WorldWideScience

Sample records for tyrosine hydroxylase transcripts

  1. [Tyrosine hydroxylase of the blood leukocytes].

    Science.gov (United States)

    Mineeva, M F

    1987-07-01

    Tyrosine hydroxylase activity has been established in blood plasma leucocytes of rat, cat and man. Tyrosine precursors and some nuclear erythroid cells. GFU-GM did hydroxylase activity in leucocytes shows the Km for tyrosine inhibited by high concentrations of L6 tyrosine (substrate inhibition), alpha-methyl-para-tyrosine dopamine. The kinetic properties of leucocyte tyrosine hydroxylase are qualitatively similar to the properties of brain tyrosine hydroxylase.

  2. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Morphological Features of Tyrosine Hydroxylase Immunoreactive ...

    African Journals Online (AJOL)

    The current immunohistochemical study used the antibody against tyrosine hydroxylase (TH) to observe the immunoreactive elements in the mouse pancreas. The results indicated the presence of immunoreactive nerve fibers and endocrine cells. The immunopositive nerve fibers appeared as thick and thin bundles; thick ...

  4. Interferon-alpha signalling in bovine adrenal chromaffin cells: involvement of signal-transducer and activator of transcription 1 and 2, extracellular signal-regulated protein kinases 1/2 and serine 31 phosphorylation of tyrosine hydroxylase.

    Science.gov (United States)

    Douglas, S A; Bunn, S J

    2009-03-01

    Adrenal medullary chromaffin cells are an integral part of the neuroendocrine system, playing an important role in the physiological adaptation to stress. In response to a wide variety of stimuli, including acetylcholine released from the splanchnic nerve, hormones such as angiotensin II or paracrine signals such as prostaglandins, chromaffin cells synthesise and secrete catecholamines and a number of biologically active peptides. This adrenal medullary output mediates a complex and diverse stress response. We report that chromaffin cells also respond both acutely and chronically to interferon (IFN)-alpha, thus providing a mechanism of interaction between the immune system and the stress response. Incubation of isolated bovine chromaffin cells maintained in culture, with IFN-alpha resulted in a rapid, transient activation of the extracellular signal-regulated protein kinase (ERK)1/2, which was maximal after 5 min. IFN-alpha mediated activation of ERK1/2 appeared to be responsible for the increased phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This tyrosine hydroxylase phosphorylation was exclusively on serine 31, with no change in the phosphorylation of serine 19 or 40. This increase in the serine 31 phosphorylation of tyrosine hydroxylase was prevented by inhibition of protein kinase C or ERK1/2 activation. Incubation with IFN-alpha also resulted in a time- and concentration-dependent phosphorylation and nuclear translocation of signal transducer and activator of transcription proteins (STAT)1 and 2. This response was maximal after approximately 60 min. Prolonged treatment with IFN-alpha (12-48 h) resulted in increased expression of STAT1 and, to a lesser extent, STAT2. Thus, these findings demonstrate that adrenal medullary chromaffin cells are responsive to IFN-alpha and provide a possible cellular mechanism by which this immune-derived signal can potentially influence and integrate with the stress response.

  5. Organization and evolution of the rat tyrosine hydroxylase gene

    International Nuclear Information System (INIS)

    Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

    1987-01-01

    This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family

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

    Science.gov (United States)

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

    2016-07-01

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

  7. Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells.

    Science.gov (United States)

    Kawahata, Ichiro; Yamakuni, Tohru

    2018-02-01

    Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT

  8. Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner.

    Science.gov (United States)

    Rönnberg, Elin; Calounova, Gabriela; Pejler, Gunnar

    2012-01-01

    Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.

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

    Directory of Open Access Journals (Sweden)

    Jian-qing Liang

    2016-01-01

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

  10. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  11. Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells.

    Science.gov (United States)

    Knowles, P J; Douglas, S A; Bunn, S J

    2011-03-01

    The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied

  12. Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice.

