we injected a recombinant adenovirus expressing pleiotrophin in the substantia nigra of 6-hydroxydopamine lesioned rats. Results The viral vector induced pleiotrophin over-expression by astrocytes in the substantia nigra pars compacta, without modifying endogenous neuronal expression. The percentage of tyrosine hydroxylase-immunoreactive cells as well as the area of their projections in the lesioned striatum was higher in pleiotrophin-treated animals than in controls. Conclusions These results indicate that pleiotrophin over-expression partially rescues tyrosine hydroxylase-immunoreactive cell bodies and terminals of dopaminergic neurons undergoing 6-hydroxydopamine-induced degeneration. PMID:21649894

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

PubMed

Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent

2017-03-01

Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Sexual dimorphism and the effects of the X-linked Tfm locus on hexobarbitone metabolism and action in mice.

PubMed

King, D K; Shapiro, B H

1981-09-01

1 Normal males of the testicular feminized strain of mice (Tfm) had longer hexobarbitone-induced sleeping times than females, and hepatic hexobarbitone hydroxylase activity different in that the Km was higher and the Vmax lower in the male. 2 Castration and androgen replacement studies indicated that testicular androgens were responsible for the sexual differences in drug metabolism found in this mouse strain. 3 Hepatic hexobarbitone metabolism and action were feminized in the intact, androgen-insensitive, genetically male Tfm mouse. Furthermore, hexobarbitone hydroxylase activities were less responsive to large doses of testosterone in Tfm mice than in normal males. 4 The Tfm mouse with a deficiency in androgen receptors responded to the enzyme-inductive effects of phenobarbitone and softwood bedding, indicating that these inducers do not act through the androgen receptors.

Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timmers, R.J.; Granneman, J.C.; Lambert, J.G.

1988-11-01

Estrogen-2-hydroxylase activity, involved in the biosynthesis of catecholestrogens, was localized in the brain of the male African catfish, Clarias gariepinus, by means of a radiometric assay using (2-TH)estradiol as substrate. Fore- and midbrain were divided in 18, 500-microns thick, transverse sections from which small defined areas were punched out and assayed. The estrogen-2-hydroxylase activity was calculated from the release of tritium during hydroxylation, and expressed in femtomole catecholestradiol.milligram-1 tissue.hour-1. The enzyme could be demonstrated throughout the brain. A high activity (greater than 350 fmol) was observed in the telencephalon, in particularly the rostral part and the area ventralis pars dorsalis;more » in the diencephalon in the preoptic region, including the magnocellular part of the preoptic nucleus and the rostral part of the anterior periventricular nucleus; and in the area tuberalis, including the nucleus lateralis tuberis, the rostral part of the nucleus anterior tuberis, the caudal part of the nucleus posterior periventricularis, and in the nucleus recessus posterioris. Also a high activity was detected in the mesencephalic tectum opticum and the dorsolateral part of the torus semicircularis. The ventral mesencephalon showed a moderate (200-350 fmol) to low (less than 200 fmol) activity, whereas the lowest activity was found in the hindbrain (118 fmol). The significance of the biosynthesis of catecholestrogens in the brain is discussed in light of the negative feedback mechanism of gonadal steroids on gonadotropin release.« less

Ablation of cytochrome P450 omega-hydroxylase 4A14 gene attenuates hepatic steatosis and fibrosis

PubMed Central

Zhang, Xiaoyan; Li, Sha; Zhou, Yunfeng; Su, Wen; Ruan, Xiongzhong; Wang, Bing; Zheng, Feng; Warner, Margaret; Gustafsson, Jan-Åke; Guan, Youfei

2017-01-01

Nonalcoholic fatty liver disease (NAFLD) is characterized by simple hepatic steatosis (SS), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and cirrhosis. Dysregulated fatty acid metabolism in the liver plays a critical role in the pathogenesis of NAFLD. Cytochrome P450 omega-hydroxylase 4A14 (CYP4A14) is a homolog of human CYP4A hydroxylase that catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid in mice. The goal of this study was to determine the role of CYP4A14 in the development and the progression of NAFLD. Here, we showed that hepatic CYP4A expression was up-regulated in the livers of patients and three murine models of NAFLD. Adenovirus-mediated overexpression of CYP4A14 in the livers of C57BL/6 mice resulted in a fatty liver phenotype with a significant increase in hepatic fatty acid translocase (FAT/CD36) expression. In contrast, CYP4A14 gene-deficient mice fed a high-fat diet or a methionine and choline-deficient (MCD) diet exhibited attenuated liver lipid accumulation and reduced hepatic FAT/CD36 expression. In addition, hepatic inflammation and fibrosis was markedly ameliorated in MCD diet-fed CYP4A14-deficient mice. Collectively, CYP4A14 plays an important role in the pathogenesis of both SS and NASH and may represent a potential therapeutic target for the treatment of NAFLD. PMID:28270609

Conditional Depletion of Hippocampal Brain-Derived Neurotrophic Factor Exacerbates Neuropathology in a Mouse Model of Alzheimer’s Disease

PubMed Central

Braun, David J.; Kalinin, Sergey

2017-01-01

Damage occurring to noradrenergic neurons in the locus coeruleus (LC) contributes to the evolution of neuroinflammation and neurodegeneration in a variety of conditions and diseases. One cause of LC damage may be loss of neurotrophic support from LC target regions. We tested this hypothesis by conditional unilateral knockout of brain-derived neurotrophic factor (BDNF) in adult mice. To evaluate the consequences of BDNF loss in the context of neurodegeneration, the mice harbored familial mutations for human amyloid precursor protein and presenilin-1. In these mice, BDNF depletion reduced tyrosine hydroxylase staining, a marker of noradrenergic neurons, in the rostral LC. BDNF depletion also reduced noradrenergic innervation in the hippocampus, the frontal cortex, and molecular layer of the cerebellum, assessed by staining for dopamine beta hydroxylase. BDNF depletion led to an increase in cortical amyloid plaque numbers and size but was without effect on plaque numbers in the striatum, a site with minimal innervation from the LC. Interestingly, cortical Iba1 staining for microglia was reduced by BDNF depletion and was correlated with reduced dopamine beta hydroxylase staining. These data demonstrate that reduction of BDNF levels in an LC target region can cause retrograde damage to LC neurons, leading to exacerbation of neuropathology in distinct LC target areas. Methods to reduce BDNF loss or supplement BDNF levels may be of value to reduce neurodegenerative processes normally limited by LC noradrenergic activities. PMID:28266222

Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.

1986-03-01

In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine storesmore » after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.« less

Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: evidence of different genetic mechanisms involved in the production of the disease.

PubMed Central

López-Gutiérrez, A U; Riba, L; Ordoñez-Sánchez, M L; Ramírez-Jiménez, S; Cerrillo-Hinojosa, M; Tusié-Luna, M T

1998-01-01

Congenital adrenal hyperplasia (CAH) is an inherited recessive disorder of adrenal steroidogenesis caused by mutations in the steroid 21-hydroxylase gene (CYP21) in more than 90% of affected patients. The CYP21 gene is located within the HLA complex locus on chromosome 6 (6p21.3). During a molecular characterisation study of a group of 47 Mexican families with 21-hydroxylase deficiency, we identified nine in which the mutation or mutations found in the patient did not appear to originate from one of the parents. Through DNA fingerprinting, paternity was established in all nine families with a probability of non-paternity in the range of 10(-19) to 10(-23). Among these families, we identified one patient with exclusive paternal inheritance of all eight markers tested on chromosome 6p, despite normal maternal and paternal contributions for eight additional markers on three different chromosomes. We did not identify duplication of paternal information for markers in the 6q region, consistent with lack of expression of transient neonatal diabetes owing to genomic imprinting in this patient. Our results substantiate evidence for the existence of different genetic mechanisms involved in the expression of this recessive condition in a substantial portion (approximately 19%) of affected Mexican families. In addition to the identification of a patient with paternal uniparental disomy, the occurrence of germline mutations may explain the unusual pattern of segregation in the majority of the remaining eight families. PMID:9863599

AAV-Mediated Gene Transfer of the Obesity-Associated Gene Etv5 in Rat Midbrain Does Not Affect Energy Balance or Motivated Behavior

PubMed Central

Boender, Arjen J.; Koning, Nivard A.; van den Heuvel, José K.; Luijendijk, Mieneke C. M.; van Rozen, Andrea J.; la Fleur, Susanne E.; Adan, Roger A. H.

2014-01-01

Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior. PMID:24710089

Molecular modelling and quantum biochemistry computations of a naturally occurring bioremediation enzyme: Alkane hydroxylase from Pseudomonas putida P1.

PubMed

de Sousa, B G; Oliveira, J I N; Albuquerque, E L; Fulco, U L; Amaro, V E; Blaha, C A G

2017-10-01

Many species of bacteria involved in degradation of n-alkanes have an important constitutional metabolic enzyme, the alkane hydroxylase called AlkB, specialized in the conversion of hydrocarbons molecules that can be used as carbon and/or energy source. This enzyme plays an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. A number of these enzymes has been biochemically characterized in detail because the potential of alkane hydroxylases to catalyse high added-value reactions is widely recognized. Nevertheless, the industrial and process bioremediation application of them is restricted, owing to their complex biochemistry, challenging process requirements, and the limited number of their three-dimensional structures. Furthermore, AlkB has great potential as biocatalysts for selective transformation of a wide range of chemically inert unreactive alkanes into reactive chemical precursors that can be used as tools for bioremediation and bioprocesses. Aiming to understand the possible ways the AlkB enzyme Pseudomonas putida P1 interacts with octane, octanol and 1-octyne, we consider its suitable biochemical structure taking into account a 3-D homology modelling. Besides, by using a quantum chemistry computational model based on the density functional theory (DFT), we determine possible protein-substrate interaction regions measured by means of its binding energy simulated throughout the Molecular Fractionation with Conjugated Caps (MFCC) approach. Copyright © 2017 Elsevier Inc. All rights reserved.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jokilehto, Terhi; Turku Graduate School of Biomedical Sciences, Turku; Hoegel, Heidi

2010-04-15

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells.more » The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.« less

Clinical manifestations and hormonal profile of two women with Cushing's disease and mild deficiency of 21-hydroxylase.

PubMed

Boronat, M; Carrillo, A; Ojeda, A; Estrada, J; Ezquieta, B; Marín, F; Nóvoa, F J

2004-06-01

The development of Cushing's disease among patients with deficiency of 21-hydroxylase has not been observed to date. The clinical manifestations and the hormonal profile of this exceptional association are herein described through the study of two cases. The first one was a 39-yr-old woman who had undergone non-curative transsphenoidal surgery for a pituitary-dependent Cushing's syndrome 12 yr before. She showed hypertension, central obesity, severe hirsutism, alopecia and hyperpigmentation. Urinary excretion of cortisol was normal, but ACTH levels were very high and hormonal dynamic studies (cortisol circadian rhythm, insulin-induced hypoglycemia and dexamethasone suppression tests) revealed the qualitative disturbances that characterize Cushing's disease. Serum concentrations of androstenedione, free testosterone and 17-hydroxyprogesterone were clearly increased. Reexamination of the tissue samples from previous surgery confirmed the presence of an ACTH-producing pituitary adenoma. CYP21 gene analysis found the splicing 655G mutation at intron 2 and the V281L mutation at exon 7. The second case was a 21-yr-old woman who was diagnosed with pituitary ACTH-dependent Cushing's syndrome according to unequivocal clinical and laboratory findings. However, hirsutism was particularly severe and both serum androgens and 17-hydroxyprogesterone were elevated. The patient was heterozygote for a large conversion of CYP21 gene. In these cases, the clinical and biochemical expression of Cushing's syndrome was determined by the different severity of 21-hydroxylase deficiency and the subsequent residual ability of adrenal cortex to synthesize cortisol.

Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

PubMed

Nagatsu, Toshiharu; Nagatsu, Ikuko

2016-11-01

Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems.

PubMed

Biondi, R; Xia, Y; Rossi, R; Paolocci, N; Ambrosio, G; Zweier, J L

2001-03-01

Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects ofmore » pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.« less

Vomeronasal inputs to the rodent ventral striatum.

PubMed

Ubeda-Bañon, I; Novejarque, A; Mohedano-Moriano, A; Pro-Sistiaga, P; Insausti, R; Martinez-Garcia, F; Lanuza, E; Martinez-Marcos, A

2008-03-18

Vertebrates sense chemical signals through the olfactory and vomeronasal systems. In squamate reptiles, which possess the largest vomeronasal system of all vertebrates, the accessory olfactory bulb projects to the nucleus sphericus, which in turn projects to a portion of the ventral striatum known as olfactostriatum. Characteristically, the olfactostriatum is innervated by neuropeptide Y, tyrosine hydroxylase and serotonin immunoreactive fibers. In this study, the possibility that a structure similar to the reptilian olfactostriatum might be present in the mammalian brain has been investigated. Injections of dextran-amines have been aimed at the posteromedial cortical amygdaloid nucleus (the putative mammalian homologue of the reptilian nucleus sphericus) of rats and mice. The resulting anterograde labeling includes the olfactory tubercle, the islands of Calleja and sparse terminal fields in the shell of the nucleus accumbens and ventral pallidum. This projection has been confirmed by injections of retrograde tracers into the ventral striato-pallidum that render retrograde labeling in the posteromedial cortical amygdaloid nucleus. The analysis of the distribution of neuropeptide Y, tyrosine hydroxylase, serotonin and substance P in the ventral striato-pallidum of rats, and the anterograde tracing of the vomeronasal amygdaloid input in the same material confirm that, similar to reptiles, the ventral striatum of mammals includes a specialized vomeronasal structure (olfactory tubercle and islands of Calleja) displaying dense neuropeptide Y-, tyrosine hydroxylase- and serotonin-immunoreactive innervations. The possibility that parts of the accumbens shell and/or ventral pallidum could be included in the mammalian olfactostriatum cannot be discarded.

Testicular adrenal rest tumors develop independently of long-term disease control: a longitudinal analysis of 50 adult men with congenital adrenal hyperplasia due to classic 21-hydroxylase deficiency.

PubMed

Reisch, Nicole; Rottenkolber, Marietta; Greifenstein, Anais; Krone, Nils; Schmidt, Heinrich; Reincke, Martin; Schwarz, Hans-Peter; Beuschlein, Felix

2013-11-01

Testicular adrenal rest tumors (TARTs) and hypogonadotropic hypogonadism are the two most common causes for male infertility in classic 21-hydroxylase deficiency. Current hypotheses suggest the quality of disease control to be one of the main pathogenic factors for TART development. The aim was to study long-term predictors for TART development in a retrospective longitudinal study. Fifty men with classic 21-hydroxylase deficiency (31 salt wasting, 19 simple virilizing) were investigated. Testicular ultrasound at a median age at investigation of 27 years detected TARTs in 28 of 50 subjects (19 salt wasting, 9 simple virilizing). TART presence was correlated with long-term parameters of disease control during childhood and adolescence obtained from patients' charts: 24-hour urine pregnanetriol, serum 17-hydroxyprogesterone, onset and stage of pubic hair development, testicular growth, and bone age in relation to chronological age. There was no difference in pregnanetriol excretion over lifetime between patients with and without TARTs. Similarly, neither development of pubic hair and testicular volume (Tanner) nor bone age in relation to chronological age differed between the two groups. Furthermore, the two groups had the same body mass index and the same impairment of final height in relation to midparental target height. Our longitudinal analysis demonstrates no association between TART presence and parameters of disease control. These data, therefore, argue for other mechanisms more relevant for TART induction including those occurring during fetal development.

A novel regio‑specific cyclosporin hydroxylase gene revealed through the genome mining of Pseudonocardia autotrophica.

PubMed

Ban, Jun-Gyu; Woo, Min-Woo; Lee, Bo-Ram; Lee, Mi-Jin; Choi, Si-Sun; Kim, Eung-Soo

2014-05-01

The regio-specific hydroxylation at the 4th N-methyl leucine of the immunosuppressive agent cyclosporin A (CsA) was previously proposed to be mediated by a unique cytochrome P450 hydroxylase (CYP), CYP-sb21 from the rare actinomycetes Sebekia benihana. Interestingly, a different rare actinomycetes species, Pseudonocardia autotrophica, was found to possess a different regio-selectivity, the preferential hydroxylation at the 9th N-methyl leucine of CsA. Through an in silico analysis of the whole genome of P. autotrophica, we describe here the classification of 31 total CYPs in P. autotrophica. Three putative CsA CYP genes, showing the highest sequence homologies with CYPsb21, were successfully inactivated using PCR-targeted gene disruption. Only one knock-out mutant, ΔCYP-pa1, failed to convert CsA to its hydroxylated forms. The hydroxylation activity of CsA by CYP-pa1 was confirmed by CYP-pa1 gene complementation as well as heterologous expression in the CsA non-hydroxylating Streptomyces coelicolor. Moreover, the cyclosporine regio-selectivity of CYP-pa1 expressed in the ΔCYP-sb21 S. benihana mutant strain was also confirmed unchanged through cross complementation. These results show that preferential regio-specific hydroxylation at the 9th N-methyl leucine of CsA is carried out by a specific P450 hydroxylase gene in P. autotrophica, CYP-pa1, setting the stage for the biotechnological application of CsA regioselective hydroxylation.

Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

PubMed

Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S

2015-01-05

Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus.

PubMed

Schlangen, Karin; Miosic, Silvija; Thill, Jana; Halbwirth, Heidi

2010-07-01

A chalcone 3-hydroxylase (CH3H) cDNA clone was isolated and characterized from Cosmos sulphureus petals accumulating butein (2',3,4,4'-tetrahydroxychalcone) derivatives as yellow flower pigments. The recombinant protein catalyses the introduction of an additional hydroxyl group in the B-ring of chalcones, a reaction with high similarity to the hydroxylation of flavonoids catalysed by the well-studied flavonoid 3'-hydroxylase (F3'H). CH3H shows high specificity for chalcones, but a low F3'H activity was also detected. By contrast, the common F3'H from C. sulphureus does not accept chalcones as substrates and is therefore unlikely to be involved in the creation of the B-ring hydroxylation pattern of the yellow flower pigments. CH3H was primarily expressed in young buds, the main tissue for chalcone pigment formation. Expression levels in open flowers and 3-d-old seedlings were lower and almost no CH3H expression was observed in leaves. F3'H, in contrast, showed the highest expression also in buds, but comparable expression rates in all other tissues tested. Recombinant hybrid proteins constructed from CH3H and F3'H fragments demonstrated that amino acid residues at a substrate recognition site and an insertion of four amino acid residues in a putative loop region have an impact on chalcone acceptance. This is the first identification of a CH3H cDNA from any plant species.

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

PubMed Central

Fernández, Cecilia; Belli, Susana; Buzzalino, Noemi; Dain, Liliana

2013-01-01

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%–95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation. PMID:23936690

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

PubMed

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

2013-01-01

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

Genetics Home Reference: tyrosine hydroxylase deficiency

MedlinePlus

... Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews® [Internet]. Seattle (WA): ... MA, Hol FA, Smeitink J, Seeger J, Grattan-Smith P, Ryan MM, Hoffmann GF, Donati MA, Blau ...

Genetics Home Reference: fatty acid hydroxylase-associated neurodegeneration

MedlinePlus

... Certain 2-hydroxylated fatty acids are important in forming normal myelin; myelin is the protective covering that ... C, Alshehhi AA, Proukakis C, Sibtain NA, Maier H, Sharifi R, Patton MA, Bashir W, Koul R, ...

Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

PubMed Central

Seeley, Todd W; Sternlicht, Mark D; Klaus, Stephen J; Neff, Thomas B; Liu, David Y

2017-01-01

The effects of pharmacological hypoxia-inducible factor (HIF) stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD) mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF), using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs), FG-4497 or roxadustat (FG-4592). In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors. PMID:28331872

Immunological detection of phenylalanine hydroxylase protein in Drosophila melanogaster.

PubMed Central

Silva, F J; Bel, Y; Botella, L M; Cotton, R G; Ferré, J

1992-01-01

A monoclonal antibody raised against monkey liver phenylalanine hydroxylase (PAH) has been used to detect this protein in Drosophila melanogaster. A cross-reacting material (CRM) band of apparent molecular mass 50-52 kDa, equivalent to that deduced for the Drosophila melanogaster PAH protein based on the pah gene cDNA sequence, has been detected. This CRM was analysed throughout development and showed an equivalent pattern to that reported for PAH activity in this insect, with maxima at pupariation and at pharate adult formation. Distribution of this CRM in larval tissues, the haemolymph and the adult body is mainly restricted to the larval fat body and the adult head. Demonstration of this CRM as the PAH protein comes from the correlation between the decreased PAH enzyme activities of two mutant strains and their decreased amounts of CRM by Western blotting. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1417795

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

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina

PubMed Central

Dang, Thu‐Thuy T.; Franke, Jakob; Tatsis, Evangelos

2017-01-01

Abstract Plants create tremendous chemical diversity from a single biosynthetic intermediate. In plant‐derived ajmalan alkaloid pathways, the biosynthetic intermediate vomilenine can be transformed into the anti‐arrhythmic compound ajmaline, or alternatively, can isomerize to form perakine, an alkaloid with a structurally distinct scaffold. Here we report the discovery and characterization of vinorine hydroxylase, a cytochrome P450 enzyme that hydroxylates vinorine to form vomilenine, which was found to exist as a mixture of rapidly interconverting epimers. Surprisingly, this cytochrome P450 also catalyzes the non‐oxidative isomerization of the ajmaline precursor vomilenine to perakine. This unusual dual catalytic activity of vinorine hydroxylase thereby provides a control mechanism for the bifurcation of these alkaloid pathway branches. This discovery highlights the unusual catalytic functionality that has evolved in plant pathways. PMID:28654178

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina.

PubMed

Dang, Thu-Thuy T; Franke, Jakob; Tatsis, Evangelos; O'Connor, Sarah E

2017-08-01

Plants create tremendous chemical diversity from a single biosynthetic intermediate. In plant-derived ajmalan alkaloid pathways, the biosynthetic intermediate vomilenine can be transformed into the anti-arrhythmic compound ajmaline, or alternatively, can isomerize to form perakine, an alkaloid with a structurally distinct scaffold. Here we report the discovery and characterization of vinorine hydroxylase, a cytochrome P450 enzyme that hydroxylates vinorine to form vomilenine, which was found to exist as a mixture of rapidly interconverting epimers. Surprisingly, this cytochrome P450 also catalyzes the non-oxidative isomerization of the ajmaline precursor vomilenine to perakine. This unusual dual catalytic activity of vinorine hydroxylase thereby provides a control mechanism for the bifurcation of these alkaloid pathway branches. This discovery highlights the unusual catalytic functionality that has evolved in plant pathways. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Acute biotoxic effect of styrene on rat liver. Correlation with enzyme-mediated mutagenicity of benzpyrene and acrylonitrile.

PubMed

Roberfroid, M; Poncelet, F; Lambotte-Vandepaer, M; Duverger-Van Bogaert, M; de Meester, C; Mercier, M

1978-01-01

Styrene is commonly used in western Europe for the manufacture of plastics suitable for packaging foodstuffs. This report demonstrates that, injected intraperitoneally at a dose as low as 10 mg/kg, styrene modifies the catalytic properties of aryl hydrocarbon hydroxylase by reducing its KM value. A similar effect is reported for two potent chemical carcinogens, 3-methylcholanthrene and benzo(a)pyrene. Ethylbenzene and benzo(e)pyrene and phenobarbital do not produce the same effect. Pretreatments of the rats with chemicals which modify aryl hydrocarbon hydroxylase also increase the capacity of the liver enzymes to activate benzopyrene to a mutagenic intermediate in vitro, as measured by the Ames test for mutagenicity. Exposure to both styrene and the other modifiers of the xenobiotic-metabolizing enzymes could thus influence the carcinogenic and toxic effects of chemicals which are activated by these enzymes. This hypothesis needs further investigation.