    Science.gov (United States)

    Vyas, Pankhuri; Wu, Jingjing Sherry; Zimmerman, Amanda; Fuchs, Paul; Glowatzki, Elisabeth

    2017-02-01

    Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.

  13. Translation of tyrosine hydroxylase from poly(A)-mRNA in pheochromocytoma cells is enhanced by dexamethasone.

    OpenAIRE

    Baetge, E E; Kaplan, B B; Reis, D J; Joh, T H

    1981-01-01

    Polysomal poly(A)-mRNA was purified from a clonal cell line of rat pheochromocytoma (PC 12) and translated in a reticulocyte cell-free protein-synthesizing system. Tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahyropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was isolated from other protein by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. The molecular weight and relative proportion of tyrosine hydroxylase to other proteins synthesi...

  14. Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells

    OpenAIRE

    1986-01-01

    We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of lamini...

  15. Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.

    OpenAIRE

    Fukami, M H; Haavik, J; Flatmark, T

    1990-01-01

    Incubation of bovine chromaffin cells with L-[14C]phenylalanine resulted in label accumulation in catecholamines at about 30% of the rate seen with L-tyrosine as precursor. Studies with purified tyrosine hydroxylase (EC 1.14.16.2) showed that the enzyme catalysed the hydroxylation of L-phenylalanine first to L-p-tyrosine and then to 3,4-dihydroxyphenylalanine (DOPA). No evidence for a significant involvement of an L-m-tyrosine intermediate in DOPA formation was found.

  16. Tyrosine hydroxylase polymorphism (C-824T) and hypertension: a population-based study

    DEFF Research Database (Denmark)

    Nielsen, Søren J; Jeppesen, Jørgen Lykke; Torp-Pedersen, Christian

    2010-01-01

    Sympathetic nervous system (SNS) overactivity is present in a large proportion of the hypertensive population and precedes the development of established hypertension. Variations in the proximal promoter of the tyrosine hydroxylase (TH) gene have been shown to influence biochemical and physiologi...

  17. Mutant torsinA interacts with tyrosine hydroxylase in cultured cells.

    Science.gov (United States)

    O'Farrell, C A; Martin, K L; Hutton, M; Delatycki, M B; Cookson, M R; Lockhart, P J

    2009-12-15

    A specific mutation (DeltaE302/303) in the torsinA gene underlies most cases of dominantly inherited early-onset torsion dystonia. This mutation causes the protein to aggregate and form intracellular inclusion bodies in cultured cells and animal models. Co-expression of the wildtype and mutant proteins resulted in the redistribution of the wildtype protein from the endoplasmic reticulum to inclusion bodies in cultured HEK293 cells, and this was associated with increased interaction between the two proteins. Expression of DeltaE302/303 but not wildtype torsinA in primary postnatal midbrain neurons resulted in the formation of intracellular inclusion bodies, predominantly in dopaminergic neurons. Tyrosine hydroxylase was sequestered in these inclusions and this process was mediated by increased protein-protein interaction between mutant torsinA and tyrosine hydroxylase. Analysis in an inducible neuroblastoma cell culture model demonstrated altered tyrosine hydroxylase activity in the presence of the mutant but not wildtype torsinA protein. Our results suggest that the interaction of tyrosine hydroxylase and mutant torsinA may contribute to the phenotype and reported dopaminergic dysfunction in torsinA-mediated dystonia.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  1. Human glioblastoma ADF cells express tyrosinase, L-tyrosine hydroxylase and melanosomes and are sensitive to L-tyrosine and phenylthiourea.

    Science.gov (United States)

    Bonfigli, Antonella; Zarivi, Osvaldo; Colafarina, Sabrina; Cimini, Anna Maria; Ragnelli, Anna Maria; Aimola, Pierpaolo; Natali, Pier Giorgio; Cerù, Maria Paola; Amicarelli, Fernanda; Miranda, Michele

    2006-06-01

    Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma. Copyright 2006 Wiley-Liss, Inc.

  2. Manganese-Mediated Decrease in Levels of c-RET and Tyrosine Hydroxylase Expression In Vitro.

    Science.gov (United States)

    Kumasaka, Mayuko Y; Yajima, Ichiro; Ohgami, Nobutaka; Ninomiya, Hiromasa; Iida, Machiko; Li, Xiang; Oshino, Reina; Tanihata, Hiroko; Yoshinaga, Masafumi; Kato, Masashi

    2017-11-01

    Previous studies showed that overexposure to manganese causes parkinsonism, a disorder of dopaminergic neurons. Previous studies also showed that activity of c-RET kinase controls dopamine production through regulation of tyrosine hydroxylase (TH) expression, suggesting the involvement of c-RET in the development of parkinsonism. To our knowledge, however, there is no report showing a correlation between manganese-mediated parkinsonism and c-RET. In this study, we examined the effect of manganese on the expression and/or activation levels of c-RET and TH in human TH-expressing cells (TGW cells). We first found that treatment with 30 and 100 μM manganese resulted in reduction of c-RET transcript level and degradation of c-RET protein through promotion of ubiquitination. We then examined the biological significance of manganese-mediated decrease of c-RET protein expression. Decreased TH expression with decreased c-RET kinase activity was observed in c-RET protein-depleted TGW cells by treatment with manganese (30 μM) as well as by c-RET siRNA transfection. Since TH protein has been shown to be involved in the dopamine-producing pathway in previous studies, our results indicate the possibility that manganese-mediated reduction of TH expression and phosphorylation via decreased expression of c-RET protein in neural cells is involved in parkinsonism induced by manganese.

  3. Transient appearance of tyrosine hydroxylase immunoreactive cells in the midline epithelial seam of the human fetal secondary palate.

    Science.gov (United States)

    Katori, Yukio; Shibata, Shunichi; Kawase, Tetsuaki; Cho, Baik Hwan; Murakami, Gen

    2012-07-01

    Transient immunoreactivity for tyrosine hydroxylase, which mediates the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine, in the midline epithelial seam between the bilateral palatal shelves was investigated in human fetuses. Horizontal or frontal paraffin sections of two human fetuses at 9 and 15 weeks of gestation were used to examine the distribution of tyrosine hydroxylase-immunoreactive cells in regions of the entire head other than the brain. Immunohistochemical staining for S100 protein, calretinin, cytokeratin 14, and vimentin was examined using adjacent or near sections. Tyrosine hydroxylase-immunoreactive cells were large and densely distributed in the midline epithelial seam at the site of palatal fusion in fetuses at 9 weeks but not in fetuses at 15 weeks, in which the midline epithelial seam had already disappeared. No expression of S100 protein, calretinin, or vimentin was detected, but the midline epithelial seam was positive for cytokeratin 14. Tyrosine hydroxylase immunoreactivity was not detected in epithelia during the process of palatal fusion in mice from E 14.0 to 15.0. These findings indicate that tyrosine hydroxylase-immunoreactive cells in the midline epithelial seams are nonneural epithelial cells and suggest that the tyrosine hydroxylase is a novel factor involved in normal palatal formation, especially the fate of the midline epithelial seam in humans.

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

    OpenAIRE

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

    1994-01-01

    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 ...

  5. Nurr1 represses tyrosine hydroxylase expression via SIRT1 in human neural stem cells.

    Science.gov (United States)

    Kim, Tae Eun; Seo, Ji-Seon; Seo, Ji Sun; Yang, Jae Won; Kim, Min Woong; Kausar, Rukhsana; Joe, Eunhye; Kim, Bo Yeon; Lee, Myung Ae