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.

Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

PubMed

Park, Nam Il; Park, Jee Hee; Park, Sang Un

2012-02-01

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

Cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase and pyranocoumarin biosynthesis in Angelica gigas.

PubMed

Park, Jee Hee; Park, Nam Il; Xu, Hui; Park, Sang Un

2010-08-27

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.

Effects of exposure to air pollution and smoking on the placental aryl hydrocarbon hydroxylase (AHH) activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hincal, F.

Aryl hydrocarbon hydroxylase (AHH) activities were determined in placental tissues of 152 nonsmoker or exsmoker women who live in Ankara and 125 nonsmoker women who live in areas surrounding Ankara. Levels of AHH were also determined in the placentas of 52 cigarette smokers. The mean AHH activity in the Ankara group was 11.17 +/- 5.41; in the control group, 6.44 +/- 5.48; and for smokers, 45.68 +/- 53.36, which indicates significant differences (p < .001). There was a strong correlation (r = 0.89) between the AHH activities of individuals who live in Ankara and smoke content of the air. Placentalmore » AHH activity did not show any relation to the age, nutritional and dietary habits, factors of indoor pollution, duration of pregnancy, nor did the weight, length and Apgar score of the babies.« less

Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley

PubMed Central

Koopmann, Edda; Logemann, Elke; Hahlbrock, Klaus

1999-01-01

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit. PMID:9880345

L-dopa-induced desensitization depends on 5-hydroxytryptamine imbalance in hemiparkinsonian rats.

PubMed

Kääriäinen, Tiina M; García-Horsman, Juan Arturo; Piltonen, Marjo; Männistö, Pekka T

2009-02-18

We have shown before that 2-week intrastriatal L-3,4-dihydroxyphenylalanine (L-dopa) infusion significantly decreased contralateral rotations induced by acute intraperitoneal L-dopa/carbidopa and increased striatal tryptophan hydroxylase in 6-hydroxydopamine-lesioned rats. Here, we examined the effect of acutely administered L-dopa (10 microg) into 6-hydroxydopamine-lesioned rat striata under the inhibition of tryptophan hydroxylase by 4-chloro-DL-phenylalanine. Acute intrastriatal L-dopa infusion significantly decreased contralateral rotations induced by intraperitoneal L-dopa/carbidopa (10/30 mg/kg) 1 and 7 days after intrastriatal L-dopa. This desensitization to L-dopa occurred only when there was a striatal 5-hydroxytryptamine (5-HT) imbalance, not when 5-HT levels in the intact and lesioned sides were similar, either very low (day 1 postinfusion) or similarly recovered (day 7 postinfusion). We conclude that 5-HT plays a significant role in the striatal dopaminergic imbalance that evokes the rotational behavior.

Transient hyper-17-OHPnemia unrelated to cross-reactions with residual fetal adrenal cortex products.

PubMed

Mizuno, Haruo; Ohro, Yoichiro; Sugiyama, Yukari; Ito, Tetsuya; Hasegawa, Tomonobu; Homma, Keiko; Ueshiba, Hajime; Ono, Makoto; Togari, Hajime

2004-01-01

To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age. Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed. Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment. Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life. Copyright 2004 S. Karger AG, Basel

Biallelic Mutations in DNAJC12 Cause Hyperphenylalaninemia, Dystonia, and Intellectual Disability.

PubMed

Anikster, Yair; Haack, Tobias B; Vilboux, Thierry; Pode-Shakked, Ben; Thöny, Beat; Shen, Nan; Guarani, Virginia; Meissner, Thomas; Mayatepek, Ertan; Trefz, Friedrich K; Marek-Yagel, Dina; Martinez, Aurora; Huttlin, Edward L; Paulo, Joao A; Berutti, Riccardo; Benoist, Jean-François; Imbard, Apolline; Dorboz, Imen; Heimer, Gali; Landau, Yuval; Ziv-Strasser, Limor; Malicdan, May Christine V; Gemperle-Britschgi, Corinne; Cremer, Kirsten; Engels, Hartmut; Meili, David; Keller, Irene; Bruggmann, Rémy; Strom, Tim M; Meitinger, Thomas; Mullikin, James C; Schwartz, Gerard; Ben-Zeev, Bruria; Gahl, William A; Harper, J Wade; Blau, Nenad; Hoffmann, Georg F; Prokisch, Holger; Opladen, Thomas; Schiff, Manuel

2017-02-02

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH 4 ) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH 4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH 4 -activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH 4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA. Copyright © 2017 American Society of Human Genetics. All rights reserved.

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

PubMed

Witsch, Thilo J; Turowski, Pawel; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D; Seeger, Werner; Morty, Rory E

2014-02-01

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.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins1[OPEN

PubMed Central

Kaufholdt, David; Broggini, Giovanni A.L.; Flachowsky, Henryk; Hänsch, Robert

2015-01-01

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H. PMID:25862456

Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001.

PubMed

Pakala, Suresh B; Gorla, Purushotham; Pinjari, Aleem Basha; Krovidi, Ravi Kumar; Baru, Rajasekhar; Yanamandra, Mahesh; Merrick, Mike; Siddavattam, Dayananda

2007-01-01

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component "A" were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component "A", typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.

The draft genome of Ruellia speciosa (Beautiful Wild Petunia: Acanthaceae).

PubMed

Zhuang, Yongbin; Tripp, Erin A

2017-04-01

The genus Ruellia (Wild Petunias; Acanthaceae) is characterized by an enormous diversity of floral shapes and colours manifested among closely related species. Using Illumina platform, we reconstructed the draft genome of Ruellia speciosa, with a scaffold size of 1,021 Mb (or ∼1.02 Gb) and an N50 size of 17,908 bp, spanning ∼93% of the estimated genome (∼1.1 Gb). The draft assembly predicted 40,124 gene models and phylogenetic analyses of four key enzymes involved in anthocyanin colour production [flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), and dihydroflavonol 4-reductase (DFR)] found that most angiosperms here sampled harboured at least one copy of F3H, F3'H, and DFR. In contrast, fewer than one-half (but including R. speciosa) harboured a copy of F3'5'H, supporting observations that blue flowers and/or fruits, which this enzyme is required for, are less common among flowering plants. Ka/Ks analyses of duplicated copies of F3'H and DFR in R. speciosa suggested purifying selection in the former but detected evidence of positive selection in the latter. The genome sequence and annotation of R. speciosa represents only one of only four families sequenced in the large and important Asterid clade of flowering plants and, as such, will facilitate extensive future research on this diverse group, particularly with respect to floral evolution. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

The draft genome of Ruellia speciosa (Beautiful Wild Petunia: Acanthaceae)

PubMed Central

Zhuang, Yongbin

2017-01-01

Abstract The genus Ruellia (Wild Petunias; Acanthaceae) is characterized by an enormous diversity of floral shapes and colours manifested among closely related species. Using Illumina platform, we reconstructed the draft genome of Ruellia speciosa, with a scaffold size of 1,021 Mb (or ∼1.02 Gb) and an N50 size of 17,908 bp, spanning ∼93% of the estimated genome (∼1.1 Gb). The draft assembly predicted 40,124 gene models and phylogenetic analyses of four key enzymes involved in anthocyanin colour production [flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and dihydroflavonol 4-reductase (DFR)] found that most angiosperms here sampled harboured at least one copy of F3H, F3′H, and DFR. In contrast, fewer than one-half (but including R. speciosa) harboured a copy of F3′5′H, supporting observations that blue flowers and/or fruits, which this enzyme is required for, are less common among flowering plants. Ka/Ks analyses of duplicated copies of F3′H and DFR in R. speciosa suggested purifying selection in the former but detected evidence of positive selection in the latter. The genome sequence and annotation of R. speciosa represents only one of only four families sequenced in the large and important Asterid clade of flowering plants and, as such, will facilitate extensive future research on this diverse group, particularly with respect to floral evolution. PMID:28431014

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.

SUPPRESSION OF HIF-1α TRANSCRIPTIONAL ACTIVITY BY THE HIF PROLYL HYDROXYLASE EGLN1*

PubMed Central

To, Kenneth K. W.; Huang, L. Eric

2005-01-01

The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor (VHL). Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which, EGLN1 and EGLN3, are hypoxia-inducible at their mRNA levels in a HIF-1α-dependent manner. In this study, we demonstrate that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a VHL-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Hence, we propose that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provides new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases. PMID:16157596

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

PubMed

Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

2004-01-01

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Impact of Unusual Fatty Acid Synthesis on Futile Cycling through β-Oxidation and on Gene Expression in Transgenic Plants1[w

PubMed Central

Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

2004-01-01

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal β-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward β-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through β-oxidation than the expression profile of genes involved in lipid metabolism. PMID:14671017

Finasteride inhibited brain dopaminergic system and open-field behaviors in adolescent male rats.

PubMed

Li, Li; Kang, Yun-Xiao; Ji, Xiao-Ming; Li, Ying-Kun; Li, Shuang-Cheng; Zhang, Xiang-Jian; Cui, Hui-Xian; Shi, Ge-Ming

2018-02-01

Finasteride inhibits the conversion of testosterone to dihydrotestosterone. Because androgen regulates dopaminergic system in the brain, it could be hypothesized that finasteride may inhibit dopaminergic system. The present study therefore investigates the effects of finasteride in adolescent and early developmental rats on dopaminergic system, including contents of dopamine and its metabolites (dihydroxy phenyl acetic acid and homovanillic acid) and tyrosine hydroxylase expressions both at gene and protein levels. Meanwhile, open-field behaviors of the rats are examined because of the regulatory effect of dopaminergic system on the behaviors. Open-field behaviors were evaluated by exploratory and motor behaviors. Dopamine and its metabolites were assayed by liquid chromatography-mass spectrometry. Tyrosine hydroxylase mRNA and protein expressions were determined by real-time qRT-PCR and western blot, respectively. It was found that in adolescent male rats, administration of finasteride at doses of 25 and 50 mg/kg for 14 days dose dependently inhibited open-field behaviors, reduced contents of dopamine and its metabolites in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and down-regulated tyrosine hydroxylase mRNA and protein expressions in substantia nigra and ventral tegmental area. However, there was no significant change of these parameters in early developmental rats after finasteride treatment. These results suggest that finasteride inhibits dopaminergic system and open-field behaviors in adolescent male rats by inhibiting the conversion of testosterone to dihydrotestosterone, and imply finasteride as a potential therapeutic option for neuropsychiatric disorders associated with hyperactivities of dopaminergic system and androgen. © 2017 John Wiley & Sons Ltd.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins.

PubMed

Sircar, Debabrata; Gaid, Mariam M; Chizzali, Cornelia; Reckwell, Dennis; Kaufholdt, David; Beuerle, Till; Broggini, Giovanni A L; Flachowsky, Henryk; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

2015-06-01

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H. © 2015 American Society of Plant Biologists. All Rights Reserved.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

PubMed Central

Toyomasu, Tomonobu; Kawaide, Hiroshi; Mitsuhashi, Wataru; Inoue, Yasunori; Kamiya, Yuji

1998-01-01

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action. PMID:9847128

Abscisic acid negatively regulates elicitor-induced synthesis of capsidiol in wild tobacco.

PubMed

Mialoundama, Alexis Samba; Heintz, Dimitri; Debayle, Delphine; Rahier, Alain; Camara, Bilal; Bouvier, Florence

2009-07-01

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8'-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8'-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants.

DBH deficiency in an elderly patient: efficacy and safety of chronic droxidopa.

PubMed

Despas, Fabien; Pathak, A; Berry, M; Cagnac, R; Massabuau, P; Liozon, E; Galinier, M; Senard, J M

2010-06-01

We describe the effects of chronic droxidopa in a patient with Dopamine beta-hydroxylase deficiency diagnosed at the age of 73. Investigations were performed to assess sympathetic activity (MIBG scintigraphy, catecholamines) and cardiovascular droxidopa safety.

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

MedlinePlus

... Shackleton C, Imperato-McGinley J. Mutations in CYP11B1 gene: phenotype-genotype correlations. Am J Med Genet A. 2003 Oct ... are genome editing and CRISPR-Cas9? What is precision medicine? What ...

Expression of Molecular Markers in Brain, Serum, and Lung Tissues Following Hypobaric Hypoxia

DTIC Science & Technology

2018-01-01

8 4.2 HIF-1α ELISA Results...9 4.3 Prolyl-4-hydroxylase Alpha Polypeptide I (P4Ha1) ELISA Results...10 4.4 Vascular Endothelial Growth Factor ELISA Results .......................................................12

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

PubMed Central

Romero-Fernandez, W.; Borroto-Escuela, D.O.; Vargas-Barroso, V.; Narváez, M.; Di Palma, M.; Agnati, L.F.; Sahd, J. Larriva

2014-01-01

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region. PMID:25308843

Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

PubMed

Romero-Fernandez, W; Borroto-Escuela, D O; Vargas-Barroso, V; Narváez, M; Di Palma, M; Agnati, L F; Larriva Sahd, J; Fuxe, K

2014-07-18

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tubero-infundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region.  Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and /or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.

17-hydroxylase/17,20-lyase deficiency due to a R96Q mutation causing hypertension and poor breast development

PubMed Central

Al Suwaidi, Hana; Attia, Salima; Al Ameri, Ahlam

2015-01-01

Summary Combined17α-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Hypertension and hypokalemia are essential presenting features. We report an Arab family with four affected XX siblings. The eldest presented with abdominal pain and was diagnosed with a retroperitoneal malignant mixed germ cell tumour. She was hypertensive and hypogonadal. One sibling presented with headache due to hypertension while the other two siblings were diagnosed with hypertension on a routine school check. A homozygous R96Q missense mutation in P450c17 was detected in the index case who had primary amenorrhea and lack of secondary sexual characters at 17 years. The middle two siblings were identical twins and had no secondary sexual characters at the age of 14. All siblings had hypokalemia, very low level of adrenal androgens, high ACTH and high levels of aldosterone substrates. Treatment was commenced with steroid replacement and puberty induction with estradiol. The index case had surgical tumor resection and chemotherapy. All siblings required antihypertensive treatment and the oldest remained on two antihypertensive medications 12 years after diagnosis. Her breast development remained poor despite adequate hormonal replacement. Combined 17α-hydroxylase/17,20-lyase deficiency is a rare condition but might be underdiagnosed. It should be considered in young patients presenting with hypertension, particularly if there is a family history of consanguinity and with more than one affected sibling. Antihypertensive medication might continue to be required despite adequate steroid replacement. Breast development may remain poor in mutations causing complete form of the disease. Learning points Endocrine hypertension due to rarer forms of CAH should be considered in children and adolescents, particularly if more than one sibling is affected and in the presence of consanguinity. 17α-hydroxylase/17,20-lyase deficiency is a rare form of CAH but might be underdiagnosed. Blood pressure measurement should be carried out in all females presenting with hypogonadism. Anti-hypertensive medications might be required despite adequate steroid replacement. Initial presenting features might vary within affected members of the same family. Adverse breast development might be seen in the complete enzyme deficiency forms of the disease. PMID:26543560

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

PubMed

Liu, G; Gelboin, H V; Myers, M J

1991-02-01

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

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.

Genetics Home Reference: dopa-responsive dystonia

MedlinePlus

... neurotransmitters called dopamine and serotonin. Among their many functions, dopamine transmits signals within the brain to produce smooth ... production of a tyrosine hydroxylase enzyme with reduced function, which leads to a decrease in dopamine production. A reduction in the amount of dopamine ...

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

PubMed Central

Hovakimyan, Marine; Haas, Stefan Jean-Pierre; Schmitt, Oliver; Gerber, Bernd; Wree, Andreas; Andressen, Christian

2006-01-01

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6-hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100 000 hNPCs (1 µL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100β, neuronal nuclei antigen, neuron-specific enolase and tyrosine hydroxylase. Compared with the apomorphine-induced rotations in the lesioned-only group (7.4 ± 0.5 min−1), lesioned and successfully transplanted animals (0.3 ± 0.1 min−1) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned-only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei-immunoreactive cells were found to survive and migrate up to 2000 µm into the host parenchyma, many containing the pan-neuronal markers neuronal nuclei antigen and neuron-specific enolase. In the striatum, tyrosine hydroxylase-immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase-immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo. PMID:17118060

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

Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.

PubMed

Walker, Linda C; Overstreet, Mayra A; Yeowell, Heather N

2005-01-01

Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.

Biochemically Silent Abdominal Paragangliomas in Patients with Mutations in the Succinate Dehydrogenase Subunit B Gene

PubMed Central

Timmers, Henri J. L. M.; Pacak, Karel; Huynh, Thanh T.; Abu-Asab, Mones; Tsokos, Maria; Merino, Maria J.; Baysal, Bora E.; Adams, Karen T.; Eisenhofer, Graeme

2008-01-01

Context: Patients with adrenal and extra-adrenal abdominal paraganglioma (PGL) almost invariably have increased plasma and urine concentrations of metanephrines, the O-methylated metabolites of catecholamines. We report four cases of biochemically silent abdominal PGL, in which metanephrines were normal despite extensive disease. Objective: Our objective was to identify the mechanism underlying the lack of catecholamine hypersecretion and metabolism to metanephrines in biochemically silent PGL. Design: This is a descriptive study. Setting: The study was performed at a referral center. Patients: One index case and three additional patients with large abdominal PGL and metastases but with the lack of evidence of catecholamine production, six patients with metastatic catecholamine-producing PGL and a mutation of the succinate dehydrogenase subunit B (SDHB) gene, and 136 random patients with catecholamine-producing PGL were included in the study. Main Outcome Measures: Plasma, urine, and tumor tissue concentrations of catecholamines and metabolites were calculated with electron microscopy and tyrosine hydroxylase immunohistochemistry. Results: All four patients with biochemically silent PGL had an underlying SDHB mutation. In the index case, the tumor tissue concentration of catecholamines (1.8 nmol/g) was less than 0.01% that of the median (20,410 nmol/g) for the 136 patients with catecholamine-producing tumors. Electron microscopy showed the presence of normal secretory granules in all four biochemically silent PGLs. Tyrosine hydroxylase immunoreactivity was negligible in the four biochemically silent PGLs but abundant in catecholamine-producing PGLs. Conclusions: Patients with SDHB mutations may present with biochemically silent abdominal PGLs due to defective catecholamine synthesis resulting from the absence of tyrosine hydroxylase. Screening for tumors in patients with SDHB mutations should not be limited to biochemical tests of catecholamine excess. PMID:18840642

Androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway: Insights into enzyme activities and steroid fluxes in healthy infants during the first year of life from the urinary steroid metabolome.

PubMed

Dhayat, Nasser A; Dick, Bernhard; Frey, Brigitte M; d'Uscio, Claudia H; Vogt, Bruno; Flück, Christa E

2017-01-01

The steroid profile changes dramatically from prenatal to postnatal life. Recently, a novel backdoor pathway for androgen biosynthesis has been discovered. However, its role remains elusive. Therefore, we investigated androgen production from birth to one year of life with a focus on minipuberty and on production of androgens through the backdoor pathway. Additionally, we assessed the development of the specific steroid enzyme activities in early life. To do so, we collected urine specimens from diapers in 43 healthy newborns (22 females) at 13 time points from birth to one year of age in an ambulatory setting, and performed in house GC-MS steroid profiling for 67 steroid metabolites. Data were analyzed for androgen production through the classic and backdoor pathway and calculations of diagnostic ratios for steroid enzyme activities were performed. Analysis revealed that during minipuberty androgen production is much higher in boys than in girls (e.g. androsterone (An)), originates largely from the testis (An boys -An girls ), and uses predominantly the alternative backdoor pathway (An/Et; Δ5<Δ4 lyase activity). Modelling of steroid enzyme activities showed age-related effects for 21-, 11-, 17-hydroxylase and P450 oxidoreductase activities as well as 3β-hydroxysteroid dehydrogenase, 11β-hydroxylase type 1/2 and 5α-reductase activities. Sex-related characteristics were found for 21-hydroxylase and 5α-reductase activities. Overall, our study shows that androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway. Calculations of specific diagnostic ratios for enzyme activities seem to allow the diagnosis of specific steroid disorders from the urinary steroid metabolome. Copyright © 2016 Elsevier Ltd. All rights reserved.

Placenta-specific Methylation of the Vitamin D 24-Hydroxylase Gene

PubMed Central

Novakovic, Boris; Sibson, Mandy; Ng, Hong Kiat; Manuelpillai, Ursula; Rakyan, Vardhman; Down, Thomas; Beck, Stephan; Fournier, Thierry; Evain-Brion, Danielle; Dimitriadis, Eva; Craig, Jeffrey M.; Morley, Ruth; Saffery, Richard

2009-01-01

Plasma concentrations of biologically active vitamin D (1,25-(OH)2D) are tightly controlled via feedback regulation of renal 1α-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled, and 1,25-(OH)2D levels are significantly elevated, suggesting a role in pregnancy progression. Epigenetic regulation of CYP27B1 and CYP24A1 has previously been described in cell and animal models, and despite emerging evidence for a critical role of epigenetics in placentation generally, little is known about the regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. In this study, we investigated the methylation status of genes regulating vitamin D bioavailability and activity in the placenta. No methylation of the VDR (vitamin D receptor) and CYP27B1 genes was found in any placental tissues. In contrast, the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue. No methylation was detected in any somatic human tissue tested. Methylation was also evident in marmoset and mouse placental tissue. All three genes were hypermethylated in choriocarcinoma cell lines, highlighting the role of vitamin D deregulation in this cancer. Gene expression analysis confirmed a reduced capacity for CYP24A1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down-regulates basal promoter activity and abolishes vitamin D-mediated feedback activation. This study strongly suggests that epigenetic decoupling of vitamin D feedback catabolism plays an important role in maximizing active vitamin D bioavailability at the fetomaternal interface. PMID:19237542

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

PubMed

Mahajan, Monika; Yadav, Sudesh Kumar

2014-08-01

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

Effect of long-term actual spaceflight on the expression of key genes encoding serotonin and dopamine system

NASA Astrophysics Data System (ADS)

Popova, Nina; Shenkman, Boris; Naumenko, Vladimir; Kulikov, Alexander; Kondaurova, Elena; Tsybko, Anton; Kulikova, Elisabeth; Krasnov, I. B.; Bazhenova, Ekaterina; Sinyakova, Nadezhda

The effect of long-term spaceflight on the central nervous system represents important but yet undeveloped problem. The aim of our work was to study the effect of 30-days spaceflight of mice on Russian biosatellite BION-M1 on the expression in the brain regions of key genes of a) serotonin (5-HT) system (main enzymes in 5-HT metabolism - tryptophan hydroxylase-2 (TPH-2), monoamine oxydase A (MAO A), 5-HT1A, 5-HT2A and 5-HT3 receptors); b) pivotal enzymes in DA metabolism (tyrosine hydroxylase, COMT, MAO A, MAO B) and D1, D2 receptors. Decreased expression of genes encoding the 5-HT catabolism (MAO A) and 5-HT2A receptor in some brain regions was shown. There were no differences between “spaceflight” and control mice in the expression of TPH-2 and 5-HT1A, 5-HT3 receptor genes. Significant changes were found in genetic control of DA system. Long-term spaceflight decreased the expression of genes encoding the enzyme in DA synthesis (tyrosine hydroxylase in s.nigra), DA metabolism (MAO B in the midbrain and COMT in the striatum), and D1 receptor in hypothalamus. These data suggested that 1) microgravity affected genetic control of 5-HT and especially the nigrostriatal DA system implicated in the central regulation of muscular tonus and movement, 2) the decrease in the expression of genes encoding key enzyme in DA synthesis, DA degradation and D1 receptor contributes to the movement impairment and dyskinesia produced by the spaceflight. The study was supported by Russian Foundation for Basic Research grant No. 14-04-00173.

Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

PubMed Central

Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M.; Liu, Jie; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

2011-01-01

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (Vmax/km) for any of the complexes is 70–6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex–mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716

Atorvastatin protects GABAergic and dopaminergic neurons in the nigrostriatal system in an experimental rat model of transient focal cerebral ischemia.

PubMed

Sabogal, Angélica María; Arango, César Augusto; Cardona, Gloria Patricia; Céspedes, Ángel Enrique

2014-01-01

Cerebral ischemia is the third leading cause of death and the primary cause of permanent disability worldwide. Atorvastatin is a promising drug with neuroprotective effects that may be useful for the treatment of stroke. However, the effects of atorvastatin on specific neuronal populations within the nigrostriatal system following cerebral ischemia are unknown. To evaluate the effects of atorvastatin on dopaminergic and GABAergic neuronal populations in exofocal brain regions in a model of transient occlusion of the middle cerebral artery. Twenty-eight male eight-week-old Wistar rats were used in this study. Both sham and ischemic rats were treated with atorvastatin (10 mg/kg) or carboxymethylcellulose (placebo) by gavage at 6, 24, 48 and 72 hours post-reperfusion. We analyzed the immunoreactivity of glutamic acid decarboxylase and tyrosine hydroxylase in the globus pallidus, caudate putamen and substantia nigra. We observed neurological damage and cell loss in the caudate putamen following ischemia. We also found an increase in tyrosine hydroxylase immunoreactivity in the medial globus pallidus and substantia nigra reticulata, as well as a decrease in glutamic acid decarboxylase immunoreactivity in the lateral globus pallidus in ischemic animals treated with a placebo. However, atorvastatin treatment was able to reverse these effects, significantly decreasing tyrosine hydroxylase levels in the medial globus pallidus and substantia nigra reticulata and significantly increasing glutamic acid decarboxylase levels in the lateral globus pallidus. Our data suggest that post-ischemia treatment with atorvastatin can have neuro-protective effects in exofocal regions far from the ischemic core by modulating the GABAergic and dopaminergic neuronal populations in the nigrostriatal system, which could be useful for preventing neurological disorders.

Homozygous Mutation G539R in the Gene for P450 Oxidoreductase in a Family Previously Diagnosed as Having 17,20-Lyase Deficiency

PubMed Central

Hershkovitz, Eli; Parvari, Ruthi; Wudy, Stefan A.; Hartmann, Michaela F.; Gomes, Larissa G.; Loewental, Neta; Miller, Walter L.

2008-01-01

Context: Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17α-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR). Objective: Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency. Patients: Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency. Methods: Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced. Results: Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17α-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17. Conclusion: POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency. PMID:18559916

Homozygous mutation G539R in the gene for P450 oxidoreductase in a family previously diagnosed as having 17,20-lyase deficiency.

PubMed

Hershkovitz, Eli; Parvari, Ruthi; Wudy, Stefan A; Hartmann, Michaela F; Gomes, Larissa G; Loewental, Neta; Miller, Walter L

2008-09-01

Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17alpha-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR). Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency. Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency. Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced. Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17alpha-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17. POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency.

Hindbrain Catecholamine Neurons Activate Orexin Neurons During Systemic Glucoprivation in Male Rats.

PubMed

Li, Ai-Jun; Wang, Qing; Elsarelli, Megan M; Brown, R Lane; Ritter, Sue

2015-08-01

Hindbrain catecholamine neurons are required for elicitation of feeding responses to glucose deficit, but the forebrain circuitry required for these responses is incompletely understood. Here we examined interactions of catecholamine and orexin neurons in eliciting glucoprivic feeding. Orexin neurons, located in the perifornical lateral hypothalamus (PeFLH), are heavily innervated by hindbrain catecholamine neurons, stimulate food intake, and increase arousal and behavioral activation. Orexin neurons may therefore contribute importantly to appetitive responses, such as food seeking, during glucoprivation. Retrograde tracing results showed that nearly all innervation of the PeFLH from the hindbrain originated from catecholamine neurons and some raphe nuclei. Results also suggested that many catecholamine neurons project collaterally to the PeFLH and paraventricular hypothalamic nucleus. Systemic administration of the antiglycolytic agent, 2-deoxy-D-glucose, increased food intake and c-Fos expression in orexin neurons. Both responses were eliminated by a lesion of catecholamine neurons innervating orexin neurons using the retrogradely transported immunotoxin, anti-dopamine-β-hydroxylase saporin, which is specifically internalized by dopamine-β-hydroxylase-expressing catecholamine neurons. Using designer receptors exclusively activated by designer drugs in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide. Clozapine-N-oxide injection increased food intake and c-Fos expression in PeFLH orexin neurons as well as in paraventricular hypothalamic nucleus neurons. In summary, catecholamine neurons are required for the activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding.

Downregulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) or cinnamate-4-hydrolylase (C4H) in Eucalyptus urophylla x Eucalyptus grandis leads to increased extractability

DOE PAGES

Ziebell, Angela; Gjersing, Erica; Hinchee, Maud; ...

2016-01-20

Lignin reduction through breeding and genetic modification has the potential to reduce costs in biomass processing in pulp and paper, forage, and lignocellulosic ethanol industries. Here, we present detailed characterization of the extractability and lignin structure of Eucalyptus urophylla x Eucalyptus grandis RNAi downregulated in p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) or cinnamate-4-hydroxylase (C4H). Both the C3'H and C4H downregulated lines were found to have significantly higher extractability when exposed to NaOH base extraction, indicating altered cell wall construction. The molecular weight of isolated lignin was measured and lignin structure was determined by HSQC NMR-based lignin subunit analysis for control and themore » C3'H and C4H downregulated lines. The slight reductions in average molecular weights of the lignin isolated from the transgenic lines (C3'H = 7000, C4H = 6500, control = 7300) does not appear to explain the difference in extractability. The HSQC NMR-based lignin subunit analysis showed increases in H lignin content for the C3'H but only slight differences in the lignin subunit structure of the C3'H and C4H downregulated lines when compared to the control. The greatest difference between the C3'H and C4H downregulated lines is the total lignin content; therefore, it appears that overall lowered lignin content contributes greatly to reduced recalcitrance and increased extractability of cell wall biopolymers. Furthermore, studies will be conducted to determine how the reduction in lignin content creates a less rigid cell wall that is more prone to extraction and sugar release.« less

Isolation and functional analysis of a homolog of flavonoid 3',5'-hydroxylase gene from Pericallis × hybrida.

PubMed

Sun, Yi; Huang, He; Meng, Li; Hu, Ke; Dai, Si-Lan

2013-10-01

As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T₀ generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T₁ generation were similar to those of T₀ generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene. © 2013 Scandinavian Plant Physiology Society.

The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

PubMed Central

Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

2012-01-01

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases

PubMed Central

2004-01-01

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively expressed β-subunit. In normoxia, HIF-1α is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1α expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-α. Using a VHL capture assay, we demonstrate that HIF-α prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1α after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1α in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia. PMID:15104534

The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei.

PubMed

Mapanao, Ratchaneegorn; Chang, Chin-Chyuan; Cheng, Winton

2017-02-01

Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate-water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

How are substrate binding and catalysis affected by mutating Glu127 and Arg161 in prolyl-4-hydroxylase? A QM/MM and MD study.

NASA Astrophysics Data System (ADS)

Timmins, Amy; de Visser, Sam P.

2017-11-01

Prolyl-4-hydroxylase is a vital enzyme for human physiology involved in the biosynthesis of 4-hydroxyproline, an essential component for collagen formation. The enzyme performs a unique stereo- and regioselective hydroxylation at the C4 position of proline despite the fact that the C5 hydrogen atom should be thermodynamically easier to abstract. To gain insight into the mechanism and find the origin of this regioselectivity, we have done a quantum mechanics/molecular mechanics (QM/MM) study on wildtype and mutant structures. In a previous study [Timmins, Saint-André and de Visser, J. Am. Chem. Soc. 2017] we identified several active site residues critical for substrate binding and positioning. In particular, the Glu127 and Arg161 were shown to form multiple hydrogen bonding and ion-dipole interactions with substrate and could thereby affect the regio- and stereoselectivity of the reaction. In this work, we decided to test that hypothesis and report a QM/MM and molecular dynamics (MD) study on prolyl-4-hydroxylase and several active site mutants where Glu127 or Arg161 are mutated for Asp, Gln or Lys. Thus, the R161D and R161Q mutants give very high barriers for hydrogen atom abstraction from any proline C–H bond and therefore will be inactive. The R161K mutant, by contrast, sees the regio- and stereoselectivity of the reaction change but still is expected to hydroxylate proline at room temperature. By contrast, the Glu127 mutants E127D and E127Q show possible changes in regioselectivity with the former being more probable to react compared to the latter.

The Role of Endogenous Serotonin in Methamphetamine-Induced Neurotoxicity to Dopamine Nerve Endings of the Striatum

PubMed Central

Thomas, David M.; Angoa-Pérez, Mariana; Francescutti-Verbeem, Dina M.; Shah, Mrudang M.; Kuhn, Donald M.

2010-01-01

Methamphetamine (METH) is a neurotoxic drug of abuse that damages the dopamine (DA) neuronal system in a highly delimited manner. The brain structure most affected by METH is the striatum where long-term DA depletion and microglial activation are maximal. Endogenous DA has been implicated as a critical participant in METH-induced neurotoxicity, most likely as a substrate for non-enzymatic oxidation by METH-generated reactive oxygen species (ROS). The striatum is also extensively innervated by serotonin (5HT) nerve endings and this neurochemical system is modified by METH in much the same manner as seen in DA nerve endings (i.e., increased release of 5HT, loss of function in tryptophan hydroxylase and the serotonin transporter, long-term depletion of 5HT stores). 5HT can also be modified by ROS to form highly reactive species that damage neurons but its role in METH neurotoxicity has not been assessed. Increases in 5HT levels with 5HTP do not change METH-induced neurotoxicity to the DA nerve endings as revealed by reductions in DA, tyrosine hydroxylase and dopamine transporter levels. Partial reductions in 5HT with p-chlorophenylalanine (PCPA) are without effect on METH toxicity, despite the fact that PCPA largely prevents METH-induced hyperthermia. Mice lacking the gene for brain tryptophan hydroxylase 2 are devoid of brain 5HT and respond to METH in the same manner as wild-type controls, despite showing enhanced drug-induced hyperthermia. Taken together, the present results indicate that endogenous 5HT does not appear to play a role in METH-induced damage to DA nerve endings of the striatum. PMID:20722968

The neurotoxic effects of methamphetamine on 5-hydroxytryptamine and dopamine in brain: evidence for the protective effect of chlormethiazole.

PubMed

Green, A R; De Souza, R J; Williams, J L; Murray, T K; Cross, A J

1992-04-01

Studies were undertaken in mice and rats on the neurotoxic effects of methamphetamine on dopaminergic and 5-hydroxytryptaminergic neurones in the brain and the neuroprotective action of chlormethiazole. In initial studies, mice were injected with methamphetamine (5 mg/kg, i.p.) at 2 hr intervals, to a total of 4 times. This procedure produced a 66% loss of striatal dopamine and a 50% loss of tyrosine hydroxylase activity 3 days later. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each dose of methamphetamine, totally prevented the methamphetamine-induced loss of tyrosine hydroxylase activity and partly prevented the loss of dopamine. Phencyclidine (20 mg/kg, i.p.), given in place of chlormethiazole, also prevented the loss of tyrosine hydroxylase. Administration to rats of 4 doses of methamphetamine (15 mg/kg, i.p.) at 3 hr intervals resulted in a 75% loss of striatal dopamine 3 days later and a similar loss of 5-HT and 5-HIAA in cortex and hippocampus. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each injection of methamphetamine, protected against the loss of dopamine and indoleamine content, in the respective regions. Pentobarbital (25 mg/kg, i.p.) also provided substantial protection but diazepam (2.5 mg/kg, i.p.) was without effect. Confirming earlier studies, dizocilpine (1 mg/kg) also provided substantial protection against the methamphetamine-induced neurotoxicity. Preliminary data indicated that chlormethiazole was not neuroprotective because of a hypothermic action. These data therefore demonstrate that chlormethiazole is an effective neuroprotective agent against methamphetamine-induced neurotoxicity and extend the evidence for the possible value of this drug in preventing neurodegeneration.

The role of endogenous serotonin in methamphetamine-induced neurotoxicity to dopamine nerve endings of the striatum.

PubMed

Thomas, David M; Angoa Pérez, Mariana; Francescutti-Verbeem, Dina M; Shah, Mrudang M; Kuhn, Donald M

2010-11-01

Methamphetamine (METH) is a neurotoxic drug of abuse that damages the dopamine (DA) neuronal system in a highly delimited manner. The brain structure most affected by METH is the striatum where long-term DA depletion and microglial activation are maximal. Endogenous DA has been implicated as a critical participant in METH-induced neurotoxicity, most likely as a substrate for non-enzymatic oxidation by METH-generated reactive oxygen species. The striatum is also extensively innervated by serotonin (5HT) nerve endings and this neurochemical system is modified by METH in much the same manner as seen in DA nerve endings (i.e., increased release of 5HT, loss of function in tryptophan hydroxylase and the serotonin transporter, long-term depletion of 5HT stores). 5HT can also be modified by reactive oxygen species to form highly reactive species that damage neurons but its role in METH neurotoxicity has not been assessed. Increases in 5HT levels with 5-hydroxytryptophan do not change METH-induced neurotoxicity to the DA nerve endings as revealed by reductions in DA, tyrosine hydroxylase and dopamine transporter levels. Partial reductions in 5HT with p-chlorophenylalanine are without effect on METH toxicity, despite the fact that p-chlorophenylalanine largely prevents METH-induced hyperthermia. Mice lacking the gene for brain tryptophan hydroxylase 2 are devoid of brain 5HT and respond to METH in the same manner as wild-type controls, despite showing enhanced drug-induced hyperthermia. Taken together, the present results indicate that endogenous 5HT does not appear to play a role in METH-induced damage to DA nerve endings of the striatum. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

Developmental neurotoxicants target neurodifferentiation into the serotonin phenotype: Chlorpyrifos, diazinon, dieldrin and divalent nickel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slotkin, Theodore A.; Seidler, Frederic J.

2008-12-01

Developmental exposure to organophosphates (OP) produces long-term changes in serotonin (5HT) synaptic function and associated behaviors, but there are disparities among the different OPs. We contrasted effects of chlorpyrifos and diazinon, as well as non-OP neurotoxicants (dieldrin, Ni{sup 2+}) using undifferentiated and differentiating PC12 cells, a well-established neurodevelopmental model. Agents were introduced at 30 {mu}M for 24 or 72 h, treatments devoid of cytotoxicity, and we evaluated the mRNAs encoding the proteins for 5HT biosynthesis, storage and degradation, as well as 5HT receptors. Chlorpyrifos and diazinon both induced tryptophan hydroxylase, the rate-limiting enzyme for 5HT biosynthesis, but chlorpyrifos had amore » greater effect, and both agents suppressed expression of 5HT transporter genes, effects that would tend to augment extracellular 5HT. However, whereas chlorpyrifos enhanced the expression of most 5HT receptor subtypes, diazinon evoked overall suppression. Dieldrin evoked even stronger induction of tryptophan hydroxylase, and displayed a pattern of receptor effects similar to that of diazinon, even though they come from different pesticide classes. In contrast, Ni{sup 2+} had completely distinct actions, suppressing tryptophan hydroxylase and enhancing the vesicular monoamine transporter, while also reducing 5HT receptor gene expression, effects that would tend to lower net 5HT function. Our findings provide some of the first evidence connecting the direct, initial mechanisms of developmental neurotoxicant action on specific transmitter pathways with their long-term effects on synaptic function and behavior, while also providing support for in vitro test systems as tools for establishing mechanisms and outcomes of related and unrelated neurotoxicants.« less

Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding.

PubMed

Repa, J J; Lund, E G; Horton, J D; Leitersdorf, E; Russell, D W; Dietschy, J M; Turley, S D

2000-12-15

Sterol 27-hydroxylase (CYP27) participates in the conversion of cholesterol to bile acids. We examined lipid metabolism in mice lacking the Cyp27 gene. On normal rodent chow, Cyp27(-/-) mice have 40% larger livers, 45% larger adrenals, 2-fold higher hepatic and plasma triacylglycerol concentrations, a 70% higher rate of hepatic fatty acid synthesis, and a 70% increase in the ratio of oleic to stearic acid in the liver versus Cyp27(+/+) controls. In Cyp27(-/-) mice, cholesterol 7alpha-hydroxylase activity is increased 5-fold, but bile acid synthesis and pool size are 47 and 27%, respectively, of those in Cyp27(+/+) mice. Intestinal cholesterol absorption decreases from 54 to 4% in knockout mice, while fecal neutral sterol excretion increases 2.5-fold. A compensatory 2.5-fold increase in whole body cholesterol synthesis occurs in Cyp27(-/-) mice, principally in liver, adrenal, small intestine, lung, and spleen. The mRNA for the cholesterogenic transcription factor sterol regulatory element-binding protein-2 (SREBP-2) and mRNAs for SREBP-2-regulated cholesterol biosynthetic genes are elevated in livers of mutant mice. In addition, the mRNAs encoding the lipogenic transcription factor SREBP-1 and SREBP-1-regulated monounsaturated fatty acid biosynthetic enzymes are also increased. Hepatic synthesis of fatty acids and accumulation of triacylglycerols increases in Cyp27(-/-) mice and is associated with hypertriglyceridemia. Cholic acid feeding reverses hepatomegaly and hypertriglyceridemia but not adrenomegaly in Cyp27(-/-) mice. These studies confirm the importance of CYP27 in bile acid synthesis and they reveal an unexpected function of the enzyme in triacylglycerol metabolism.

Abscisic Acid Negatively Regulates Elicitor-Induced Synthesis of Capsidiol in Wild Tobacco1[W

PubMed Central

Mialoundama, Alexis Samba; Heintz, Dimitri; Debayle, Delphine; Rahier, Alain; Camara, Bilal; Bouvier, Florence

2009-01-01

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8′-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8′-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants. PMID:19420326

OMEGA-3 LONG-CHAIN POLYUNSATURATED FATTY ACIDS IN OLDER CHILDREN

USDA-ARS?s Scientific Manuscript database

Phenylketonuria (PKU), the most prevalent inborn error of metabolism, is usually secondary to low hepatic activity of phenylalanine hydroxylase, the enzyme that catalyzes conversion of phenylalanine to tyrosine. Growth and development of infants and children with PKU who are managed by mandatory n...

ADHD Candidate Gene Study in a Population-Based Birth Cohort: Association with DBH and DRD2

ERIC Educational Resources Information Center

Nyman, Emma S.; Ogdie, Matthew N.; Loukola, Anu; Varilo, Teppo; Taanila, Anja; Hurtig, Tuula; Moilanen, Irma K.; Loo, Sandra K.; McGough, James J.; Jarvelin, Marjo-Riitta; Smalley, Susan L.

2007-01-01

A study aims to examine the genetic contribution if any to attention-deficit/hyperactivity disorder (ADHD). The results confirm the hypothesis and the association of dopamine [beta]-hydroxylase and dopamine receptor D2 genes with ADHD.

Clinical, biochemical and genetic features with nonclassical 21-hydroxylase deficiency and final height.

PubMed

Savaş-Erdeve, Şenay; Çetinkaya, Semra; Abalı, Zehra Yavaş; Poyrazoğlu, Şükran; Baş, Firdevs; Berberoğlu, Merih; Sıklar, Zeynep; Korkmaz, Özlem; Buluş, Derya; Akbaş, Emine Demet; Güran, Tülay; Böber, Ece; Akın, Onur; Yılmaz, Gülay Can; Aycan, Zehra

2017-07-26

The clinical, laboratory, genetic properties and final height of a large cohort of patients with nonclassical 21-hydroxylase deficiency (NC21OHD) in Turkey were analyzed. This multicenter, nationwide web-based study collected data. The mean age was 9.79±4.35 years (229 girls, 29 boys). The most common symptoms were premature pubarche (54.6%) and hirsutism (28.6%). The peak cortisol was found below 18 μg/dL in three (15.45%) patients. A mutation was detected in the CYP21A2 gene of 182 (87.5%) patients. The most common mutation was V281L. Final height in female patients who were diagnosed and treated before attaining final height or near final height was found to be shorter than the final height in female patients who were diagnosed after attaining final height or near final height. The final height of the patients who were treated during childhood was found to be shorter than the final height of patients during the adolescent period.

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

PubMed

Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

2015-12-30

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

In-crystal reaction cycle of a toluene-bound diiron hydroxylase

NASA Astrophysics Data System (ADS)

Acheson, Justin F.; Bailey, Lucas J.; Brunold, Thomas C.; Fox, Brian G.

2017-03-01

Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe2+/Fe3+ species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.

The discovery of the pressor effect of DOPS and its blunting by decarboxylase inhibitors.

PubMed

Kaufmann, H

2006-01-01

In the 1950s it was found that an artificial aminoacid, 3,4-threo-dihydroxyphenylserine (DOPS), was converted to norepinephrine (NE) in a single step by the enzyme L-aromatic amino acid decarboxylase (AADC), bypassing the need for the rate limiting enzyme dopamine beta hydroxylase. Trying to replicate the success of dihydroxyphenylalanine (DOPA) in the treatment of Parkinson disease, treatment with DOPS was attempted in patients with autonomic failure who have impaired NE release. DOPS improved orthostatic hypotension in patients with familial amyloid polyneuropathy, congenital deficiency of dopamine beta hydroxylase, pure autonomic failure and multiple system atrophy. DOPS pressor effect is due to its conversion to NE outside the central nervous system because concomitant administration of carbidopa, an inhibitor of AADC that does not cross the blood-brain barrier, blunted both the increase in plasma NE and the pressor response. DOPS pressor response is not dependent on intact sympathetic terminals because its conversion to NE also occurs in non-neuronal tissues.

A patient with PMP22-related hereditary neuropathy and DBH-gene-related dysautonomia.

PubMed

Bartoletti-Stella, Anna; Chiaro, Giacomo; Calandra-Buonaura, Giovanna; Contin, Manuela; Scaglione, Cesa; Barletta, Giorgio; Cecere, Annagrazia; Garagnani, Paolo; Tieri, Paolo; Ferrarini, Alberto; Piras, Silvia; Franceschi, Claudio; Delledonne, Massimo; Cortelli, Pietro; Capellari, Sabina

2015-10-01

Recurrent focal neuropathy with liability to pressure palsies is a relatively frequent autosomal-dominant demyelinating neuropathy linked to peripheral myelin protein 22 (PMP22) gene deletions. The combination of PMP22 gene mutations with other genetic variants is known to cause a more severe phenotype than expected. We present the case of a patient with severe orthostatic hypotension since 12 years of age, who inherited a PMP22 gene deletion from his father. Genetic double trouble was suspected because of selective sympathetic autonomic disturbances. Through exome-sequencing analysis, we identified two novel mutations in the dopamine beta hydroxylase gene. Moreover, with interactome analysis, we excluded a further influence on the origin of the disease by variants in other genes. This case increases the number of unique patients presenting with dopamine-β-hydroxylase deficiency and of cases with genetically proven double trouble. Finding the right, complete diagnosis is crucial to obtain adequate medical care and appropriate genetic counseling.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

PubMed Central

Henze, Anne-Theres; Garvalov, Boyan K.; Seidel, Sascha; Cuesta, Angel M.; Ritter, Mathias; Filatova, Alina; Foss, Franziska; Dopeso, Higinio; Essmann, Clara L.; Maxwell, Patrick H.; Reifenberger, Guido; Carmeliet, Peter; Acker-Palmer, Amparo; Acker, Till

2014-01-01

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability. PMID:25420773

Syringyl-rich lignin renders poplars more resistant to degradation by wood decay fungi.