    2013-01-01

    Nurr1 is an orphan nuclear receptor best known for its essential role in the development and maintenance of midbrain dopaminergic (DA) neurons. During DA neurogenesis, Nurr1 directly targets human tyrosine hydroxylase (hTH). Here we investigated this targeting to identify the molecular mechanisms by which Nurr1 regulates DA neurogenesis. We previously cloned the hTH promoter and found three consensus elements for Nurr1 binding: NBRE-A, -B, and -C. In the present study, gel retardation and luciferase assays using hTH constructs showed that Nurr1 preferentially bound to NBRE-A, through which it mediated transcriptional activity. Furthermore, Nurr1 displayed dual-function transcriptional activities depending on the cell type. In DA-like SH-SY5Y cells, Nurr1 dose-dependently stimulated hTH-3174 promoter activity by 7- to 11-fold. However, in the human neural stem cell (hNSC) line HB1.F3, Nurr1 strongly repressed transcription from the same promoter. This repression was relieved by mutation of only the NBRE-A element and by nicotinamide [an inhibitor of class III histone deacetylases (HDACs), such as SIRT1], but not by trichostatin A (an inhibitor of class I and II HDACs). SIRT1 was strongly expressed in the nucleus of HB1.F3 cells, while it was localized in the cytoplasm in SH-SY5Y cells. ChIP assays of HB1.F3 cells showed that Nurr1 overexpression significantly increased the SIRT1 occupancy of the NBRE-A hTH promoter region, while low SIRT1 levels were observed in control cells. In contrast, no significant SIRT1 recruitment was observed in SH-SY5Y cells. These results indicate that differential SIRT1 localization may be involved in hTH gene regulation. Overall, our findings suggest that Nurr1 exists in dual transcriptional complexes, including co-repressor complexes that can be remodeled to become co-activators and can fine-tune hTH gene transcription during human DA neurogenesis.

  6. COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons.

    Science.gov (United States)

    Bovetti, Serena; Bonzano, Sara; Garzotto, Donatella; Giannelli, Serena Gea; Iannielli, Angelo; Armentano, Maria; Studer, Michèle; De Marchis, Silvia

    2013-12-01

    COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.

  7. Phase Difference in the Induction of Tyrosine Hydroxylase in Cell Body and Nerve Terminals of Sympathetic Neurones

    Science.gov (United States)

    Thoenen, Hans; Mueller, Robert A.; Axelrod, Julius

    1970-01-01

    The induction of tyrosine hydroxylase in the nerve terminals of the rat heart by reserpine lags behind that in the stellate ganglion by two to three days. Cycloheximide given three days after reserpine blocks the further rise of the enzyme in the nerve terminals. The increase in tyrosine hydroxylase activity of the lumbar ganglion is as marked as that in the stellate ganglion. The increase of enzyme activity in the sciatic nerve after reserpine administration resembles that found in the heart nerve terminals. Determination of enzyme activity in segments of sciatic nerves indicates a two-day lag and then a proximal-distal transport of enzyme, but the apparent rate is not sufficient to account for the increase in enzyme in the nerve terminals. These findings are compatible with the local synthesis of induced tyrosine hydroxylase in the nerve terminals rather than the peripheral movement of the completed enzyme. PMID:4189989

  8. Identification by Hydrogen/Deuterium Exchange of Structural Changes in Tyrosine Hydroxylase Associated with Regulation

    Science.gov (United States)

    Wang, Shanzhi; Sura, Giri R.; Dangott, Lawrence J.; Fitzpatrick, Paul F.

    2009-01-01

    The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by three orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrates access; this conformation is favored by phosphorylation of Ser40. PMID:19371093

  9. Protein phosphatase 2A is involved in the tyrosine hydroxylase phosphorylation regulated by α-synuclein.

    Science.gov (United States)

    Hua, Gao; Xiaolei, Lan; Weiwei, Yang; Hao, Wang; Yuangang, Zhu; Dongmei, Liu; Yazhuo, Zhang; Hui, Yang