PubMed

Skyba, Oleksandr; Douglas, Carl J; Mansfield, Shawn D

2013-04-01

In order to elucidate the effects of lignin composition on the resistance of wood to degradation by decay fungi, wood specimens from two transgenic poplar lines expressing an Arabidopsis gene encoding ferulate 5-hydroxylase (F5H) driven by the cinnimate-4-hydroxylase promoter (C4H::F5H) that increased syringyl/guaiacyl (S/G) monolignol ratios relative to those in the untransformed control wood were incubated with six different wood decay fungi. Alterations in wood weight and chemical composition were monitored over the incubation period. The results showed that transgenic poplar lines extremely rich in syringyl lignin exhibited a drastically improved resistance to degradation by all decay fungi evaluated. Lignin monomer composition and its distribution among cell types and within different cell layers were the sole wood chemistry parameters determining wood durability. Since transgenic poplars with exceedingly high syringyl contents were recalcitrant to degradation, where wood durability is a critical factor, these genotypes may offer improved performance.

Syringyl-Rich Lignin Renders Poplars More Resistant to Degradation by Wood Decay Fungi

PubMed Central

Skyba, Oleksandr; Douglas, Carl J.

2013-01-01

In order to elucidate the effects of lignin composition on the resistance of wood to degradation by decay fungi, wood specimens from two transgenic poplar lines expressing an Arabidopsis gene encoding ferulate 5-hydroxylase (F5H) driven by the cinnimate-4-hydroxylase promoter (C4H::F5H) that increased syringyl/guaiacyl (S/G) monolignol ratios relative to those in the untransformed control wood were incubated with six different wood decay fungi. Alterations in wood weight and chemical composition were monitored over the incubation period. The results showed that transgenic poplar lines extremely rich in syringyl lignin exhibited a drastically improved resistance to degradation by all decay fungi evaluated. Lignin monomer composition and its distribution among cell types and within different cell layers were the sole wood chemistry parameters determining wood durability. Since transgenic poplars with exceedingly high syringyl contents were recalcitrant to degradation, where wood durability is a critical factor, these genotypes may offer improved performance. PMID:23396333

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.

PubMed

Mitchell, Andrew J; Dunham, Noah P; Martinie, Ryan J; Bergman, Jonathan A; Pollock, Christopher J; Hu, Kai; Allen, Benjamin D; Chang, Wei-Chen; Silakov, Alexey; Bollinger, J Martin; Krebs, Carsten; Boal, Amie K

2017-10-04

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

PubMed

Sim, Jingwei; Cowburn, Andrew S; Palazon, Asis; Madhu, Basetti; Tyrakis, Petros A; Macías, David; Bargiela, David M; Pietsch, Sandra; Gralla, Michael; Evans, Colin E; Kittipassorn, Thaksaon; Chey, Yu C J; Branco, Cristina M; Rundqvist, Helene; Peet, Daniel J; Johnson, Randall S

2018-04-03

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Electrochemical Hydroxylation of C3-C12 n-Alkanes by Recombinant Alkane Hydroxylase (AlkB) and Rubredoxin-2 (AlkG) from Pseudomonas putida GPo1.

PubMed

Tsai, Yi-Fang; Luo, Wen-I; Chang, Jen-Lin; Chang, Chun-Wei; Chuang, Huai-Chun; Ramu, Ravirala; Wei, Guor-Tzo; Zen, Jyh-Myng; Yu, Steve S-F

2017-08-21

An unprecedented method for the efficient conversion of C 3 -C 12 linear alkanes to their corresponding primary alcohols mediated by the membrane-bound alkane hydroxylase (AlkB) from Pseudomonas putida GPo1 is demonstrated. The X-ray absorption spectroscopy (XAS) studies support that electrons can be transferred from the reduced AlkG (rubredoxin-2, the redox partner of AlkB) to AlkB in a two-phase manner. Based on this observation, an approach for the electrocatalytic conversion from alkanes to alcohols mediated by AlkB using an AlkG immobilized screen-printed carbon electrode (SPCE) is developed. The framework distortion of AlkB-AlkG adduct on SPCE surface might create promiscuity toward gaseous substrates. Hence, small alkanes including propane and n-butane can be accommodated in the hydrophobic pocket of AlkB for C-H bond activation. The proof of concept herein advances the development of artificial C-H bond activation catalysts.

Methotrexate in Atherogenesis and Cholesterol Metabolism

PubMed Central

Coomes, Eric; Chan, Edwin S. L.; Reiss, Allison B.

2011-01-01

Methotrexate is a disease-modifying antirheumatic drug commonly used to treat inflammatory conditions such as rheumatoid arthritis which itself is linked to increased cardiovascular risk. Treatments that target inflammation may also impact the cardiovascular system. While methotrexate improves cardiovascular risk, inhibition of the cyclooxygenase (COX)-2 enzyme promotes atherosclerosis. These opposing cardiovascular influences may arise from differing effects on the expression of proteins involved in cholesterol homeostasis. These proteins, ATP-binding cassette transporter (ABC) A1 and cholesterol 27-hydroxylase, facilitate cellular cholesterol efflux and defend against cholesterol overload. Methotrexate upregulates expression of cholesterol 27-hydroxylase and ABCA1 via adenosine release, while COX-2 inhibition downregulates these proteins. Adenosine, acting through the A2A and A3 receptors, may upregulate proteins involved in reverse cholesterol transport by cAMP-PKA-CREB activation and STAT inhibition, respectively. Elucidating underlying cardiovascular mechanisms of these drugs provides a framework for developing novel cardioprotective anti-inflammatory medications, such as selective A2A receptor agonists. PMID:21490773

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

5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation

PubMed Central

Aik, WeiShen; Che, Ka Hing; Li, Xuan Shirley; Kristensen, Jan B. L.; King, Oliver N. F.; Chan, Mun Chiang; Yeoh, Kar Kheng; Choi, Hwanho; Walport, Louise J.; Thinnes, Cyrille C.; Bush, Jacob T.; Lejeune, Clarisse; Rydzik, Anna M.; Rose, Nathan R.; Bagg, Eleanor A.; McDonough, Michael A.; Krojer, Tobias; Yue, Wyatt W.; Ng, Stanley S.; Olsen, Lars; Brennan, Paul E.; Oppermann, Udo; Muller-Knapp, Susanne; Klose, Robert J.; Ratcliffe, Peter J.; Schofield, Christopher J.; Kawamura, Akane

2015-01-01

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement PMID:26682036

Association study of the tryptophan hydroxylase gene and bipolar affective disorder using family-based internal controls.

PubMed

Rietschel, M; Schorr, A; Albus, M; Franzek, E; Kreiner, R; Held, T; Knapp, M; Müller, D J; Schulze, T G; Propping, P; Maier, W; Nöthen, M M

2000-06-12

The tryptophan hydroxylase (TPH) gene encodes for the rate-limiting enzyme of the serotonin metabolism and, therefore, has to be considered a major candidate for association studies in affective disorders. Recently, an association between this gene and bipolar affective disorder has been reported in a French population. We sought to replicate this finding in a German sample. Allele frequencies of a biallelic polymorphism (A218C) of the TPH gene were determined in 95 bipolar I patients and their parents. Preferential transmission of alleles from heterozygous parents to bipolar offspring was tested with the "transmission disequilibrium test" (TDT), which eliminates the contribution of population stratification to an association finding. Our sample yielded a power >90% to detect the originally reported effect. Neither allele 218A nor allele 218C were preferentially transmitted from heterozygous parents to bipolar offspring. Our results, therefore, do not support the hypothesis that the TPH gene is involved in the etiology of bipolar disorder.

Activity of the sympathetic-adrenomedullary system in rats after space flight on the COSMOS biosatellites

NASA Astrophysics Data System (ADS)

Kvetňanský, R.; Vigaš, M.; Németh, Š.; Macho, L.; Tigranyan, R. A.

The indicators of adrenomedullary activity (catecholamine content (CA) and the activity of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH)) were measured in the adrenal glands of rats living in a state of weightlessness for 18.5-19.5 days on board the biosatellites COSMOS 936 and COSMOS 1129. None of these indicators was significantly changed by space flight, neither in the group living in a state of weightlessness nor in the group living in a centrifuge on board the spacecraft and exposed to artificial gravity of 1 g (COSMOS 936). Animals exposed after space flight to repeated immobilization stress on Earth showed a significant decrease of adrenal adrenaline and an appreciable increase in adrenal TH activity compared to stressed animals which were not in space. These results suggest that a prolonged state of weightlessness during space flight does not by itself represent an intensive stressful stimulus for the adrenomedullary system but potentiates the response of cosmonauts to stress after return to Earth.

NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward.

PubMed

Logan, Ryan W; Parekh, Puja K; Kaplan, Gabrielle N; Becker-Krail, Darius D; Williams, Wilbur P; Yamaguchi, Shintaro; Yoshino, Jun; Shelton, Micah A; Zhu, Xiyu; Zhang, Hui; Waplinger, Spencer; Fitzgerald, Ethan; Oliver-Smith, Jeffrey; Sundarvelu, Poornima; Enwright, John F; Huang, Yanhua H; McClung, Colleen A

2018-05-04

The diurnal regulation of dopamine is important for normal physiology and diseases such as addiction. Here we find a novel role for the CLOCK protein to antagonize CREB-mediated transcriptional activity at the tyrosine hydroxylase (TH) promoter, which is mediated by the interaction with the metabolic sensing protein, Sirtuin 1 (SIRT1). Additionally, we demonstrate that the transcriptional activity of TH is modulated by the cellular redox state, and daily rhythms of redox balance in the ventral tegmental area (VTA), along with TH transcription, are highly disrupted following chronic cocaine administration. Furthermore, CLOCK and SIRT1 are important for regulating cocaine reward and dopaminergic (DAergic) activity, with interesting differences depending on whether DAergic activity is in a heightened state and if there is a functional CLOCK protein. Taken together, we find that rhythms in cellular metabolism and circadian proteins work together to regulate dopamine synthesis and the reward value for drugs of abuse.

Salicylate and catechol levels are maintained in nahG transgenic poplar

Treesearch

Alison M. Morse; Timothy J. Tschaplinski; Christopher Dervinis; Paula M. Pijut; Eric A. Schmelz; Wendy Day; John M. Davis

2007-01-01

Metabolic profiling was used to investigate the molecular phenotypes of a transgenic Populus tremula × P. alba hybrid expressing the nahG transgene, a bacterial gene encoding salicylate hydroxylase that converts salicylic acid to catechol. Despite the efficacy of this transgenic approach to reduce...

Bioinorganic Chemical Modeling of Dioxygen-Activating Copper Proteins.

ERIC Educational Resources Information Center

Karlin, Kenneth D.; Gultneh, Yilma

1985-01-01

Discusses studies done in modeling the copper centers in the proteins hemocyanin (a dioxygen carrier), tyrosinase, and dopamine beta-hydroxylase. Copper proteins, model approach in copper bioinorganic chemistry, characterization of reversible oxygen carriers and dioxygen-metal complexes, a copper mono-oxygenase model reaction, and other topics are…

The ventrolateral medulla and medullary raphe in sudden unexpected death in epilepsy.

PubMed

Patodia, Smriti; Somani, Alyma; O'Hare, Megan; Venkateswaran, Ranjana; Liu, Joan; Michalak, Zuzanna; Ellis, Matthew; Scheffer, Ingrid E; Diehl, Beate; Sisodiya, Sanjay M; Thom, Maria

2018-06-01

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of premature death in patients with epilepsy. One hypothesis proposes that sudden death is mediated by post-ictal central respiratory depression, which could relate to underlying pathology in key respiratory nuclei and/or their neuromodulators. Our aim was to investigate neuronal populations in the ventrolateral medulla (which includes the putative human pre-Bötzinger complex) and the medullary raphe. Forty brainstems were studied comprising four groups: 14 SUDEP, six epilepsy controls, seven Dravet syndrome cases and 13 non-epilepsy controls. Serial sections through the medulla (from obex 1 to 10 mm) were stained for Nissl, somatostatin, neurokinin 1 receptor (for pre-Bötzinger complex neurons) and galanin, tryptophan hydroxylase and serotonin transporter (neuromodulatory systems). Using stereology total neuronal number and densities, with respect to obex level, were measured. Whole slide scanning image analysis was used to quantify immunolabelling indices as well as co-localization between markers. Significant findings included reduction in somatostatin neurons and neurokinin 1 receptor labelling in the ventrolateral medulla in sudden death in epilepsy compared to controls (P < 0.05). Galanin and tryptophan hydroxylase labelling was also reduced in sudden death cases and more significantly in the ventrolateral medulla region than the raphe (P < 0.005 and P < 0.05). With serotonin transporter, reduction in labelling in cases of sudden death in epilepsy was noted only in the raphe (P ≤ 0.01); however, co-localization with tryptophan hydroxylase was significantly reduced in the ventrolateral medulla. Epilepsy controls and cases with Dravet syndrome showed less significant alterations with differences from non-epilepsy controls noted only for somatostatin in the ventrolateral medulla (P < 0.05). Variations in labelling with respect to obex level were noted of potential relevance to the rostro-caudal organization of respiratory nuclear groups, including tryptophan hydroxylase, where the greatest statistical difference noted between all epilepsy cases and controls was at obex 9-10 mm (P = 0.034), the putative level of the pre-Bötzinger complex. Furthermore, there was evidence for variation with duration of epilepsy for somatostatin and neurokinin 1 receptor. Our findings suggest alteration to neuronal populations in the medulla in SUDEP with evidence for greater reduction in neuromodulatory neuropeptidergic and mono-aminergic systems, including for galanin, and serotonin. Other nuclei need to be investigated to evaluate if this is part of more widespread brainstem pathology. Our findings could be a result of previous seizures and may represent a pathological risk factor for SUDEP through impaired respiratory homeostasis during a seizure.

The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium.

PubMed Central

Morandini, P; Offer, J; Traynor, D; Nayler, O; Neuhaus, D; Taylor, G W; Kay, R R

1995-01-01

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development. Images Figure 3 Figure 4 PMID:7702568

Maltreatment, Conscience Functioning and Dopamine Beta Hydroxylase in Emotionally Disturbed Boys.

ERIC Educational Resources Information Center

Galvin, Matthew R.; And Others

1997-01-01

Nineteen hospitalized, emotionally disturbed boys screened for maltreatment either before or after age 3 were compared with a normal control group for enzyme activity and conscience functions in moral valuation. Subjects who experienced early maltreatment had more developmental delays and more interferences with conscience functions than other…

76 FR 82310 - Cardiovascular and Renal Drugs Advisory Committee; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-30

... (Parkinson's Disease, Multiple System Atrophy, and Pure Autonomic Failure), Dopamine Beta-Hydroxylase... recommendations to the Agency on FDA's regulatory issues. Date and Time: The meeting will be held on February 23... Colesville Rd., Silver Spring, MD 20910. The hotel's telephone number is (301) 589-5200. Contact Person...

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.

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.

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.

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.

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.

Evolution of Enzymes Required for Biodegradation of Pentachlorophenol in Sphingobium Chlorophenolicum

DTIC Science & Technology

2007-06-15

1883-1888. 3 Schopfer, L. M.; Massey, V. "Kinetic and mechanistic studies on the oxidation of the melilotate hydroxylase-2-OH- cinnamate complex by...del Cardayré. 2002. Genome shuffling of Lactobacillus for improved acid tolerance. Nature Biotechnol. 20: 707-712. 14 Zhang, Y.-X., K. Perry, V

Defense Response in Slash Pine: Chitosan Treatment Alters the Abundance of Specific mRNAs

Treesearch

Mary E. Mason; John M. Davis

1997-01-01

We used differential display to identify chitosan responsive cDNAs in slashpine cell cultures. Two clones that showed increased mRNA abundance had sequence similarity to genes with roles in major plant defense responses, clone 18 to cinnamic acid 4-hydroxylase, and clone 30 to chitinase.

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.

Infant formula feeding increases hepatic cholesterol 7 alpha hydroxylase (CYP7A1) expression and fecal bile acid loss in neonatal piglets

USDA-ARS?s Scientific Manuscript database

During the postnatal feeding period, formula-fed infants have higher cholesterol synthesis rates, and lower circulating cholesterol concentrations as compared to their breastfed counterparts. Although this disparity has been attributed to the uniformly low dietary cholesterol content of typical inf...

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.

The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

PubMed

Schuster, I

1985-06-01

Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

NASA Technical Reports Server (NTRS)

Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

2003-01-01

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

Caudal dysplasia sequence with penile enlargement: case report and a potential pathogenic hypothesis.

PubMed

Makhoul, I R; Aviram-Goldring, A; Paperna, T; Sujov, P; Rienstein, S; Smolkin, T; Epelman, M; Gershoni-Baruch, R

2001-02-15

The clinical spectrum of caudal dysplasia sequence (CDS) is noted for its diversity. The origin of CDS remains unknown, though poorly controlled gestational diabetes has been implicated in some cases. Here we describe the case of a newborn with CDS associated with penile enlargement (PE). The main anomalies included anal atresia, agenesis of the kidneys and of the sacrococcygeal vertebrae, dysgenesis of lumbar vertebrae, and bilateral cryptorchidism. Penile enlargement (7 cm), a rather unusual finding, has so far not been reported in association with CDS. Chromosomal analysis failed, and the neonate died 30 min after birth. Comparative genomic hybridization analysis using stored DNA showed a balanced normal male DNA content, which negates chromosomal losses or gains as a cause of CDS and/or PE. PE due to virilizing-type adrenal hyperplasia, caused by common mutations in the genes encoding for the adrenal enzymes 21-hydroxylase and 11-hydroxylase, was ruled out. We report on a previously unpublished case of the coexistence of PE and severe CDS and propose a possible pathogenetic hypothesis of this association. Copyright Wiley-Liss. Inc.

Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

PubMed

Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

2014-05-16

Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

A study of the epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison's disease

PubMed Central

Volpato, M; Prentice, L; Chen, S; Betterle, C; Rees Smith, B; Furmaniak, J

1998-01-01

Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH. PMID:9486414

Metabolism of 2,4-dichlorophenol in tobacco engineered with bacterial degradative genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkins, E.J.; Sekine, M.; Gordon, M.P.

1990-05-01

The potential use of plants in toxic waste remediation has been overlooked. While chlorophenols are relatively slowly metabolized in Nicotiana tabacum var. Xanthi leaf extracts, chlorocatechols are rapidly metabolized, presumably by polyphenol oxidases. Our initial focus has been the fate of 2,4-dichlorophenol (2,4DCP) in var. Xanthi plants which express a bacterial 2,4DCP hydroxylase, which converts 2,4DCP to 3,5-dichlorocatechol. The roots of wild type and 2,4DCP hydroxylase transgenic plants growing in hydroponics were exposed to {sup 14}C-2,4DCP. Approximately 95% of {sup 14}C-2,4DCP metabolites remained in the roots when exposed to 2,4DCP. Upon extraction of root tissue, three major metabolites were foundmore » in untransformed plants and four major metabolites in transformed plants. Upon digestion with beta-D-glucosidase, these metabolites disappeared concomitant with the appearance of free 2,4DCP in wild type plants and 2,4DCP and 3,5-dichlorocatechol in transgenic plants. It is apparent that the chlorophenols are not readily available substrates for polyphenol oxidases in whole plants.« less

Steroidal 5α-reductase and 17α-hydroxylase/17,20-lyase (CYP17) inhibitors useful in the treatment of prostatic diseases.

PubMed

Salvador, Jorge A R; Pinto, Rui M A; Silvestre, Samuel M

2013-09-01

The role of steroidal inhibitors of androgen biosynthesis as potential weapons in the treatment of prostatic diseases, such as benign prostatic hyperplasia and prostatic cancer will be reviewed. Two enzymes have been targeted in the development of inhibitors that potentially could be useful in the management of such conditions. 5α-Reductase is primarily of interest in benign prostatic disease, though some role in the chemoprevention of prostatic carcinoma have been considered, whereas the 17α-hydroxylase/17,20-lyase (CYP17) enzyme is of interest in the treatment of malignant disease. An overview of the main achievements obtained during the past years will be presented, however special focus will be made on steroidal molecules that reached clinical trials or have been commercially launched. Relevant examples of such drugs are finasteride, dutasteride, abiraterone acetate and galeterone (TOK-001, formerly known as VN/124-1). This article is part of a Special Issue entitled "Synthesis and biological testing of steroid derivatives as inhibitors". Copyright © 2013 Elsevier Ltd. All rights reserved.

PKCδ inhibition enhances tyrosine hydroxylase phosphorylation in mice after methamphetamine treatment

PubMed Central

Nguyen, Xuan-Khanh Thi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean L.; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2014-01-01

The present study was designed to evaluate the specific role of protein kinase C (PKC) δ in methamphetamine (MA)-induced dopaminergic toxicity. A multiple-dose administration regimen of MA significantly increases PKCδ expression, while rottlerin, a PKCδ inhibitor, significantly attenuates MA-induced hyperthermia and behavioural deficits. These behavioural effects were not significantly observed in PKCδ antisense oligonucleotide (ASO)-treated- or PKCδ knockout (−/−)-mice. There were no MA-induced significant decreases of dopamine (DA) content or tyrosine hydroxylase (TH) expression in the striatum in rottlerin-treated-, ASO-treated- or PKCδ (−/−)-mice. The administration of MA also results in a significant decrease of TH phosphorylation at ser 40, but not ser 31, while the inhibition of PKCδ consistently and significantly attenuates MA-induced reduction in the phosphorylation of TH at ser 40. Therefore, these results suggest that the MA-induced enhancement of PKCδ expression is a critical factor in the impairment of TH phosphorylation at ser 40 and that pharmacological or genetic inhibition of PKCδ may be protective against MA-induced dopaminergic neurotoxicity in vivo. PMID:21672585

Discovery of novel 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid derivatives as HIF prolyl hydroxylase inhibitors for treatment of renal anemia.

PubMed

Hamada, Makoto; Takayama, Tetsuo; Shibata, Tsuyoshi; Hiratate, Akira; Takahashi, Masato; Yashiro, Miyoko; Takayama, Noriko; Okumura-Kitajima, Lisa; Koretsune, Hiroko; Kajiyama, Hiromitsu; Naruse, Takumi; Kato, Sota; Takano, Hiroki; Kakinuma, Hiroyuki

2018-06-01

Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3-5.6 h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

PubMed

Gelband, C H; Sumners, C; Lu, D; Raizada, M K

1997-10-31

The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

PubMed

Gelband, C H; Sumners, C; Lu, D; Raizada, M K

1998-02-27

The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

Dopamine-Secreting Paraganglioma in the Retroperitoneum.

PubMed

Matsuda, Yusuke; Kimura, Noriko; Yoshimoto, Takanobu; Sekiguchi, Yoshihiro; Tomoishi, Junzo; Kasahara, Ichiro; Hara, Yoshihito; Ogawa, Yoshihiro

2017-03-01

Pheochromocytomas and paragangliomas, which exclusively produce dopamine, are very rare. Herein, we report for the first time a Japanese case of an exclusively dopamine-producing paraganglioma accompanied by detailed immunohistochemical analyses. A 70-year-old Japanese woman was referred to our hospital for functional examination of her left retroperitoneal mass. Her adrenal functions were normal, except for excessive dopamine secretion. After the tumorectomy, her dopamine level normalized. The histopathological diagnosis of the tumor was paraganglioma; this was confirmed by positive immunostaining of chromogranin A (CgA), tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and succinate dehydrogenase gene subunit B (SDHB). However, the immunostaining of CgA in the tumor cells showed peculiar dot-like staining located corresponding to Golgi complex in the perinuclear area, rather than the diffuse cytoplasmic staining usually observed in epinephrine- or norepinephrine-producing functional pheochromocytomas and paragangliomas. The immunohistochemical results suggested that the tumor cells had sparse neuroendocrine granules in the cytoplasm, resulting in inhibition of catecholamine synthesis from dopamine to norepinephrine in neurosecretory granules. This may be the mechanism responsible for exclusive dopamine secretion in the present case.

[Congenital adrenal hyperplasia due to lack of 17α-hydroxylase: a report of a new mutation in the gene CYP17A1].

PubMed

Perales Martínez, J I; Pina Marqués, B; de Arriba Muñoz, A; Mayayo Dehesa, E; Labarta Aizpún, J I; Loidi Fernández, L

2015-01-01

P450c17 enzyme catalyses two different reactions: the 17α-hydroxylation of progesterone and pregnenolone, and segmenting the carbon 17-20 binding from the 17,20lyase producing adrenal androgens. This enzyme is coded by the CYP17A1 gene. The case is presented of a 14 year old patient with delayed pubertal development and a high blood pressure for height and age. 46,XX karyotype. Hormonal studies highlighted hypergonadotropic hypogonadism, adrenal insufficiency and mineralocorticoid excess. Subsequent genetic studies showed a homozygous mutation in the CYP17A1 gene (c.753+G>A), not previously described, which is responsible for the pathophysiology of 17α-hydroxylase deficiency. This entity is a rare form of congenital adrenal hyperplasia. The disease often goes unnoticed until adolescence or early adult life, and should be suspected in 46,XY individuals with ambiguous genitalia or 46,XX with delayed puberty associated with hypertension and/or hypokalaemia. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Long-term follow-up of a female with congenital adrenal hyperplasia due to P450-oxidoreductase deficiency.

PubMed

Bonamichi, Beatriz D S F; Santiago, Stella L M; Bertola, Débora R; Kim, Chong A; Alonso, Nivaldo; Mendonca, Berenice B; Bachega, Tania A S S; Gomes, Larissa G

2016-10-01

P450 oxidoreductase deficiency (PORD) is a variant of congenital adrenal hyperplasia that is caused by POR gene mutations. The POR gene encodes a flavor protein that transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 type II (including 21-hydroxylase, 17α-hydroxylase 17,20 lyase and aromatase), which is fundamental for their enzymatic activity. POR mutations cause variable impairments in steroidogenic enzyme activities that result in wide phenotypic variability ranging from 46,XX or 46,XY disorders of sexual differentiation, glucocorticoid deficiency, with or without skeletal malformations similar to Antley-Bixler syndrome to asymptomatic newborns diagnosed during neonatal screening test. Little is known about the PORD long-term evolution. We described a 46,XX patient with mild atypical genitalia associated with severe bone malformation, who was diagnosed after 13 years due to sexual infantilism. She developed large ovarian cysts and late onset adrenal insufficiency during follow-up, both of each regressed after hormone replacement therapies. We also described a late surgical approach for the correction of facial hypoplasia in a POR patient.

Seventeen Alpha-Hydroxylase Deficiency Associated with Absent Gonads and Myelolipoma: A Case Report and Review of Literature.

PubMed

Soveid, Mahmood; Rais-Jalali, Ghanbar Ali

2016-11-01

Congenital adrenal hyperplasia comprises a group of disorders resulting from defects in enzymes required for the synthesis of cortisol. The clinical presentation depends on the specific enzyme defect. We report a rare case of congenital adrenal hyperplasia due to 17 alpha-hydroxylase deficiency. A 26-year-old female patient referred with hypertension and hypokalemia. She also had primary amenorrhea and lack of sexual development. The karyotype was 46, XY. Hormonal evaluation showed low serum levels of all steroid hormones, requiring alpha-hydroxylation, which included cortisol, 17 alpha-hydroxy progesterone, dehydroepiandrosterone sulfate, estradiol, and testosterone. The levels of adrenocorticotropic, follicle-stimulating, and luteinizing hormones were high. Radiological and surgical investigations failed to show a gonad. She also had a large myelolipoma. Treatment was commenced with low-dose dexamethasone and conjugated estrogen. Her hypertension and hypokalemia were resolved. The myelolipoma was removed by laparoscopy due to pain and sensation of heaviness. Our review of literature revealed that a combination of this disorder with either agonadism or myelolipoma is very rare and that only 2 previous cases have been reported for each entity.

Metabolic Engineering of Escherichia coli for Producing Astaxanthin as the Predominant Carotenoid

PubMed Central

Lu, Qian; Bu, Yi-Fan; Liu, Jian-Zhong

2017-01-01

Astaxanthin is a carotenoid of significant commercial value due to its superior antioxidant potential and wide applications in the aquaculture, food, cosmetic and pharmaceutical industries. A higher ratio of astaxanthin to the total carotenoids is required for efficient astaxanthin production. β-Carotene ketolase and hydroxylase play important roles in astaxanthin production. We first compared the conversion efficiency to astaxanthin in several β-carotene ketolases from Brevundimonas sp. SD212, Sphingomonas sp. DC18, Paracoccus sp. PC1, P. sp. N81106 and Chlamydomonas reinhardtii with the recombinant Escherichia coli cells that synthesize zeaxanthin due to the presence of the Pantoea ananatis crtEBIYZ. The B. sp. SD212 crtW and P. ananatis crtZ genes are the best combination for astaxanthin production. After balancing the activities of β-carotene ketolase and hydroxylase, an E. coli ASTA-1 that carries neither a plasmid nor an antibiotic marker was constructed to produce astaxanthin as the predominant carotenoid (96.6%) with a specific content of 7.4 ± 0.3 mg/g DCW without an addition of inducer. PMID:28937591

Click Chemistry-based Discovery of [3-Hydroxy-5-(1H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of Anemia.

PubMed

Wu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qi-Dong; Zhang, Xiaojin

2018-06-01

As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assay. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.

Enriched environment protects the nigrostriatal dopaminergic system and induces astroglial reaction in the 6-OHDA rat model of Parkinson's disease.

PubMed

Anastasía, Agustín; Torre, Luciana; de Erausquin, Gabriel A; Mascó, Daniel H

2009-05-01

Enriched environment (EE) is neuroprotective in several animal models of neurodegeneration. It stimulates the expression of trophic factors and modifies the astrocyte cell population which has been said to exert neuroprotective effects. We have investigated the effects of EE on 6-hydroxydopamine (6-OHDA)-induced neuronal death after unilateral administration to the medial forebrain bundle, which reaches 85-95% of dopaminergic neurons in the substantia nigra after 3 weeks. Continuous exposure to EE 3 weeks before and after 6-OHDA injection prevents neuronal death (assessed by tyrosine hydroxylase staining), protects the nigrostriatal pathway (assessed by Fluorogold retrograde labeling) and reduces motor impairment. Four days after 6-OHDA injection, EE was associated with a marked increase in glial fibrillary acidic protein staining and prevented neuronal death (assessed by Fluoro Jade-B) but not partial loss of tyrosine hydroxylase staining in the anterior substantia nigra. These results robustly demonstrate that EE preserves the entire nigrostriatal system against 6-OHDA-induced toxicity, and suggests that an early post-lesion astrocytic reaction may participate in the neuroprotective mechanism.

Polymorphism of the Tryptophan Hydroxylase 2 (TPH2) Gene Is Associated with Chimpanzee Neuroticism

PubMed Central

Morimura, Naruki; Udono, Toshifumi; Hayasaka, Ikuo; Humle, Tatyana; Murayama, Yuichi; Ito, Shin'ichi; Inoue-Murayama, Miho

2011-01-01

In the brain, serotonin production is controlled by tryptophan hydroxylase 2 (TPH2), a genotype. Previous studies found that mutations on the TPH2 locus in humans were associated with depression and studies of mice and studies of rhesus macaques have shown that the TPH2 locus was involved with aggressive behavior. We previously reported a functional single nucleotide polymorphism (SNP) in the form of an amino acid substitution, Q468R, in the chimpanzee TPH2 gene coding region. In the present study we tested whether this SNP was associated with neuroticism in captive and wild-born chimpanzees living in Japan and Guinea, respectively. Even after correcting for multiple tests (Bonferroni p = 0.05/6 = 0.008), Q468R was significantly related to higher neuroticism (β = 0.372, p = 0.005). This study is the first to identify a genotype linked to a personality trait in chimpanzees. In light of the prior studies on humans, mice, and rhesus macaques, these findings suggest that the relationship between neuroticism and TPH2 has deep phylogenetic roots. PMID:21765945

A giant ovarian cyst in a neonate with classical 21-hydroxylase deficiency with very high testosterone levels demonstrating a high-dose hook effect.

PubMed

Güran, Tülay; Yeşil, Gözde; Güran, Ömer; Cesur, Suna; Bosnalı, Oktav; Celayir, Ayşenur; Topçuoğlu, Sevilay; Bereket, Abdullah

2012-09-01

Congenital adrenal hyperplasia (CAH) is a group of disorders affecting the adrenal steroid synthesis. The most common form, 21-hydroxylase deficiency (21-OHD), leads to decreased production of cortisol and aldosterone with increased androgen secretion. In classic CAH, glucocorticoid treatment can be life-saving and serves to bring the symptoms under control. However, the treatment challenge is to effectively control the excess androgen effect by using the lowest possible glucocorticoid dose. Previous studies suggested a relationship between ovarian cyst formation and adrenal androgen excess, but neonatal large ovarian cysts have been very rarely reported in newborns with CAH. Here, we present the unique case of a neonate with classical 21-OHD who underwent surgery for a giant (10x8x7 cm) unilateral solitary ovarian follicular cyst on the 2nd postnatal day. Hormonal evaluation of the patient revealed high-dose hook effect for serum testosterone levels for the first time by a two-site immunoradiometric assay. Possible mechanisms by which androgen excess may cause ovarian cyst formation are discussed.

Metabolic Engineering of Wheat Provitamin A by Simultaneously Overexpressing CrtB and Silencing Carotenoid Hydroxylase (TaHYD).

PubMed

Zeng, Jian; Wang, Xiatian; Miao, Yingjie; Wang, Cheng; Zang, Mingli; Chen, Xi; Li, Miao; Li, Xiaoyan; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2015-10-21

Increasing the provitamin A content in staple crops via carotenoid metabolic engineering is one way to address vitamin A deficiency. In this work a combination of methods was applied to specifically increase β-carotene content in wheat by metabolic engineering. Endosperm-specific silencing of the carotenoid hydroxylase gene (TaHYD) increased β-carotene content 10.5-fold to 1.76 μg g(-1) in wheat endosperm. Overexpression of CrtB introduced an additional flux into wheat, accompanied by a β-carotene increase of 14.6-fold to 2.45 μg g(-1). When the "push strategy" (overexpressing CrtB) and "block strategy" (silencing TaHYD) were combined in wheat metabolic engineering, significant levels of β-carotene accumulation were obtained, corresponding to an increase of up to 31-fold to 5.06 μg g(-1). This is the first example of successful metabolic engineering to specifically improve β-carotene content in wheat endosperm through a combination of methods and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat.

Phenylalanine hydroxylase deficiency in Mexico: genotype-phenotype correlations, BH4 responsiveness and evidence of a founder effect.

PubMed

Vela-Amieva, M; Abreu-González, M; González-del Angel, A; Ibarra-González, I; Fernández-Lainez, C; Barrientos-Ríos, R; Monroy-Santoyo, S; Guillén-López, S; Alcántara-Ortigoza, M A

2015-07-01

The mutational spectrum of the phenylalanine hydroxylase gene (PAH) in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype-phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype-phenotype correlations and genotype-based prediction of BH4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5G>T being the most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1A>T and c.969+6T>C. The genotype-phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH4 testing and therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transient inhibition of connective tissue infiltration and collagen deposition into porous poly(lactic-co-glycolic acid) discs.

PubMed

Love, Ryan J; Jones, Kim S

2013-12-01

Connective tissue rapidly proliferates on and around biomaterials implanted in vivo, which impairs the function of the engineered tissues, biosensors, and devices. Glucocorticoids can be utilized to suppress tissue ingrowth, but can only be used for a limited time because they nonselectively arrest cell proliferation in the local environment. The present study examined use of a prolyl-4-hydroxylase inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), to suppress connective tissue ingrowth in porous PLGA discs implanted in the peritoneal cavity for 28 days. The prolyl-4-hydroxylase inhibitor was found to be effective at inhibiting collagen deposition within and on the outer surface of the disc, and also limited connective tissue ingrowth, but not to the extent of glucocorticoid inhibition. Finally, it was discovered that 1,4-DPCA suppressed Scavenger Receptor A expression on a macrophage-like cell culture, which may account for the drug's ability to limit connective tissue ingrowth in vivo. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Neurotoxicity of cerebro-spinal fluid from patients with Parkinson's disease on mesencephalic primary cultures as an in vitro model of dopaminergic neurons.

PubMed

Kong, Ping; Zhang, Ben-Shu; Lei, Ping; Kong, Xiao-Dong; Zhang, Shi-Shuang; Li, Dai; Zhang, Yun

2015-08-01

Parkinson's disease is a degenerative disorder of the central nervous system. In spite of extensive research, neither the cause nor the mechanisms have been firmly established thus far. One assumption is that certain toxic substances may exist in the cerebro-spinal fluid (CSF) of Parkinson's disease patients. To confirm the neurotoxicity of CSF and study the potential correlation between neurotoxicity and the severity of Parkinson's disease, CSF was added to cultured cells. By observation of cell morphology, changes in the levels of lactate dehydrogenase, the ratio of tyrosine hydroxylase-positive cells, and the expression of tyrosine hydroxylase mRNA and protein, the differences between the two groups were shown. The created in vitro model of dopaminergic neurons using primary culture of mouse embryonic mesencephalic tissue is suitable for the study of neurotoxicity. The observations of the present study indicated that CSF from Parkinson's disease patients contains factors that can cause specific injury to cultured dopaminergic neurons. However, no obvious correlation was found between the neurotoxicity of CSF and the severity of Parkinson's disease.

High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase.

PubMed

Bordeaux, Mélanie; de Girval, Diane; Rullaud, Robin; Subileau, Maeva; Dubreucq, Eric; Drone, Jullien

2014-01-01

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.

A novel integration system of magnetically immobilized cells and a pair of graphite plate-stainless iron mesh electrodes for the bioremediation of coking wastewater.

PubMed

Jiang, Bei; Tan, Liang; Ning, Shuxiang; Shi, Shengnan

2016-09-01

Magnetically immobilized cells of Comamonas sp. JB coupling with electrode reaction was developed to enhance the treatment efficiency of coking wastewater containing phenol, carbazole (CA), dibenzofuran (DBF), and dibenzothiophene (DBT). The pair of graphite plate-stainless iron mesh electrodes was chosen as the most suitable electrodes. Magnetically immobilized cells coupling with graphite plate-stainless iron mesh electrodes (coupling system) exhibited high degradation activity for all the compounds, which were significantly higher than the sum by single magnetically immobilized cells and electrode reaction at the optimal voltage. Recycling experiments demonstrated that the degradation activity of coupling system increased gradually during eight recycles, indicating that there was a coupling effect between the biodegradation and electrode reaction. Phenol hydroxylase and qPCR assays confirmed that appropriate electrical stimulation could improve phenol hydroxylase activity and promote cells growth. Toxicity assessment suggested the treatment of the coking wastewater by coupling system led to less toxicity than untreated wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

Morphological, biochemical, and histopathological indices and contaminant burdens of cotton rats (Sigmodon hispidus) at three hazardous waste sites near Houston, Texas, USA

USGS Publications Warehouse

Rattner, B.A.; Flickinger, Edward L.; Hoffman, D.J.

1993-01-01

Male cotton rats (Sigmodon hispidus) were studied at three industrial waste sites near Houston, Texas, to determine whether various morphological, biochemical, and histopathological indices provided evidence of contaminant exposure and toxic insult. Only modest changes were detected in cotton rats residing at waste sites compared with reference sites. No single parameter was consistently altered, except hepatic cytochrome P-450 concentration which was lower ( [Formula: see text] ) at two waste sites, and tended to be lower ( [Formula: see text] ) at a third waste site. Elevated petroleum hydrocarbon concentrations were detected in rats at one waste site, but contaminant burdens of rats from the other sites were unremarkable. Unlike rats captured in summer, those trapped in winter exhibited hepatocellular hypertrophy and up to a 65% increase in liver: body weight ratio, cytochrome P-450 concentration, and activities of aniline hydroxylase, aryl hydrocarbon hydroxylase, and glutathione S-transferase. Although genotoxicity has been previously documented in cotton rats residing at two of the waste sites, biomarkers in the present study provided little evidence of exposure and damage

Post-transcriptional Regulation of Tyrosine Hydroxylase Expression in Adrenal Medulla and Brain

PubMed Central

Tank, A. William; Xu, Lu; Chen, Xiqun; Radcliffe, Pheona; Sterling, Carol R.

2009-01-01

It is well-established that long-term stress leads to induction of tyrosine hydroxylase (TH) mRNA and TH protein in adrenal medulla and brain. This induction is usually associated with stimulation of TH gene transcription rate. However, a number of studies have reported major discrepancies between the stress-induced changes in TH gene transcription, TH mRNA and TH protein. These discrepancies suggest that post-transcriptional mechanisms also play an important role in regulating TH expression in response to stress and other stimuli. In this report we summarize some of our findings and literature reports that demonstrate these discrepancies in adrenal medulla, locus coeruleus and midbrain dopamine neurons. We then describe our recent work investigating the molecular mechanisms that mediate this post-transcriptional regulation in adrenal medulla and midbrain. Our results suggest that trans-acting factors binding to the polypyrimidine-rich region of the 3′UTR of TH mRNA play a role in these post-transcriptional mechanisms. A hypothetical cellular model describing this post-transcriptional regulation is proposed. PMID:19120116

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen*

PubMed Central

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A.; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M.; Marini, Joan C.; Yamauchi, Mitsuo

2016-01-01

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

DIFFERENTIAL POSTPARTUM SENSITIVITY TO THE ANXIETY-MODULATING EFFECTS OF OFFSPRING CONTACT IS ASSOCIATED WITH INNATE ANXIETY AND BRAINSTEM LEVELS OF DOPAMINE BETA-HYDROXYLASE IN FEMALE LABORATORY RATS

PubMed Central

RAGAN, C. M.; LONSTEIN, J. S.

2014-01-01

In female mammals, the postpartum period involves dramatic shifts in many socioemotional behaviors. This includes a suppression of anxiety-related behaviors that requires recent physical contact with offspring. Factors contributing to differences among females in their susceptibility to the anxiety-modulating effect of offspring contact are unknown, but could include their innate anxiety and brain monoaminergic activity. Anxiety behavior was assessed in a large group of nulliparous female rats and the least-anxious and most-anxious tertiles were mated. Anxiety was assessed again postpartum after females were permitted or prevented from contacting their offspring 4 h before testing. Levels of dopamine β-hydroxylase (DBH, norepinephrine synthesizing enzyme) and tryptophan hydroxylase- 2 (TPH2, serotonin synthesizing enzyme) were measured in the brainstem and dorsal raphe, respectively. It was found that anxiety-related behavior in the two groups did not differ when dams were permitted contact with offspring before testing. Removal of the offspring before testing, however, differentially affected anxiety based on dams’ innate anxiety. Specifically, dams reverted back to their pre-mating levels of anxiety such that offspring removal slightly increased anxiety in the most-anxious females but greatly lowered anxiety in the least-anxious females. This reduction in anxiety in the least-anxious females after litter removal was associated with lower brainstem DBH. There was no relationship between females’ anxiety and dorsal raphe TPH2. Thus, a primary effect of recent contact with offspring on anxiety-related behavior in postpartum rats is to shift females away from their innate anxiety to a more moderate level of responding. This effect is particularly true for females with the lowest anxiety, may be mediated by central noradrenergic systems, and has implications for their ability to attend to their offspring. PMID:24161285

Fatty acid ω-hydroxylases from Solanum tuberosum.

PubMed

Bjelica, Anica; Haggitt, Meghan L; Woolfson, Kathlyn N; Lee, Daniel P N; Makhzoum, Abdullah B; Bernards, Mark A

2016-12-01

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1. Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

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

PubMed

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

2009-04-01

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

Double Knockdown of Prolyyl Hydroxylase and Factor Inhibiting HIF with Non-Viral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

PubMed Central

Huang, Mei; Nguyen, Patricia; Jia, Fangjun; Hu, Shijun; Gong, Yongquan; de Almeida, Patricia E.; Wang, Li; Nag, Divya; Kay, Mark A.; Giaccia, Amato J; Robbins, Robert C.; Wu, Joseph C.

2011-01-01

Background Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation may be an effective therapeutic option for ischemic heart failure. Methods and Results PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double knockdown group. Fluorescence activated cell sorting (FACS) showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser capture microdissection (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively. Conclusions We demonstrated that HIF-1α up-regulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. PMID:21911818

The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

PubMed Central

Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

2012-01-01

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

Systemic administration of the propargylamine CGP 3466B prevents behavioural and morphological deficits in rats with 6-hydroxydopamine-induced lesions in the substantia nigra.

PubMed

Andringa, G; van Oosten, R V; Unger, W; Hafmans, T G; Veening, J; Stoof, J C; Cools, A R

2000-08-01

The ability of CGP 3466B to attenuate the behavioural and morphological consequences of experimentally induced cell death was investigated in a recently updated animal model of Parkinson's disease. 6-Hydroxydopamine was infused bilaterally into the substantia nigra pars compacta of rats that were pretreated with desimipramine. Treatment with CGP 3466B (0.0014-1.4 mg/kg, injected subcutaneously) or its solvent was begun 2 h after the 6-OHDA injection, and maintained twice daily for 14 days. After a washout period of 14 days, changes in motor behaviour were evaluated, using the open field test (analysis of normal and abnormal stepping, e.g.) and the paw test (analysis of retraction time of limbs). Changes in learning and memory were evaluated with the help of the Morris water maze task. Following immunocytochemical staining of tyrosine hydroxylase, the extent of the lesion was quantified using a computerized system. CGP 3466B prevented all deficits produced by 6-hydroxydopamine (6-OHDA), though at different doses. It prevented: abnormal stepping (0.0014-0.014 mg/kg); increased forelimb and hindlimb retraction time (0.014-0.14 mg/kg and 0.0014-0.14 mg/kg, respectively); delayed learning (1.4 mg/kg); and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra (0.0014-0.014 mg/kg). CGP 3466B (0.0014-0.14 mg/kg) induced no deficits in sham-treated rats. CGP 3466B (1.4 mg/kg), however, did not show any benefit on motor deficits in 6-OHDA-lesioned rats, and induced abnormal movements and decreased the tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and the ventral tegmental area of sham-lesioned animals. It is concluded that CGP 3466B prevents all 6-OHDA-induced behavioural and immunocytochemical deficits, though at different doses. CGP 3466B is suggested to be a valuable agent for inhibiting the dopaminergic degeneration in patients with Parkinson's disease.

1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gan, L.S.L.; Acebo, A.L.; Alworth, W.L.

The preparation of 1-ethynylpyrene (EP) by incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products briefly. Addition of microsomes restores the original rate. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in those from phenobarbital (PB) pretreated rats or controls. Ep inhibits the hydroxylation of benzo(a)pyrene (BP) by liver microsomes. Ep more effectively inhibits the oxidation of BP in liver microsomes from BF rats than from PB rats or from controls. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparentlymore » irreversible. Kinetic analyses show that the inhibition of BP hydroxylation is due to loss of the activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. A self-catalyzed inhibition of the cytochrome P-450 dependent BP hydroxylation may occur in the presence of EP. Incubation with EP under conditions that result in loss of BP hydroxylase activity in microsomes from BF rats and 66% of the activity from PB rats causes the loss of 6 and 12% of the cytochrome P-450, respectively. Thus the loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. Selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content. In vivo EP does not cause a significant change in the cytochrome P-450 content in the microsomes isolated, or result in the change in BP hydroxylation.« less

A missense mutation in the human cytochrome b5 gene causes 46,XY disorder of sex development due to true isolated 17,20 lyase deficiency.