    2015-03-01

    α-Synuclein (α-Syn) plays a crucial role in the pathophysiology of Parkinson's disease (PD), the degeneration of dopaminergic neurons. Previous studies have shown that α-Syn regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase (TH). In neurons, protein phosphatases (PPs) play a prominent role in directing signaling toward survival or degeneration. This study was to re-evaluate whether α-Syn could regulate the tyrosine hydroxylase phosphorylation by protein phosphatase-2A (PP2A) in dopaminergic MN9D cells and cortex neurons. Our data demonstrated for the first time that α-Syn stimulates PP2A activity and reduces phosphorylation of TH through regulating the methylation of PP2A in dopaminergic MN9D cells and primary cortex neurons. Increased PP2A activity and reduced phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in α-Syn overexpression dopaminergic cells (Syn) and primary cortex neurons, and the TH phosphorylation relieved by enhancing PP2A methylation in Syn group could be abated by using PP inhibitors, okadaic acid (OKA). OKA could reduce the cell damage and cell apoptosis induced by α-Syn. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies targeting at PP2A.

  10. Tyrosine hydroxylase positive nerves and mast cells in the porcine gallbladder

    Directory of Open Access Journals (Sweden)

    I. Stefanov

    2017-03-01

    Full Text Available The aim of this study was to detect the localisation of tyrosine hydroxylase (TH positive nerve fibres (THN and distribution of tyrosine hydroxylase positive mast cells (THMC in the wall of porcine gallbladder. THN were observed as single fibres, nerve fibres forming perivascular plexuses and nerve fibres grouped within the nerve fascicles. In the gallbladder`s fundus, body and neck, the TH+ fibres formed mucosal, muscular and serosal nonganglionated nerve plexuses. Toluidine blue (TB staining was used to confirm that the TH positive cells were mast cells. The number of THMC in the propria of gallbladder`s fundus, body and neck was significantly higher than in the muscular and serosal layers in both genders. The number of mast cells in the musculature was higher than in the serosa. The density and location of the THMC were similar to the TB positive (with gamma meta-chromasia mast cells in both males and females, and statistically significant difference was not established. In conclusion, original data concerning the existence and localisation of catecholaminergic nerves and THMC distribution in the porcine gallbladder’s wall are presented. The results could con-tribute to the body of knowledge of functional communication between autonomic nerves and mast cells in the gallbladder.

  11. An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l-DOPA from Cultured Rat Striatal Cells

    OpenAIRE

    Geller, Alfred I.; During, Matthew J.; Oh, Young J.; Freese, Andrew; O’Malley, Karen

    1995-01-01

    In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l-DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSV...

  12. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    International Nuclear Information System (INIS)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-01-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35 S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed

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

    DEFF Research Database (Denmark)

    Huebener, Nicole; Fest, Stefan; Strandsby, Anne Bystrup

    2008-01-01

    hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA...... following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we......Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine...

  14. The locus coeruleus complex of the bottlenose dolphin (Tursiops truncatus) as revealed by tyrosine hydroxylase immunohistochemistry.

    Science.gov (United States)

    Manger, Paul R; Ridgway, Sam H; Siegel, Jerome M

    2003-06-01

    Using tyrosine hydroxylase immunohistochemistry we examined the structure of the pontine, or rostral rhombencephalic, catecholaminergic cells groups, which may be collectively termed the locus coeruleus complex (LC), in the bottlenose dolphin. The present study is the first to describe the LC in a cetacean species and, at 1.3 kg, represents the largest non-human brain to date in which the LC has been investigated. We identified four catecholaminergic cell groups in the dorsal pontine tegementum and peri-aqueductal gray matter: A6 dorsal (locus coeruleus), A6 ventral (locus coeruleus alpha), A7 (subcoeruleus), and A5 (fifth arcuate nucleus). No patterns of cellular distribution, nuclear subdivision, or cellular morphology indicate specialization of the LC, which might have been anticipated because of the large absolute brain size and unihemispheric sleep phenomenology of cetaceans.

  15. Hypoxia-inducible factor-1α upregulates tyrosine hydroxylase and dopamine transporter by nuclear receptor ERRγ in SH-SY5Y cells.