PubMed

Idkowiak, Jan; Randell, Tabitha; Dhir, Vivek; Patel, Pushpa; Shackleton, Cedric H L; Taylor, Norman F; Krone, Nils; Arlt, Wiebke

2012-03-01

Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations.

Vitamin D-Mediated Hypercalcemia: Mechanisms, Diagnosis, and Treatment

PubMed Central

Tebben, Peter J.; Singh, Ravinder J.

2016-01-01

Hypercalcemia occurs in up to 4% of the population in association with malignancy, primary hyperparathyroidism, ingestion of excessive calcium and/or vitamin D, ectopic production of 1,25-dihydroxyvitamin D [1,25(OH)2D], and impaired degradation of 1,25(OH)2D. The ingestion of excessive amounts of vitamin D3 (or vitamin D2) results in hypercalcemia and hypercalciuria due to the formation of supraphysiological amounts of 25-hydroxyvitamin D [25(OH)D] that bind to the vitamin D receptor, albeit with lower affinity than the active form of the vitamin, 1,25(OH)2D, and the formation of 5,6-trans 25(OH)D, which binds to the vitamin D receptor more tightly than 25(OH)D. In patients with granulomatous disease such as sarcoidosis or tuberculosis and tumors such as lymphomas, hypercalcemia occurs as a result of the activity of ectopic 25(OH)D-1-hydroxylase (CYP27B1) expressed in macrophages or tumor cells and the formation of excessive amounts of 1,25(OH)2D. Recent work has identified a novel cause of non-PTH-mediated hypercalcemia that occurs when the degradation of 1,25(OH)2D is impaired as a result of mutations of the 1,25(OH)2D-24-hydroxylase cytochrome P450 (CYP24A1). Patients with biallelic and, in some instances, monoallelic mutations of the CYP24A1 gene have elevated serum calcium concentrations associated with elevated serum 1,25(OH)2D, suppressed PTH concentrations, hypercalciuria, nephrocalcinosis, nephrolithiasis, and on occasion, reduced bone density. Of interest, first-time calcium renal stone formers have elevated 1,25(OH)2D and evidence of impaired 24-hydroxylase-mediated 1,25(OH)2D degradation. We will describe the biochemical processes associated with the synthesis and degradation of various vitamin D metabolites, the clinical features of the vitamin D-mediated hypercalcemia, their biochemical diagnosis, and treatment. PMID:27588937

Tetrodotoxin-insensitive electrical field stimulation-induced contractions on Crotalus durissus terrificus corpus cavernosum.

PubMed

Campos, Rafael; Mónica, Fabíola Z; Rodrigues, Renata Lopes; Rojas-Moscoso, Julio Alejandro; Moreno, Ronilson Agnaldo; Cogo, José Carlos; de Oliveira, Marco Antonio; Antunes, Edson; De Nucci, Gilberto

2017-01-01

Reptiles are the first amniotes to develop an intromitent penis, however until now the mechanisms involved in the electrical field stimulation-induced contraction on corpora cavernosa isolated from Crotalus durissus terrificus were not investigated. Crotalus and rabbit corpora cavernosa were mounted in 10 mL organ baths for isometric tension recording. Electrical field stimulation (EFS)-induced contractions were performed in presence/absence of phentolamine (10 μM), guanethidine (30 μM), tetrodotoxin (1 μM and 1mM), A-803467 (10 μM), 3-iodo-L-Tyrosine (1 mM), salsolinol (3 μM) and a modified Krebs solution (equimolar substitution of NaCl by N-methyl-D-glucamine). Immuno-histochemistry for tyrosine hydroxylase was also performed. Electrical field stimulation (EFS; 8 Hz and 16 Hz) caused contractions in both Crotalus and rabbit corpora cavernosa. The contractions were abolished by previous incubation with either phentolamine or guanethidine. Tetrodotoxin (1 μM) also abolished the EFS-induced contractions of rabbit CC, but did not affect EFS-induced contractions of Crotalus CC. Addition of A-803467 (10 μM) did not change the EFS-induced contractions of Crotalus CC but abolished rabbit CC contractions. 3-iodo-L-Tyrosine and salsolinol had no effect on EFS-induced contractions of Crotalus CC and Rabbit CC. Replacement of NaCl by N- Methyl-D-glucamine (NMDG) abolished EFS-induced contractions of rabbit CC, but did not affect Crotalus CC. The presence of tyrosine hydroxylase was identified in endothelial cells only of Crotalus CC. Since the EFS-induced contractions of Crotalus CC is dependent on catecholamine release, insensitive to TTX, insensitive to A803467 and to NaCl replacement, it indicates that the source of cathecolamine is unlikely to be from adrenergic terminals. The finding that tyrosine hydroxylase is present in endothelial cells suggests that these cells can modulate Crotalus CC tone.

Four-Week Studies of Oral Hypoxia-Inducible Factor–Prolyl Hydroxylase Inhibitor GSK1278863 for Treatment of Anemia

PubMed Central

Meadowcroft, Amy M.; Maier, Rayma; Johnson, Brendan M.; Jones, Delyth; Rastogi, Anjay; Zeig, Steven; Lepore, John J.; Cobitz, Alexander R.

2016-01-01

Hypoxia-inducible factor prolyl hydroxylase inhibitors stabilize levels of hypoxia-inducible factor that upregulate transcription of multiple genes associated with the response to hypoxia, including production of erythropoietin. We conducted two phase 2a studies to explore the relationship between the dose of the hypoxia-inducible factor–prolyl hydroxylase inhibitor GSK1278863 and hemoglobin response in patients with anemia of CKD (baseline hemoglobin 8.5–11.0 g/dl) not undergoing dialysis and not receiving recombinant human erythropoietin (nondialysis study) and in patients with anemia of CKD (baseline hemoglobin 9.5–12.0 g/dl) on hemodialysis and being treated with stable doses of recombinant human erythropoietin (hemodialysis study). Participants were randomized 1:1:1:1 to a once-daily oral dose of GSK1278863 (0.5 mg, 2 mg, or 5 mg) or control (placebo for the nondialysis study; continuing on recombinant human erythropoietin for the hemodialysis study) for 4 weeks, with a 2-week follow-up. In the nondialysis study, GSK1278863 produced dose-dependent effects on hemoglobin, with the highest dose resulting in a mean increase of 1 g/dl at week 4. In the hemodialysis study, treatment with GSK1278863 in the 5-mg arm maintained mean hemoglobin concentrations after the switch from recombinant human erythropoietin, whereas mean hemoglobin decreased in the lower-dose arms. In both studies, the effects on hemoglobin occurred with elevations in endogenous erythropoietin within the range usually observed in the respective populations and markedly lower than those in the recombinant human erythropoietin control arm in the hemodialysis study, and without clinically significant elevations in plasma vascular endothelial growth factor concentrations. GSK1278863 was generally safe and well tolerated at the doses and duration studied. GSK1278863 may prove an effective alternative for managing anemia of CKD. PMID:26494831

Disruption of Mouse Cytochrome P450 4f14 (Cyp4f14 Gene) Causes Severe Perturbations in Vitamin E Metabolism*

PubMed Central

Bardowell, Sabrina A.; Duan, Faping; Manor, Danny; Swanson, Joy E.; Parker, Robert S.

2012-01-01

Vitamin E is a family of naturally occurring and structurally related lipophilic antioxidants, one of which, α-tocopherol (α-TOH), selectively accumulates in vertebrate tissues. The ω-hydroxylase cytochrome P450–4F2 (CYP4F2) is the only human enzyme shown to metabolize vitamin E. Using cDNA cloning, cell culture expression, and activity assays, we identified Cyp4f14 as a functional murine ortholog of CYP4F2. We then investigated the effect of Cyp4f14 deletion on vitamin E metabolism and status in vivo. Cyp4f14-null mice exhibited substrate-specific reductions in liver microsomal vitamin E-ω-hydroxylase activity ranging from 93% (γ-TOH) to 48% (γ-tocotrienol). In vivo data obtained from metabolic cage studies showed whole-body reductions in metabolism of γ-TOH of 90% and of 68% for δ- and α-TOH. This metabolic deficit in Cyp4f14−/− mice was partially offset by increased fecal excretion of nonmetabolized tocopherols and of novel ω-1- and ω-2-hydroxytocopherols. 12′-OH-γ-TOH represented 41% of whole-body production of γ-TOH metabolites in Cyp4f14−/− mice fed a soybean oil diet. Despite these counterbalancing mechanisms, Cyp4f14-null mice fed this diet for 6 weeks hyper-accumulated γ-TOH (2-fold increase over wild-type littermates) in all tissues and appeared normal. We conclude that CYP4F14 is the major but not the only vitamin E-ω-hydroxylase in mice. Its disruption significantly impairs whole-body vitamin E metabolism and alters the widely conserved phenotype of preferential tissue deposition of α-TOH. This model animal and its derivatives will be valuable in determining the biological actions of specific tocopherols and tocotrienols in vivo. PMID:22665481

S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanjun; Zhao, Li; Zhao, Jiangzhe

The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant 6, At5g24530) has been proven essential in plant immunity of Arabidopsis, but its biochemical properties are not well understood. Here in this paper, we report the discovery and functional characterization of DMR6 as a SA 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBAmore » by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K cat/K m=4.96×10 4 M -1s -1) than the previously reported SA 3-hydroxylase (S3H, K cat/K m=6.09 × 10 3 M -1s -1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant over accumulate SA and display phenotypes such as a smaller growth size, early senescence and a loss of susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). S5H/DMR6 is sensitively induced by SA/pathogen treatment and is widely expressed from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.« less

Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas.

PubMed

Fallo, F; Pezzi, V; Barzon, L; Mulatero, P; Veglio, F; Sonino, N; Mathis, J M

2002-12-01

The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

PubMed

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

2016-04-29

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis

DOE PAGES

Zhang, Yanjun; Zhao, Li; Zhao, Jiangzhe; ...

2017-09-12

The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant 6, At5g24530) has been proven essential in plant immunity of Arabidopsis, but its biochemical properties are not well understood. Here in this paper, we report the discovery and functional characterization of DMR6 as a SA 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBAmore » by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K cat/K m=4.96×10 4 M -1s -1) than the previously reported SA 3-hydroxylase (S3H, K cat/K m=6.09 × 10 3 M -1s -1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant over accumulate SA and display phenotypes such as a smaller growth size, early senescence and a loss of susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). S5H/DMR6 is sensitively induced by SA/pathogen treatment and is widely expressed from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.« less

Nogo-receptor 1 antagonization in combination with neurotrophin-4/5 is not superior to single factor treatment in promoting survival and morphological complexity of cultured dopaminergic neurons.

PubMed

Seiler, Stefanie; Di Santo, Stefano; Sahli, Sebastian; Andereggen, Lukas; Widmer, Hans Rudolf

2017-08-01

Cell transplantation using ventral mesencephalic tissue is an experimental approach to treat Parkinson's disease. This approach is limited by poor survival of the transplants and the high number of dopaminergic neurons needed for grafting. Increasing the yield of dopaminergic neurons in donor tissue is of great importance. We have previously shown that antagonization of the Nogo-receptor 1 by NEP1-40 promoted survival of cultured dopaminergic neurons and exposure to neurotrophin-4/5 increased dopaminergic cell densities in organotypic midbrain cultures. We investigated whether a combination of both treatments offers a novel tool to further improve dopaminergic neuron survival. Rat embryonic ventral mesencephalic neurons grown as organotypic free-floating roller tube or primary dissociated cultures were exposed to neurotrophin-4/5 and NEP1-40. The combined and single factor treatment resulted in significantly higher numbers of tyrosine hydroxylase positive neurons compared to controls. Significantly stronger tyrosine hydroxylase signal intensity was detected by Western blotting in the combination-treated cultures compared to controls but not compared to single factor treatments. Neurotrophin-4/5 and the combined treatment showed significantly higher signals for the neuronal marker microtubule-associated protein 2 in Western blots compared to control while no effects were observed for the astroglial marker glial fibrillary acidic protein between groups, suggesting that neurotrophin-4/5 targets mainly neuronal cells. Finally, NEP1-40 and the combined treatment significantly augmented tyrosine hydroxylase positive neurite length. Summarizing, our findings substantiate that antagonization of the Nogo-receptor 1 promotes dopaminergic neurons but does not further increase the yield of dopaminergic neurons and their morphological complexity when combined with neurotrophin-4/5 hinting to the idea that these treatments might exert their effects by activating common downstream pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

PubMed

Majdi, Mohammad; Abdollahi, Mohammad Reza; Maroufi, Asad

2015-11-01

Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

Reconstructive surgery for females with congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a review from the Prince of Wales Hospital.

PubMed

Houben, C H; Tsui, S Y; Mou, J W; Chan, K W; Tam, Y H; Lee, K H

2014-12-01

To present the results of feminising genitoplasty done in female patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Case series. A tertiary referral centre in Hong Kong. Female patients with congenital adrenal hyperplasia undergoing corrective surgery for virilisation between 1993 and 2012. The operative result was judged with a scoring system (1-3) for four areas: appearance of clitoris, labia and vagina, plus requirement for revision surgery. A total of 23 female patients with congenital adrenal hyperplasia with a median age of 17.5 (range, 1.5-33.8) years were identified. Of these individuals, 17 presented in the neonatal period and early infancy, of which four had an additional salt-losing crisis. Six patients-including four migrants from mainland China-were late presenters at a median age of 2 (range, 0.5-14) years. Twenty-two patients had corrective surgery at a median age of 2 (range, 1-14) years. Clitoral reduction was performed in all, and further surgery in 21 patients. The additional surgery was flap vaginoplasty in 10 patients, a modified Passerini procedure in six, and a labial reconstruction in five; one patient with prominent clitoris was for observation only. Minor revision surgery (eg mucosal trimming) was required in three patients; a revision vaginoplasty was done in one individual. Of the 23 patients, 18 (78%) with a median age of 20 (range, 9.3-33.8) years participated in the outcome evaluation: a 'good' outcome (4 points) was seen in 12 patients and a 'satisfactory' (5-9 points) result in five patients. Nearly three quarters of our cohort (n=17) presented with classic virilising form of 21-hydroxylase deficiency. Only four (25%) patients experienced a salt-losing crisis. Female gender assignment at birth was maintained for all individuals in this group. 'Good' and 'satisfactory' outcomes of surgery were reported in nearly all participants.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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]. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnicker, Nicholas J.; Razzaghi, Mortezaali; Guha Thakurta, Sanjukta

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant,more » and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.« less

Developmental nicotine exposure affects larval brain size and the adult dopaminergic system of Drosophila melanogaster.

PubMed

Morris, Melanie; Shaw, Ariel; Lambert, Madison; Perry, Haley Halperin; Lowenstein, Eve; Valenzuela, David; Velazquez-Ulloa, Norma Andrea

2018-06-14

Pregnant women may be exposed to nicotine if they smoke or use tobacco products, nicotine replacement therapy, or via e-cigarettes. Prenatal nicotine exposure has been shown to have deleterious effects on the nervous system in mammals including changes in brain size and in the dopaminergic system. The genetic and molecular mechanisms for these changes are not well understood. A Drosophila melanogaster model for these effects of nicotine exposure could contribute to faster identification of genes and molecular pathways underlying these effects. The purpose of this study was to determine if developmental nicotine exposure affects the nervous system of Drosophila melanogaster, focusing on changes to brain size and the dopaminergic system at two developmental stages. We reared flies on control or nicotine food from egg to 3rd instar larvae or from egg to adult and determined effectiveness of the nicotine treatment. We used immunohistochemistry to visualize the whole brain and dopaminergic neurons, using tyrosine hydroxylase as the marker. We measured brain area, tyrosine hydroxylase fluorescence, and counted the number of dopaminergic neurons in brain clusters. We detected an increase in larval brain hemisphere area, a decrease in tyrosine hydroxylase fluorescence in adult central brains, and a decrease in the number of neurons in the PPM3 adult dopaminergic cluster. We tested involvement of Dα7, one of the nicotinic acetylcholine receptor subunits, and found it was involved in eclosion, as previously described, but not involved in brain size. We conclude that developmental nicotine exposure in Drosophila melanogaster affects brain size and the dopaminergic system. Prenatal nicotine exposure in mammals has also been shown to have effects on brain size and in the dopaminergic system. This study further establishes Drosophila melanogaster as model organism to study the effects of developmental nicotine exposure. The genetic and molecular tools available for Drosophila research will allow elucidation of the mechanisms underlying the effects of nicotine exposure during development.

Recommendations for Treatment of Nonclassic Congenital Adrenal Hyperplasia (NCCAH): an Update

PubMed Central

Trapp, Christine M.; Oberfield, Sharon E.

2013-01-01

Congenital adrenal hyperplasia (CAH) is a family of autosomal recessive disorders. 21-hydroxylase deficiency, in which there are mutations in CYP21A2 (the gene encoding the adrenal 21-hydroxylase enzyme), is the most common form (90%) of CAH. In classic CAH there is impaired cortisol production with diagnostic increased levels of 17-OH progesterone. Excess androgen production results in virilization and in the newborn female may cause development of ambiguous external genitalia. Three-fourths of patients with classic CAH also have aldosterone insufficiency, which can result in salt-wasting; in infancy this manifests as shock, hyponatremia and hyperkalemia. CAH has a reported incidence of 1:10,000–1:20,000 births although there is an increased prevalence in certain ethnic groups. Nonclassic CAH (NCCAH) is a less severe form of the disorder, in which there is 20–50% of 21-hydroxylase enzyme activity (vs. 0–5% in classic CAH) and no salt wasting. The degree of symptoms related to androgen excess is variable and may be progressive with age, although some individuals are asymptomatic. NCCAH has an incidence of 1:1000–1:2000 births (0.1–0.2% prevalence) in the White population; an even higher prevalence is noted in certain ethnic groups such as Ashkenazi Jews (1–2%). As many as two-thirds of persons with NCCAH are compound heterozygotes and carry a severe and mild mutation on different alleles. This paper discusses the genetics of NCCAH, along with its variable phenotypic expression, and reviews the clinical course in untreated patients, which includes rapid early childhood growth, advanced skeletal age, premature adrenarche, acne, impaired reproductive function in both sexes and hirsutism as well as menstrual disorders in females. Finally, it addresses treatment with glucocorticoids vs. and other alternatives, particularly with respect to long term issues such as adult metabolic disease including insulin resistance, cardiovascular disease, metabolic syndrome, and bone mineral density. PMID:22186144

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

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum

PubMed Central

2010-01-01

Background Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. Results The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. Conclusions CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism. PMID:20128892

Interactions between Brainstem Noradrenergic Neurons and the Nucleus Accumbens Shell in Modulating Memory for Emotionally Arousing Events

ERIC Educational Resources Information Center

Kerfoot, Erin C.; Williams, Cedric L.

2011-01-01

The nucleus accumbens shell (NAC) receives axons containing dopamine-[beta]-hydroxylase that originate from brainstem neurons in the nucleus of the solitary tract (NTS). Recent findings show that memory enhancement produced by stimulating NTS neurons after learning may involve interactions with the NAC. However, it is unclear whether these…

Neurofibromin and Neuronal Apoptosis

DTIC Science & Technology

2006-07-01

role of familial pheochromocytoma genes, including succinate dehydrogenase (SDH) and Nf1, in modulating neuronal apoptosis following neurotrophin...gene products, in Nf1-/- sensory and sympathetic neurons; this work will also have relevance to the biology of familial pheochromocytoma . "So what...Schlisio, S. (2005). Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: Developmental culling and cancer. Cancer

Supression of the steroid-primed luteinizing hormone surge in the female rat by sodium dimethyldithiocarbamate: Relationship to hypothalamic catecholamines and GnRH neuronal activation

EPA Science Inventory

In female rodents, hypothalamic norepinephrine (NE) has a role in stimulating the secretion of gonadotropin-releasing hormone (GnRH) that triggers the ovulatory surge of luteinizing hormone (LH). NE synthesis from dopamine requires the presence of dopamine--hydroxylase (DH) an...

Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

USDA-ARS?s Scientific Manuscript database

Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

Identification of Two Suppressors of CSG2 Calcium Sensitivity, SCS7 and SUR2, as Genes Encoding Hydroxylases of the Sphingolipid Biosynthetic Pathway of Saccharomyces cerevisiae

DTIC Science & Technology

1997-12-10

process can adjusted by experimental alteration of thyroid status. Hyperthyroidism speeds up the time table while hypothyroidism retards it. When 88... canine kidney MDCK cells. The relative amounts ofthe labeled products can be differentially recovered from the basolateral and apical surfaces of these

Exhaustive Analysis of BH4 and Dopamine Biosynthesis Genes in Patients with Dopa-Responsive Dystonia

ERIC Educational Resources Information Center

Clot, Fabienne; Grabli, David; Cazeneuve, Cecile; Roze, Emmanuel; Castelnau, Pierre; Chabrol, Brigitte; Landrieu, Pierre; Nguyen, Karine; Ponsot, Gerard; Abada, Myriem; Doummar, Diane; Damier, Philippe; Gil, Roger; Thobois, Stephane; Ward, Alana J.; Hutchinson, Michael; Toutain, Annick; Picard, Fabienne; Camuzat, Agnes; Fedirko, Estelle; San, Chankannira; Bouteiller, Delphine; LeGuern, Eric; Durr, Alexandra; Vidailhet, Marie; Brice, Alexis

2009-01-01

Dopa-responsive dystonia is a childhood-onset dystonic disorder, characterized by a dramatic response to low dose of L-Dopa. Dopa-responsive dystonia is mostly caused by autosomal dominant mutations in the "GCH1" gene (GTP cyclohydrolase1) and more rarely by autosomal recessive mutations in the "TH" (tyrosine hydroxylase) or "SPR" (sepiapterin…

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, Charles L.; Thilly, William G.

1985-01-01

A line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity) is disclosed. Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. Mutation assays using these cells, and other cells with similar characteristics, are also disclosed.

Effects of early cholesterol intake on cholesterol 7 alpha hydroxylase (Cyp7a1) expression in piglets receiving sow's breast milk or infant formula until weaning

USDA-ARS?s Scientific Manuscript database

Unlike breast milk, infant formulas are not rich in cholesterol. To compensate for the dietary loss, hepatic cholesterol synthesis is increased in formula-fed infants. Observational studies have reported significant increases in serum cholesterol and triglycerides in adults that received formula dur...

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

PubMed

Vedler, Eve; Heinaru, Eeva; Jutkina, Jekaterina; Viggor, Signe; Koressaar, Triinu; Remm, Maido; Heinaru, Ain

2013-12-01

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees. Copyright © 2013 Elsevier GmbH. All rights reserved.

Seventeen {alpha}-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshiro, Chikara; Takasu, Nobuyuki; Wakugami, Tamio

1995-08-01

Mutation of the cytochrome P450c17 (CYP17) gene causes 17{alpha}-hydroxylase deficiency (170HD). Recently, several researchers have elucidated the molecular basis of 170HD by gene analysis. We experienced a case of 170HD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 170HD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. Anmore » indentical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient`s clinical manifestations of 170HD. This is the first case of 170HD with only one base pair deletion of the CYP17 gene. 18 refs., 3 figs.« less

Utility of a precursor-to-product ratio in the evaluation of presumptive positives in newborn screening of congenital adrenal hyperplasia.

PubMed

Tieh, P Y; Yee, J K; Hicks, R A; Mao, C S; Lee, W-Np

2017-03-01

Screening for congenital adrenal hyperplasia (CAH) caused by 21-α-hydroxylase deficiency is challenging because factors such as prematurity and stress increase intermediate steroid metabolite levels in newborn infants. The objective of this study was to explore the use of the 17-α-hydroxyprogesterone (17-OHP)/11-deoxycortisol ratio as an adjunct measure in the follow-up evaluation of infants with presumptive positive newborn screens for CAH to distinguish between infants with no disorder and those with CAH. This was a retrospective cohort study of infants with presumptive positive newborn screens for CAH. The precursor-to-product ratio of 17-OHP/11-deoxycortisol was compared between infants with no disorder (n=47) and infants with CAH (n=5). The CAH infants had higher 17-OHP/11-deoxycortisol ratios than infants with no disorder: 26 (18 to 58) and 1.05 (0.69 to 1.46), respectively (P<0.05). Among infants with no disorder, higher levels of serum 17-OHP did not reflect higher ratios, indicating sufficient enzyme activity. The results suggest that a low 17-OHP/11-deoxycortisol ratio represents 21-α-hydroxylase sufficiency among presumptive positives in newborn screening of CAH.

Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair.

PubMed

Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

2014-01-01

Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair.

Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

PubMed

Tong, Zongyong; Li, Heng; Zhang, Rongxue; Ma, Lei; Dong, Jiangli; Wang, Tao

2015-10-01

Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

Prenatal ethanol exposure alters steroidogenic enzyme activity in newborn rat testes.

PubMed

Kelce, W R; Rudeen, P K; Ganjam, V K

1989-10-01

We have examined the in utero effects of ethanol exposure on testicular steroidogenesis in newborn male pups. Pregnant Sprague-Dawley rats were fed a liquid ethanol diet (35% ethanol-derived calories), a pair-fed isocaloric liquid diet, or a standard laboratory rat chow and water diet beginning on Day 12 of gestation and continuing through parturition. Although there were no significant differences in the enzymatic activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase or C17,20-lyase, the enzymatic activity of 17 alpha-hydroxylase was significantly (p less than 0.01) reduced (i.e., approximately 36%) in the ethanol-exposed pups compared to those from the pair-fed and chow treatment groups. This lesion in testicular steroidogenic enzyme activity in newborn male pups exposed to alcohol in utero was transient as 17 alpha-hydroxylase activity from the ethanol-exposed animals returned to control levels by postnatal Day 20 and remained at control levels through adulthood (postnatal Day 60). These data suggest that the suppression of the perinatal testosterone surge in male rats exposed to alcohol in utero and the associated long term demasculinizing effects of prenatal ethanol exposure might be the result of reduced testicular steroidogenic enzyme activity in the perinatal animal.

Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.

PubMed

Kerscher, B; Nzukou, E; Kaiser, A

2010-02-01

Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Watt, Stephen A.; Dayal, Jasbani H. S.; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R.; Kimble, Roy M.; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W. H. Irwin; Leigh, Irene M.; McGrath, John A.; Salas-Alanis, Julio C.; Tolar, Jakub; South, Andrew P.

2015-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

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

Genetic engineering of novel bluer-colored chrysanthemums produced by accumulation of delphinidin-based anthocyanins.

PubMed

Noda, Naonobu; Aida, Ryutaro; Kishimoto, Sanae; Ishiguro, Kanako; Fukuchi-Mizutani, Masako; Tanaka, Yoshikazu; Ohmiya, Akemi

2013-10-01

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

Synergistic effects of melatonin and deprenyl against MPTP-induced mitochondrial damage and DA depletion.

PubMed

Khaldy, Hoda; Escames, Germaine; León, Josefa; Bikjdaouene, Leila; Acuña-Castroviejo, Darío

2003-01-01

Previous studies showed a synergistic effect of melatonin and deprenyl against dopamine (DA) autoxidation in vitro. Since oxidative stress is implicated in Parkinson's disease (PD), we explored the effects of melatonin plus deprenyl administration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in C57/Bl6 mice. Melatonin, but not deprenyl prevents the inhibition of mitochondrial complex I and the oxidative damage in nigrostriatal neurons induced by MPTP. With the dose used deprenyl recovers 50% DA levels and tyrosine hydroxylase activity depressed by the neurotoxin, normalizing locomotor activity of mice. Melatonin, which was unable to counteract MPTP-induced DA depletion and inhibition of tyrosine hydroxylase activity, potentiates the effect of deprenyl on catecholamine turnover and mice ambulatory activity. These results suggest a dissociation of complex I inhibition from DA depletion in this model of Parkinson's disease. The data also support that a combination of melatonin, which improves mitochondrial electron transport chain and reduces oxidative damage, and deprenyl, which promotes the specific function of the rescued neurons, i.e. DA turnover, may be a promising strategy for the treatment of PD.

Evidence for involvement of protein kinases in the regulation of serotonin synthesis and turnover in the mouse brain in vivo.

PubMed

Stenfors, C; Ross, S B

2002-11-01

Inhibition of cAMP-dependent protein kinase (PKA) with N-[2-methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) almost completely antagonized the increase in 5-HTP accumulation and 5-HIAA/5-HT ratio in hypothalamus induced by NAS-181, a 5-HT(1B) receptor antagonist, but had no effect when the mice were treated with NAS-181 together with WAY-100,635, a selective 5-HT(1A) receptor antagonist. Inhibition of Ca(2+)-calmodulin-dependent protein kinase (CaM kinase II) with the calmodulin antagonist N-(4-aminobutyl)-5-chloro-2-naphtalenesulfonamide (W-13) did not antagonise the effect of NAS-181 alone, but counteracted that evoked by the combined treatment with NAS-181 and WAY-100,635. The results indicate that activation of tryptophan hydroxylase by reducing the tone from terminal 5-HT(1B) receptors involves PKA whereas the depolarisation-induced activation of tryptophan hydroxylase involves CaM kinase II. The increase in the 5-HIAA/5-HT ratio may under the experimental conditions used suggest CaM kinase II-induced phosphorylation of synapsin I resulting in increased 5-HT release.

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

PubMed Central

Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.; Bourassa, Megan W.; Sleiman, Sama F.; John, Roseleen; Thinnes, Cyrille C.; Yeh, Tzu-Lan; Demetriades, Marina; Neitemeier, Sandra; Cruz, Dana; Gazaryan, Irina; Killilea, David W.; Morgenstern, Lewis; Xi, Guohua; Keep, Richard F.; Schallert, Timothy; Tappero, Ryan V.; Zhong, Jian; Cho, Sunghee; Maxfield, Frederick R.; Holman, Theodore R.; Culmsee, Carsten; Fong, Guo-Hua; Su, Yijing; Ming, Guo-li; Song, Hongjun; Cave, John W.; Schofield, Christopher J.; Colbourne, Frederick; Coppola, Giovanni; Ratan, Rajiv R.

2017-01-01

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models. PMID:26936506

L-Arogenate is a chemoattractant which can be utilized as the sole source of carbon and nitrogen by Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fischer, R.S.; Song, Jian; Gu, Wei

L-Arogenate is a commonplace amino acid in nature in consideration of its role as a ubiquitous precursor of L-phenylalanine and/or L-tyrosine. However, the questions of whether it serves as a chemoattractant molecule and whether it can serve as a substrate for catabolism have never been studied. We found that Pseudomonas aeruginosa recognizes L-arogenate as a chemoattractant molecule which can be utilized as a source of both carbon and nitrogen. Mutants lacking expression of either cyclohexadienyl dehydratase or phenylalanine hydroxylase exhibited highly reduced growth rates when utilizing L-arogenate as a nitrogen source. Utilization of L-arogenate as a source of either carbonmore » or nitrogen was dependent upon {sub S}{sup 54}, as revealed by the use of an rpoN null mutant. The evidence suggests that catabolism of L-arogenate proceeds via alternative pathways which converge at 4-hydroxyphenylpyruvate. In one pathway, prephenate formed in the periplasm by deamination of L-arogenate is converted to 4-hydroxyphenylpyruvate by cyclohexadienyl dehydrogenase. The second route depends upon the sequential action of periplasmic cyclohexadienyl dehydratase, phenylalanine hydroxylase, and aromatic aminotransferase. 32 refs., 5 figs., 4 tabs.« less

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17).

PubMed

Blass, Benjamin E; Iyer, Pravin; Abou-Gharbia, Magid; Childers, Wayne E; Gordon, John C; Ramanjulu, Mercy; Morton, George; Arumugam, Premkumar; Boruwa, Joshodeep; Ellingboe, John; Mitra, Sayan; Reddy Nimmareddy, Rajashekar; Paliwal, Shalini; Rajasekhar, Jamallamudi; Shivakumar, Savithiri; Srivastava, Pratima; Tangirala, Raghuram S; Venkataramanaiah, Konda; Bobbala, Ramreddy; Yanamandra, Mahesh; Krishnakanth Reddy, L

2018-07-15

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation. Copyright © 2018. Published by Elsevier Ltd.

Congenital Adrenal Hyperplasia: Unresolved Issues.

PubMed

Yau, Mabel; Khattab, Ahmed; Poppas, Dix; Ghizzoni, Lucia; New, Maria

2016-01-01

Congenital adrenal hyperplasia (CAH) describes a family of disorders that comes from enzymatic deficiencies in cortisol production, with 21-hydroxylase deficiency causing ∼90% of cases. Distinction is made between the severe classical form and milder nonclassical form of CAH. Molecular genetic analysis is used to confirm the hormonal diagnosis. A high rate of genotype-phenotype disconcordance has been found in 21-hydroxylase deficiency. The goal of treatment is to replace with synthetic glucocorticoids and mineralocorticoids and suppress adrenal androgen production. The treatment of patients affected with nonclassical CAH, particularly males, remains controversial. Variable synthetic glucocorticoids are used and new modes of glucocorticoid delivery are under investigation. To improve height, growth hormone and other adjuvant therapies are employed. Long-term outcomes of genital surgery using modern techniques in females affected with classical CAH continue to be investigated. Prenatal treatment with dexamethasone is available to avoid ambiguous genitalia in these females. Although studies have shown its safety to mother and fetus, prenatal treatment is still regarded as experimental. Currently, prenatal diagnosis of CAH can only be obtained through invasive methods. Recently, the detection of cell-free fetal DNA in maternal plasma has made it possible to make this diagnosis earlier and noninvasively. © 2016 S. Karger AG, Basel.

Genetic manipulation of carotenoid biosynthesis and photoprotection.

PubMed

Pogson, B J; Rissler, H M

2000-10-29

There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon-cyclase and beta-carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon-cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon-cyclase gene and demonstrated that lutein content can be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ. The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta-hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size.

Genetic manipulation of carotenoid biosynthesis and photoprotection.

PubMed Central

Pogson, B J; Rissler, H M

2000-01-01

There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon-cyclase and beta-carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon-cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon-cyclase gene and demonstrated that lutein content can be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ. The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta-hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size. PMID:11127994

Management issues of congenital adrenal hyperplasia during the transition from pediatric to adult care.

PubMed

Choi, Jin-Ho; Yoo, Han-Wook

2017-02-01

Steroid 21-hydroxylase deficiency is the most prevalent form of congenital adrenal hyperplasia (CAH), accounting for approximately 95% of cases. With the advent of newborn screening and hormone replacement therapy, most children with CAH survive into adulthood. Adolescents and adults with CAH experience a number of complications, including short stature, obesity, infertility, tumor, osteoporosis, and reduced quality of life. Transition from pediatric to adult care and management of long-term complications are challenging for both patients and health-care providers. Psychosocial issues frequently affect adherence to glucocorticoid treatment. Therefore, the safe transition of adolescents to adult care requires regular follow-up of patients by a multidisciplinary team including pediatric and adult endocrinologists. The major goals for management of adults with 21-hydroxylase deficiency are to minimize the long-term complications of glucocorticoid therapy, reduce hyperandrogenism, prevent adrenal or testicular adrenal rest tumors, maintain fertility, and improve quality of life. Optimized medical or surgical treatment strategies should be developed through coordinated care, both during transition periods and throughout patients' lifetimes. This review will summarize current knowledge on the management of adults with CAH, and suggested appropriate approaches to the transition from pediatric to adult care.

11β-Hydroxylation of Cortexolone (Reichstein Compound S) to Hydrocortisone by Curvularia lunata Entrapped in Photo-Cross-Linked Resin Gels

PubMed Central

Sonomoto, Kenji; Hoq, M. Mozammel; Tanaka, Atsuo; Fukui, Saburo

1983-01-01

Spores of Curvularia lunata were immobilized by entrapment with photo-cross-linkable resin prepolymers and incubated to form mycelium in potato dextrose broth containing cortexolone (Reichstein compound S) as an inducer of steroid 11β-hydroxylase. In a buffer system containing 2.5% dimethyl sulfoxide, this immobilized mycelium hydroxylated cortexolone to hydrocortisone. The activity of this mycelium was comparable to the activity of free mycelium. Dimethyl sulfoxide did not inhibit hydroxylase activity at the concentration used and was effective in dissolving the product. Of the various photo-cross-linkable resin prepolymers examined, use of ENT-4000, whose main chain was polyethylene glycol 4000 (chain length, approximately 40 nm), resulted in maximum hydroxylation activity of the entrapped mycelium. The chain length of prepolymers affected markedly mycelial growth in the gels and, subsequently, the activity of the entrapped mycelium. The immobilized hydroxylation system was more stable than the system in free mycelium and could be reactivated by incubation of the entrapped mycelium in potato dextrose broth containing cortexolone. The system was tested 50 times during 100 days of operation and was found to carry out the desired transformation with overall yields of 60%. Images PMID:16346194

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Mariko; Hiromoto, Takeshi; Hosokawa, Keiichi

2007-10-19

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

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, Charles L.; Thilly, William G.

1999-01-01

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics.

Enteric serotonin and oxytocin: endogenous regulation of severity in a murine model of necrotizing enterocolitis.

PubMed

Gross Margolis, Kara; Vittorio, Jennifer; Talavera, Maria; Gluck, Karen; Li, Zhishan; Iuga, Alina; Stevanovic, Korey; Saurman, Virginia; Israelyan, Narek; Welch, Martha G; Gershon, Michael D

2017-11-01

Necrotizing enterocolitis (NEC), a gastrointestinal inflammatory disease of unknown etiology that may also affect the liver, causes a great deal of morbidity and mortality in premature infants. We tested the hypothesis that signaling molecules, which are endogenous to the bowel, regulate the severity of intestinal and hepatic damage in an established murine NEC model. Specifically, we postulated that mucosal serotonin (5-HT), which is proinflammatory, would exacerbate experimental NEC and that oxytocin (OT), which is present in enteric neurons and is anti-inflammatory, would oppose it. Genetic deletion of the 5-HT transporter (SERT), which increases and prolongs effects of 5-HT, was found to increase the severity of systemic manifestations, intestinal inflammation, and associated hepatotoxicity of experimental NEC. In contrast, genetic deletion of tryptophan hydroxylase 1 (TPH1), which is responsible for 5-HT biosynthesis in enterochromaffin (EC) cells of the intestinal mucosa, and TPH inhibition with LP-920540 both decrease the severity of experimental NEC in the small intestine and liver. These observations suggest that 5-HT from EC cells helps to drive the inflammatory damage to the gut and liver that occurs in the murine NEC model. Administration of OT decreased, while the OT receptor antagonist atosiban exacerbated, the intestinal inflammation of experimental NEC. Data from the current investigation are consistent with the tested hypotheses-that the enteric signaling molecules, 5-HT (positively) and OT (negatively) regulate severity of inflammation in a mouse model of NEC. Moreover, we suggest that mucosally restricted inhibition of 5-HT biosynthesis and/or administration of OT may be useful in the treatment of NEC. NEW & NOTEWORTHY Serotonin (5-HT) and oxytocin reciprocally regulate the severity of intestinal inflammation and hepatotoxicity in a murine model of necrotizing enterocolitis (NEC). Selective depletion of mucosal 5-HT through genetic deletion or inhibition of tryptophan hydroxylase-1 ameliorates, while deletion of the 5-HT uptake transporter, which increases 5-HT availability, exacerbates the severity of NEC. In contrast, oxytocin reduces, while the oxytocin receptor antagonist atosiban enhances, NEC severity. Peripheral tryptophan hydroxylase inhibition may be useful in treatment of NEC. Copyright © 2017 the American Physiological Society.

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.

PubMed

Treves, S; Feriotto, G; Moccagatta, L; Gambari, R; Zorzato, F

2000-12-15

Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).

Role of cytochrome P450 hydroxylase in the decreased accumulation of vitamin E in muscle from turkeys compared to that from chickens

DOE PAGES

Perez, Dale M.; Richards, Mark E.; Parker, Robert; ...

2015-12-13

Turkeys and chickens reared to 5-weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). AcT ingestion per kg body weight was statistically similar between chicken and turkey only when the RRR-AcT containing diet was fed. Metabolites of alpha tocopherol (AT) and gamma tocopherol (GT) were measured in bile. When fed the RRR AcT diet, AT- and GT-metabolites were 8.3 and 5.3-fold elevated in turkey compared to chicken,more » respectively (p<0.001). When fed all-racemic AcT at 58 mg/kg feed, AT- and GT-metabolites were 2.2 and 2.4-fold elevated in turkey compared to chicken, respectively (p<0.01) with AcT ingestion per kg body weight 1.29-fold higher in the chickens (p<0.001). Turkey cytochrome P450 2C29 (CYP2C29) was increased relative to its chicken ortholog based on both RNA-Seq (p<0.001) and activity-based protein profiling (p<0.01) of liver tissue. Turkey CYP3A9 and CYP4F22 were increased compared to their chicken orthologs using as determined by Activity- Based Protein Profiling (p<0.05). The 3A and 4F family are noted as tocopherol hydroxylases in mammals. Alpha tocopherol concentrations in plasma, liver and muscle from turkey were 1.7 to 4.7-fold lower than the respective tissues from chicken (p<0.05). Lipid oxidation occurred more rapidly in turkey thigh compared to chicken thigh (p<0.05). These studies suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylases in turkey liver contribute to the decreased accumulation of tocopherols in turkey tissues compared to that of chicken.« less

Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

PubMed

Seth, Puneet; Yeowell, Heather N

2010-04-01

Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively 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 the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays 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.

Effects of chemical sympathectomy on the increases in plasma catecholamines and dopamine-beta-hydroxylase induced by forced immobilization and insulin-induced hypoglycemia: origin and fate of plasma dopamine-beta-hydroxylase.

PubMed

Israel, A S; Barbella, Y R; Cubeddu, L X

1982-06-01

The effect of acute stresses on plasma norepinephrine, epinephrine and dopamine-beta-hydroxylase (DBH) were evaluated in control and 6-hydroxydopamine-treated, awake cannulated guinea pigs. Forced immobolization for 1 hr caused a 3- and 5-fold increase in plasma DBH and norepinephrine, respectively. Pretreatment with 6-hydroxydopamine (23 mg/kg b.wt.i.a., 72 and 48 hr before stress) reduced by 70% the increase in plasma DBH and totally prevented the rise in plasma catecholamines evoked by the restraining stress. Injection of insulin (5 U/kg b.wt.i.a.) induced a 60% decrease in blood glucose, a 1-fold increase in plasma DBH and a selective 4-fold increase in plasma epinephrine; these effects were not modified by chemical sympathectomy. Our results indicate that forced immobilization and hypoglycemia produce a preferential activation of the sympathetic postganglionic nerves and of the adrenal medulla, respectively, and that in guinea pigs both stresses increase plasma DBH. The kinetics of disappearance of plasma DBH were studied after subjecting the guinea pigs for 1 hr to forced immobilization. Although 7 of 12 animals showed a biphasic rate of fall of plasma DBH, in each case there was a rapid initial fall possibly due to the "distribution" of the enzyme with a T1/2 of 1.65 hr. Similar findings were observed in 6-hydroxydopamine-treated guinea pigs. These results suggest that the distribution of DBH is the most important process in reducing the augmented plasma DBH levels elicited by a short-term stress and that this process is not dependent on the integrity of the sympathetic nerves nor on the adrenal or sympathetic origin of the enzyme. This study supports the view that the ratio, content of releasable DBH present in sympathetic nerves and adrenal glands/total circulating pool of DBH, is the factor that determines whether an increase in plasma DBH would occur in animals exposed to an acute stress.

HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

PubMed

Nixon, Anne M; Meadowcroft, Mark D; Neely, Elizabeth B; Snyder, Amanda M; Purnell, Carson J; Wright, Justin; Lamendella, Regina; Nandar, Wint; Huang, Xuemei; Connor, James R

2018-05-01

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R 2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R 2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants. © 2018 International Society for Neurochemistry.

Role of cytochrome P450 hydroxylase in the decreased accumulation of vitamin E in muscle from turkeys compared to that from chickens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez, Dale M.; Richards, Mark E.; Parker, Robert

Turkeys and chickens reared to 5-weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). AcT ingestion per kg body weight was statistically similar between chicken and turkey only when the RRR-AcT containing diet was fed. Metabolites of alpha tocopherol (AT) and gamma tocopherol (GT) were measured in bile. When fed the RRR AcT diet, AT- and GT-metabolites were 8.3 and 5.3-fold elevated in turkey compared to chicken,more » respectively (p<0.001). When fed all-racemic AcT at 58 mg/kg feed, AT- and GT-metabolites were 2.2 and 2.4-fold elevated in turkey compared to chicken, respectively (p<0.01) with AcT ingestion per kg body weight 1.29-fold higher in the chickens (p<0.001). Turkey cytochrome P450 2C29 (CYP2C29) was increased relative to its chicken ortholog based on both RNA-Seq (p<0.001) and activity-based protein profiling (p<0.01) of liver tissue. Turkey CYP3A9 and CYP4F22 were increased compared to their chicken orthologs using as determined by Activity- Based Protein Profiling (p<0.05). The 3A and 4F family are noted as tocopherol hydroxylases in mammals. Alpha tocopherol concentrations in plasma, liver and muscle from turkey were 1.7 to 4.7-fold lower than the respective tissues from chicken (p<0.05). Lipid oxidation occurred more rapidly in turkey thigh compared to chicken thigh (p<0.05). These studies suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylases in turkey liver contribute to the decreased accumulation of tocopherols in turkey tissues compared to that of chicken.« less

[Changes in brain serotonin biosynthesis in rats with diabetes mellitus induced by streptozocin: effect of insulin treatment].

PubMed

Manjarrez-Gutiérrez, G; Rocío Herrera-Márquez, J R; Bueno-Santoyo, S; González-Ramírez, M; Hernández, J

2000-01-01

To investigate if the changes in the activity of the tryptophan-5-hydroxylase and in brain serotonin synthesis provoked by diabetes mellitus persist or return to normal in the diabetic rats submitted to treatment with insulin. Diabetes induced by the administration of streptozotocin in rats and their treatment with insulin was the paradigm used. At days 7, 14 and 21 of evolution, the brain serotonergic biosynthetic activity was evaluated. The diabetic rats showed a significant decrease of body weight. Also, they showed a low concentration of I-tryptophan, as well as a diminution in the activity of the key enzyme tryptophan-5-hydroxylase and its product serotonin in the cerebral cortex and brainstem. Interestingly, the activity of the enzyme was higher in the brainstem from day 14, accompanied with an elevation of the neurotransmitter. The diabetic rats submitted to treatment with insulin showed a complete physical recovery and a return to normal of plasma and brain I-tryptophan. The activity of the enzyme not only normalized but was elevated and with an increase of serotonin in the brainstem and cerebral cortex. The present findings confirm that diabetes mellitus produced a chronic anabolic deficit and a decrease in some brain regions of serotonin synthesis. Also, demonstrate that the diabetic rats under specific treatment with insulin had a complete physical recovery and a return to normal of the serotonin precursor in the blood and brain. However, the activity of the limiting enzyme TrpOH case was elevated with an increase of the neurotransmitter in all regions studied. Since the diabetic animal, insulin treated, does recover metabolically, the mechanism of activation of the serotonin biosynthetic path in the brain may not be dependent on the decreased availability of its precursor the free plasma I-tryptophan. Instead, it might be due to a change in the kinetics of tryptophan-5-hydroxylase, since its activity remains significantly increased in spite of plasma and brain normalization of its substrate. Altogether these changes in the biosynthesis of an important brain neurotransmitter may be of relevance in the pathophysiology of the psychoneurological complications in diabetic patients.