    Science.gov (United States)

    Lim, Juhee; Kim, Hyo-In; Bang, Yeojin; Seol, Wongi; Choi, Hueng-Sik; Choi, Hyun Jin

    2015-04-15

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor relevant to the development of many mammalian organs including the brain. However, the molecular mechanisms by which signaling events mediate neuronal differentiation have not been fully elucidated. In the present study, we show for the first time that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) is upregulated by HIF-1α and plays essential roles in HIF-1α-induced upregulation of dopaminergic marker molecules such as tyrosine hydroxylase and dopamine transporter. We found that deferoxamine upregulated HIF-1α and enhanced the dopaminergic phenotype and neurite outgrowth of SH-SY5Y cells. Deferoxamine activated transcription and protein expression of ERRγ, and deferoxamine-induced upregulation of tyrosine hydroxylase and dopamine transporter was attenuated by using the ERRγ inverse agonist or silencing ERRγ. Altogether, these results suggest that HIF-1α can positively regulate the dopaminergic phenotype through ERRγ. This study could provide new perspectives for understanding the mechanisms underlying the promotion of dopaminergic neuronal differentiation by hypoxia.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hundahl, C.A., E-mail: c.hundahl@gmail.com [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Fahrenkrug, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Luuk, H. [Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Hay-Schmidt, A. [Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Hannibal, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark)

    2012-08-17

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

  17. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora

    2017-08-25

    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Cyclic AMP-dependent regulation of tyrosine hydroxylase mRNA and immunofluorescence levels in rat retinal precursor cells.

    Science.gov (United States)

    Voisin, Pierre; Bernard, Marianne

    2013-05-01

    Stimulation of tyrosine hydroxylase (TH) gene transcription by cyclic AMP (cAMP) has been clearly established in adrenal medula cells and neural-crest-derived cell lines but information on this mechanism is still lacking in dopaminergic neurons. Because they are easily amenable to in vitro experiments, dopaminergic amacrine cells of the retina might constitute a valuable model system to study this mechanism. We have used real-time reverse transcription with the polymerase chain reaction to quantify TH mRNA levels in the rat retina during post-natal development and in retinal precursor cells obtained from neonatal rats and cultured for 3 days in serum-free medium. Whereas the TH mRNA concentration remains consistantly low in control cultures, treatment with cAMP-increasing agents (forskolin, membrane depolarization, phosphodiesterase inhibitors) is sufficient to raise it to the level observed in adult retina (15-fold increase). Treatment of the cultured cells can be delayed by up to 2 days with identical results at the TH mRNA level, thus ruling out a survival-promoting effect of cAMP. TH immunofluorescence has confirmed cAMP-dependent regulation of TH expression at the protein level and indicates that the frequency of TH-positive cells in the cultures is similar to that observed in the adult retina. Selective phosphodiesterase inhibitors suggest that PDE4 is the major subtype involved in the dopaminergic amacrine cell response. Our data clearly establish the cAMP-dependent regulation of TH mRNA and immunofluorescence levels in retinal precursor cells. The possible role of this regulation mechanism in the developmental activation of TH gene expression is discussed.

  19. Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells.

    Science.gov (United States)

    Gelain, Daniel P; Moreira, Jose C F; Bevilaqua, Lia R M; Dickson, Phillip W; Dunkley, Peter R

    2007-12-01

    Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.

  20. Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Samuel O Adeosun

    Full Text Available Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc are greatly needed to effectively change the debilitating course of Parkinson's disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinson's disease.

  1. Putaminal mosaic visualized by tyrosine hydroxylase immunohistochemistry in the human neostriatum.

    Directory of Open Access Journals (Sweden)

    Ryoma eMorigaki

    2016-04-01

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

  2. A Tyrosine-Hydroxylase Characterization of Dopaminergic Neurons in the Honey Bee Brain

    Directory of Open Access Journals (Sweden)

    Stevanus R. Tedjakumala

    2017-07-01

    Full Text Available Dopamine (DA plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US. Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH immunoreactivity (ir to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1–C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES and the antennal lobe (AL; the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL of the mushroom body (MB; the C3 cluster is located below the calyces (CA of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning.