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice

PubMed Central

Kumar, Ramiya; Mota, Linda C.; Litoff, Elizabeth J.; Rooney, John P.; Boswell, W. Tyler; Courter, Elliott; Henderson, Charles M.; Hernandez, Juan P.; Corton, J. Christopher; Moore, David D.

2017-01-01

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice. PMID:28350814

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice.

PubMed

Kumar, Ramiya; Mota, Linda C; Litoff, Elizabeth J; Rooney, John P; Boswell, W Tyler; Courter, Elliott; Henderson, Charles M; Hernandez, Juan P; Corton, J Christopher; Moore, David D; Baldwin, William S

2017-01-01

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice.

Familial dopa-responsive cervical dystonia.

PubMed

Schneider, S A; Mohire, M D; Trender-Gerhard, I; Asmus, F; Sweeney, M; Davis, M; Gasser, T; Wood, N W; Bhatia, K P

2006-02-28

The authors present four cases from two unrelated families with young-onset predominant cervical dystonia with a dramatic sustained response to levodopa. Onset age was 12 years (range 9 to 15). Additional symptoms included postural hand tremor and laryngeal dystonia. Genetic testing for GTP cyclohydrolase I, tyrosine hydroxylase, and sepiapterin reductase was negative. These cases may represent new forms of dopa-responsive dystonia. Levodopa is advisable in all patients with young-onset cervical dystonia.

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, C.L.; Thilly, W.G.

1999-08-10

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics. 3 figs.

Reduced nursing frequency during prolonged lactation in the mouse decreases milk production and increases mammary expression of tryptophan hydroxylase 1 (TPH1), but does not accelerate mammary gland remodeling

USDA-ARS?s Scientific Manuscript database

We have observed that lactating mouse dams nursed 4 times per day (4X) maintained lactation, but had lower milk yields by the weigh-suckle-weigh method, than dams nursed ad libitum (AL). Therefore, we hypothesized that decreased nursing frequency would also decrease lactation persistence, increase m...

[Characterisation of three polymorphisms of the tryptophan hydroxylase 2 gene in a sample of Colombian population with major depressive disorder].

PubMed

Martínez-Idárraga, Adriana; Riveros-Barrera, Irene; Sánchez, Ricardo; Jaramillo, Luis Eduardo; Calvo-Gómez, José Manuel; Yunis-Londoño, Juan José

Identify whether rs11179000, rs136494 and rs4570625 polymorphisms of the tryptophan hydroxylase 2 gene, are associated with a major depressive disorder in a sample of the Colombian population. Case-control study was conducted in which a comparison was made between subjects diagnosed with major depressive disorder at some point in adulthood or active symptoms at the time of evaluation, and subjects with no psychiatric disease. Subjects were studied in the Department of Psychiatry, Faculty of Medicine and the Institute of Genetics at the National University of Colombia. Polymorphisms were genotyped using Taqman probes in real time PCR. As well as studying the association between major depressive disorder and these (single nucleotide polymorphisms (SNPs), the association with other factors previously associated with depression were also analysed. No statistically significant association between genotypic and allelic frequencies of each polymorphism and major depressive disorder was found. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. There was no association between any polymorphism and major depressive disorder. Copyright © 2016 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

α-Synuclein induced toxicity in brain stem serotonin neurons mediated by an AAV vector driven by the tryptophan hydroxylase promoter.

PubMed

Wan, Oi Wan; Shin, Eunju; Mattsson, Bengt; Caudal, Dorian; Svenningsson, Per; Björklund, Anders

2016-05-23

We studied the impact of α-synuclein overexpression in brainstem serotonin neurons using a novel vector construct where the expression of human wildtype α-synuclein is driven by the tryptophan hydroxylase promoter, allowing expression of α-synuclein at elevated levels, and with high selectivity, in serotonergic neurons. α-Synuclein induced degenerative changes in axons and dendrites, displaying a distorted appearance, suggesting accumulation and aggregation of α-synuclein as a result of impaired axonal transport, accompanied by a 40% loss of terminals, as assessed in the hippocampus. Tissue levels of serotonin and its major metabolite 5-HIAA remained largely unaltered, and the performance of the α-synuclein overexpressing rats in tests of spatial learning (water maze), anxiety related behavior (elevated plus maze) and depressive-like behavior (forced swim test) was not different from control, suggesting that the impact of the developing axonal pathology on serotonin neurotransmission was relatively mild. Overexpression of α-synuclein in the raphe nuclei, combined with overexpression in basal forebrain cholinergic neurons, resulted in more pronounced axonal pathology and significant impairment in the elevated plus maze. We conclude that α-synuclein pathology in serotonergic or cholinergic neurons alone is not sufficient to impair non-motor behaviors, but that it is their simultaneous involvement that determines severity of such symptoms.

Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases.

PubMed

Kamel, Marwa; Wagih, Mohamed; Kilic, Gokhan S; Diaz-Arrastia, Concepcion R; Baraka, Mohamed A; Salama, Salama A

2017-01-01

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker ( α -SMA), growth-promoting markers (PCNA; pERK1/2; FAK pY397 ; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer.

PubMed

Choi, Sul Ki; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung

2017-02-14

Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.

CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo

PubMed Central

Zhu, Jinge G.; Ochalek, Justin T.; Kaufmann, Martin; Jones, Glenville; DeLuca, Hector F.

2013-01-01

Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α,25-dihydroxyvitamin D3. It is clear that the second or 1α-hydroxylation step is carried out by a single enzyme, 25-hydroxyvitamin D 1α-hydroxylase CYP27B1. However, it is not certain what enzyme or enzymes are responsible for the initial 25-hydroxylation. An excellent case has been made for vitamin D 25-hydroxylase CYP2R1, but this hypothesis has not yet been tested. We have now produced Cyp2r1−/− mice. These mice had greater than 50% reduction in serum 25-hydroxyvitamin D3. Curiously, the 1α,25-dihydroxyvitamin D3 level in the serum remained unchanged. These mice presented no health issues. A double knockout of Cyp2r1 and Cyp27a1 maintained a similar circulating level of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Our results support the idea that the CYP2R1 is the major enzyme responsible for 25-hydroxylation of vitamin D, but clearly a second, as-yet unknown, enzyme is another contributor to this important step in vitamin D activation. PMID:24019477

Immunotoxic destruction of distinct catecholaminergic neuron populations disrupts the reproductive response to glucoprivation in female rats.

PubMed

I'Anson, Helen; Sundling, Lois A; Roland, Shannon M; Ritter, Sue

2003-10-01

We tested the hypothesis that hindbrain catecholamine (norepinephrine or epinephrine) neurons, in addition to their essential role in glucoprivic feeding, are responsible for suppressing estrous cycles during chronic glucoprivation. Normally cycling female rats were given bilateral injections of the retrogradely transported ribosomal toxin, saporin, conjugated to monoclonal dopamine beta-hydroxylase antibody (DSAP) into the paraventricular nucleus (PVN) of the hypothalamus to selectively destroy norepinephrine and epinephrine neurons projecting to the PVN. Controls were injected with unconjugated saporin. After recovery, we assessed the lesion effects on estrous cyclicity under basal conditions and found that DSAP did not alter estrous cycle length. Subsequently, we examined effects of chronic 2-deoxy-d-glucose-induced glucoprivation on cycle length. After two normal 4- to 5-d cycles, rats were injected with 2-deoxy-d-glucose (200 mg/kg every 6 h for 72 h) beginning 24 h after detection of estrus. Chronic glucoprivation increased cycle length in seven of eight unconjugated saporin rats but in only one of eight DSAP rats. Immunohistochemical results confirmed loss of dopamine beta-hydroxylase immunoreactivity in PVN. Thus, hindbrain catecholamine neurons with projections to the PVN are required for inhibition of reproductive function during chronic glucose deficit but are not required for normal estrous cyclicity when metabolic fuels are in abundance.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-11-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

PubMed Central

Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T

1988-01-01

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla. Images PMID:2903502

Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

PubMed

Leandro, João; Stokka, Anne J; Teigen, Knut; Andersen, Ole A; Flatmark, Torgeir

2017-07-01

Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l-phenylalanine (l-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 -loop to the active site in the presence of l-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l-Phe concentration. The new data support a similar functional importance of the Tyr 138 -loop in the catalytic domain and the full-length enzyme homotetramer.

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

New metabolic pathway for degradation of 2-nitrobenzoate by Arthrobacter sp. SPG

PubMed Central

Arora, Pankaj K.; Sharma, Ashutosh

2015-01-01

Arthrobacter sp. SPG utilized 2-nitrobenzoate as its sole source of carbon and energy and degraded it with accumulation of stoichiometric amounts of nitrite ions. Salicylate and catechol were detected as metabolites of the 2-nitrobenzoate degradation using high performance liquid chromatography and gas chromatography–mass spectrometry. Enzyme activities for 2-nitrobenzoate-2-monooxygenase, salicylate hydroxylase, and catechol-1,2-dioxygenase were detected in the crude extracts of the 2-nitrobenzoate-induced cells of strain SPG. The 2-nitrobenzoate-monooxygenase activity resulted in formation of salicylate and nitrite from 2-nitrobenzoate, whereas salicylate hydroxylase catalyzed the conversion of salicylate to catechol. The ring-cleaving enzyme, catechol-1,2-dioxygenase cleaved catechol to cis,cis-muconic acid. Cells of strain SPG were able to degrade 2-nitrobenzoate in sterile as well as non-sterile soil microcosms. The results of microcosm studies showed that strain SPG degraded more than 90% of 2-nitrobenzoate within 10–12 days. This study clearly shows that Arthrobacter sp. SPG degraded 2-nitrobenzoate via a new pathway with formation of salicylate and catechol as metabolites. Arthrobacter sp. SPG may be used for bioremediation of 2-nitrobenzoate-contaminated sites due to its ability to degrade 2-nitrobenzoate in soil. PMID:26082768

[Effects of perinatal exposure to bisphenol A inducing dopaminergic neuronal cell to apoptosis happening in midbrain of male rat offspring].

PubMed

Lin, Yong; Zhang, Hao; Wang, Wen-dong; Wu, De-sheng; Jiang, Song-hui; Qu, Wei-dong

2006-07-01

To investigate the mechanism and effect of rat perinatal exposure to bisphenol A (BPA) resulting in midbrain dopaminergic neuronal cell apoptosis and tyrosine hydroxylase expression of male offspring. Rat dams were randomLy divided into 4 groups on gestational day(GD) 10 and given orally the bisphenol A doses as 0, 0.5, 5, 50 mg/kg x d from GD10 to weaning. The brains of male offspring were obtained for detecting, with immunohistochemistry protocol, the Caspase-3, Bcl-2 and tyrosine hydroxylase expression in the midbrain on postnatal day 21 or 30 respectively, and the midbrain apoptotic neuronal cell were detected by TUNEL on PND21. The expression of Caspase-3 in the midbrain of rat male offspring were increased but bcl-2 were decreased on PND21 and 30, respectively. On PND21, apoptotic neuronal cell were found in the midbrain of high and medium doses groups. TH protein expression was decreased. Perinatal exposure to bisphenol A can induce the apoptosis of midbrain dopaminergic neuron in the male rat offspring even after weaning, and concomitantly decrease the midbrain TH immunoreactivity, this may cause the abnormal function of dopaminergic pathway of rat male offspring.

Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3'-hydroxylase from potato (Solanum tuberosum).

PubMed

Knollenberg, Benjamin J; Liu, Jingjing; Yu, Shu; Lin, Hong; Tian, Li

2018-02-05

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato. Copyright © 2018 Elsevier Inc. All rights reserved.

Novel therapeutic approach targeting the HIF-HRE system in the kidney.

PubMed

Nangaku, Masaomi

2009-01-01

Recent studies emphasize the role of chronic hypoxia in the tubulointerstitium as a final common pathway to end-stage renal disease. Therefore, therapeutic approaches which target the chronic hypoxia should prove effective against a broad range of renal diseases. Many of hypoxia-triggered protective mechanisms are hypoxia inducible factor (HIF)-dependent. Although HIF-1 alpha and HIF-2 alpha share both structural and functional similarity, they have different localization and can contribute in a non-redundant manner. While gene transfer of constitutively active HIF has been shown effective, pharmacological approaches to activate HIF are more desirable. Oxygen-dependent activation of prolyl hydroxylases (PHD) regulates the amount of HIF by degradation of this transcription factor. Therefore, PHD inhibitors have been the focus of recent studies on novel strategies to stabilize HIF. Cobalt is one of the inhibitors of PHD, and stimulation of HIF with cobalt is effective in a variety of kidney disease models. Furthermore, crystal structures of the catalytic domain of human prolyl hydroxylase 2 have been clarified recently. The structure aids in the design of PHD selective inhibitors for the treatment of hypoxic tissue injury. Current advance has elucidated the detailed mechanism of hypoxia-induced transcription, giving hope for the development of novel therapeutic approaches against hypoxia.

Dopamine and Mushroom Bodies in Drosophila: Experience-Dependent and -Independent Aspects of Sexual Behavior

PubMed Central

Neckameyer, Wendi S.

1998-01-01

Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminshes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5′ upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli β-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males. However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity. These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning. PMID:10454380

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

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.

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

DOE PAGES

Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.; ...

2016-03-02

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficitsmore » following ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, together these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier permeable inhibitor adaptaquin can improve functional outcomes following ICH in multiple rodent species.« less

Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa

2007-11-01

PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collectedmore » to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.« less

Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization.

PubMed

Sham, Adeline; Martinez, Eliana C; Beyer, Sebastian; Trau, Dieter W; Raghunath, Michael

2015-03-01

Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.

Leaf extract of Rhus verniciflua Stokes protects dopaminergic neuronal cells in a rotenone model of Parkinson's disease.

PubMed

Kim, Seung; Park, Se-Eun; Sapkota, Kumar; Kim, Myung-Kon; Kim, Sung-Jun

2011-10-01

The present study investigated the neuroprotective effects of Rhus verniciflua Stokes (RVS) leaf extract on rotenone-induced apoptosis in human dopaminergic cells, SH-SY5Y. Cells were pretreated with RVS extract for 1 h then treated with vehicle or rotenone for 24 h. Cell viability, cell cytotoxicity, cell morphology and nuclear morphology were examined by MTT assay, lactate dehydrogenase release assay, phase contrast microscopy and staining with Hoechast 33342, respectively. Reactive oxygen species were measured by 2'7'-dichlorofluorescein diacetate and fragmented DNA was observed by TUNEL assay. Mitochondrial membrane potential was determined by Rhodamine 123. Pro-apoptotic and anti-apoptotic proteins and tyrosine hydroxylase were analysed by Western blotting. Results showed that RVS suppressed rotenone-induced reactive oxygen species generation, cellular injury and apoptotic cell death. RVS also prevented rotenone-mediated changes in Bax/Bcl-2 levels, mitochondrial membrane potential dissipation and Caspase 3 activation. Moreover, RVS pretreatment increased the tyrosine hydroxylase levels in SH-SY5Y cells. These findings demonstrate that RVS protects SH-SY5Y cells against rotenone-induced injury and suggest that RVS may have potential therapeutic value for neurodegenerative disease associated with oxidative stress. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficitsmore » following ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, together these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier permeable inhibitor adaptaquin can improve functional outcomes following ICH in multiple rodent species.« less

Acute encephalopathy with unilateral cortical-subcortical lesions in two unrelated kindreds treated with glucocorticoids prenatally for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: established facts and novel insight.

PubMed

Grunt, Sebastian; Steinlin, Maja; Weisstanner, Christian; Schöning, Martin; Mullis, Primus E; Flück, Christa E

2013-01-01

Prenatal glucocorticoid (GC) treatment of the female fetus with 21-hydroxylase deficiency (21-OHD) may prevent genital virilization and androgen effects on the brain, but prenatal GC therapy is controversial because of possible adverse effects on fetal programming, the cardiovascular system and the brain. We report 2 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD who were treated prenatally with dexamethasone, suffered from an acute encephalopathy and showed focal and multifocal cortical and subcortical diffusion restrictions in early MRI and signs of permanent alterations in the follow-up neuroimaging studies. Both patients recovered from the acute episode. Whereas the first patient recovered without neurological sequelae the second patient showed hemianopsia and spastic hemiplegia in the neurological follow-up examination. These are 2 children with CAH, both treated prenatally with high doses of dexamethasone to prevent virilization. The question arises whether prenatal high-dose GC treatment in patients with CAH might represent a risk factor for brain lesions in later life. Adverse effects/events should be reported systematically in patients undergoing prenatal GC treatment and long-term follow-up studies involving risk factors for cerebrovascular disease should be performed. Copyright © 2013 S. Karger AG, Basel.

Renal denervation decreases blood pressure and renal tyrosine hydroxylase but does not augment the effect of hypotensive drugs.

PubMed

Skrzypecki, Janusz; Gawlak, Maciej; Huc, Tomasz; Szulczyk, Paweł; Ufnal, Marcin

2017-01-01

The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.

Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

PubMed

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

2007-03-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities▿

PubMed Central

Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.

2007-01-01

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487

Production of methoxylated flavonoids in yeast using ring A hydroxylases and flavonoid O-methyltransferases from sweet basil.

PubMed

Berim, Anna; Gang, David R

2018-07-01

Numerous methoxylated flavonoids exhibit pronounced bioactivities. Their biotechnological production and diversification are therefore of interest to pharmaceutical and nutraceutical industries. We used a set of enzymes from sweet basil (Ocimum basilicum) to construct five strains of Saccharomyces cerevisiae producing 8- and/or 6-substituted, methoxylated flavones from their natural precursor apigenin. After identifying several growth parameters affecting the overall yields and flux, we applied optimized conditions and explored the ability of the generated strains to utilize alternative substrates. The yeast cells produced substantial amounts of 6-hydroxylated, methylated derivatives of naringenin and luteolin while the corresponding derivatives of flavonol kaempferol were only detected in trace amounts. Analysis of the intermediates and by-products of the different bioconversions suggested that the substrate specificity of both the hydroxylases and the flavonoid O-methyltransferases is imposing barriers on yields obtained with alternative substrates and highlighted steps that appear to represent bottlenecks en route to increasing the strains' efficiencies. Additionally, analysis of flavonoid localization during fermentation revealed unequal distribution with strong intracellular accumulation of a number of methylated flavonoids and extracellular enrichment of several pathway intermediates. This work establishes a platform for the production of complex methoxylated flavonoids and discusses strategies for its improvement.

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

PubMed Central

Zhang, Shengnan; Fitzpatrick, Paul F.

2016-01-01

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. PMID:26823465

The Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single Turnover Experiments†

PubMed Central

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

2013-01-01

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH4) and O2. 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-BH4-phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 µM) is approximately ten-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O2 rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, detectable 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 ten-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation. PMID:23327364

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

Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

PubMed Central

Kamel, Marwa; Wagih, Mohamed; Diaz-Arrastia, Concepcion R.; Baraka, Mohamed A.

2017-01-01

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA), growth-promoting markers (PCNA; pERK1/2; FAKpY397; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF. PMID:29082249

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity.

PubMed

Bai, Chao; Berman, Judit; Farre, Gemma; Capell, Teresa; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2017-02-01

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

Nucleotide sequence variation at two genes of the phenylpropanoid pathway, the FAH1 and F3H genes, in Arabidopsis thaliana.

PubMed

Aguadé, M

2001-01-01

The FAH1 and F3H genes encode ferulate-5-hydroxylase and flavanone-3-hydroxylase, which are enzymes in the pathways leading to the synthesis of sinapic acid esters and flavonoids, respectively. Nucleotide variation at these genes was surveyed by sequencing a sample of 20 worldwide Arabidopsis thaliana ecotypes and one Arabidopsis lyrata spp. petraea stock. In contrast with most previously studied genes, the percentage of singletons was rather low in both the FAH1 and the F3H gene regions. There was, therefore, no footprint of a recent species expansion in the pattern of nucleotide variation in these regions. In both FAH1 and F3H, nucleotide variation was structured into two major highly differentiated haplotypes. In both genes, there was a peak of silent polymorphism in the 5' part of the coding region without a parallel increase in silent divergence. In FAH1, the peak was centered at the beginning of the second exon. In F3H, nucleotide diversity was highest at the beginning of the gene. The observed pattern of variation in both FAH1 and F3H, although suggestive of balancing selection, was compatible with a neutral model with no recombination.

Norepinephrine signaling through beta-adrenergic receptors is critical for expression of cocaine-induced anxiety.

PubMed

Schank, Jesse R; Liles, L Cameron; Weinshenker, David

2008-06-01

Cocaine is a widely abused psychostimulant that has both rewarding and aversive properties. While the mechanisms underlying cocaine's rewarding effects have been studied extensively, less attention has been paid to the unpleasant behavioral states induced by cocaine, such as anxiety. In this study, we evaluated the performance of dopamine beta-hydroxylase knockout (Dbh -/-) mice, which lack norepinephrine (NE), in the elevated plus maze (EPM) to examine the contribution of noradrenergic signaling to cocaine-induced anxiety. We found that cocaine dose-dependently increased anxiety-like behavior in control (Dbh +/-) mice, as measured by a decrease in open arm exploration. The Dbh -/- mice had normal baseline performance in the EPM but were completely resistant to the anxiogenic effects of cocaine. Cocaine-induced anxiety was also attenuated in Dbh +/- mice following administration of disulfiram, a dopamine beta-hydroxylase (DBH) inhibitor. In experiments using specific adrenergic antagonists, we found that pretreatment with the beta-adrenergic receptor antagonist propranolol blocked cocaine-induced anxiety-like behavior in Dbh +/- and wild-type C57BL6/J mice, while the alpha(1) antagonist prazosin and the alpha(2) antagonist yohimbine had no effect. These results indicate that noradrenergic signaling via beta-adrenergic receptors is required for cocaine-induced anxiety in mice.

Hypoxia-Inducible Factor and Its Role in the Management of Anemia in Chronic Kidney Disease

PubMed Central

Kaplan, Joshua M.; Sharma, Neeraj

2018-01-01

Hypoxia-inducible factor (HIF) plays a crucial role in the response to hypoxia at the cellular, tissue, and organism level. New agents under development to pharmacologically manipulate HIF may provide new and exciting possibilities in the treatment of anemia of chronic kidney disease (CKD) as well as in multiple other disease states involving ischemia–reperfusion injury. This article provides an overview of recent studies describing current standards of care for patients with anemia in CKD and associated clinical issues, and those supporting the clinical potential for targeting HIF stabilization with HIF prolyl-hydroxylase inhibitors (HIF-PHI) in these patients. Additionally, articles reporting the clinical potential for HIF-PHIs in ‘other’ putative therapeutic areas, the tissue and intracellular distribution of HIF- and prolyl-hydroxylase domain (PHD) isoforms, and HIF isoforms targeted by the different PHDs, were identified. There is increasing uncertainty regarding the optimal treatment for anemia of CKD with poorer outcomes associated with treatment to higher hemoglobin targets, and the increasing use of iron and consequent risk of iron imbalance. Attainment and maintenance of more physiologic erythropoietin levels associated with HIF stabilization may improve the management of patients resistant to treatment with erythropoiesis-stimulating agents and improve outcomes at higher hemoglobin targets. PMID:29382128