  3. Tyrosine hydroxylase protein expression in ventral nerve cord of Neotropical freshwater crab.

    Science.gov (United States)

    Ponzoni, Silvia

    2014-12-01

    Given the importance of catecholamines in coordinating physiological and behavioral responses in brachyurans, the present study was designed to investigate the distribution of tyrosine hydroxylase (TH)-positive cells and fibers in the ventral nerve cord of Dilocarcinus pagei the Neotropical freshwater crab. TH immunoreactivity was visualized in adult crabs of both sexes, during the intermolt period. We found TH-positive cells that have not been previously described in brachyurans. Specifically, we found a pair of TH-positive cells in the ventral region of the thoracic ganglion, and in ventral and dorsal regions of the abdominal (pleonic) ganglion, suggesting catecholaminergic modulation of claws' function and abdominal structures. In addition, great population of TH-positive cells was observed in the subesophageal ganglion, indicating conservation during evolution of catecholamines in this ganglion of decapods. Dopamine is present in cells and fiber tracts of brachyuran ventral nerve cord, projecting to endocrine, cardiac and digestive structures, suggesting widespread modulation and control of physiological functions and behavior. Dopamine plays a central role in movement and psychiatric disorders in humans. Information on dopaminergic function in the nervous system of invertebrates should improve the understanding of its function in more complex systems, such as human beings. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Hypothalamic arcuate nucleus tyrosine hydroxylase neurons play orexigenic role in energy homeostasis.

    Science.gov (United States)

    Zhang, Xiaobing; van den Pol, Anthony N

    2016-10-01

    Energy homeostasis, food intake, and body weight are regulated by specific brain circuits. Here we introduce an unexpected neuron, the tyrosine hydroxylase (TH) neuron of the arcuate nucleus (ARC), that we show makes an orexigenic contribution. Optogenetic stimulation of mouse ARC TH neurons increased food intake; attenuating transmitter release reduced body weight. Optogenetic stimulation of ARC TH cells inhibited pro-opiomelanocortin (POMC) neurons through synaptic mechanisms. ARC TH cells project to the hypothalamic paraventricular nucleus; optogenetic stimulation of ARC TH axons inhibited paraventricular nucleus neurons by dopamine and GABA co-release. Dopamine excited orexigenic neurons that synthesize agouti-related peptide and neuropeptide Y but inhibited anorexigenic neurons that synthesize POMC, as determined by whole cell recording. Food deprivation increased c-fos expression and spike frequency in ARC TH neurons. The gut peptide ghrelin evoked direct excitatory effects, suggesting these neurons monitor metabolic cues. Together these data support the view that ARC TH cells play an unrecognized and influential positive role in energy homeostasis.

  5. Compartment-specific tyrosine hydroxylase-positive innervation to AII amacrine cells in the rabbit retina.

    Science.gov (United States)

    Völgyi, B; Debertin, G; Balogh, M; Popovich, E; Kovács-Öller, T

    2014-06-13

    Tyrosine-hydroxylase-positive (TH(+)) amacrine cells release dopamine in a paracrine manner and also form GABA-ergic contact sites with inner retinal neurons. The best known sites are formed by TH(+) fibrous rings and AII amacrine cell somata in stratum 1 of the inner plexiform layer (IPL). An AII amacrine cell is a highly compartmentalized neuron with relatively large soma, a stout dendritic stalk and two sets of processes, one showing lobular appearance and extending horizontally in stratum 1 and a second transversally elongated group of fibers in strata 4 and 5. Although, all of these compartments have been reported as tic sites, it is uncertain if TH(+) amacrine cell inputs are homogeneously distributed or they rather target specific AII cell compartments. In this study we investigated the TH(+)/AII cell system by immunohistochemistry to map the potential synaptic contacts in the rabbit retina. We found numerous intimate contacts between the two amacrine cell populations throughout the IPL. However, TH(+) fibers favored the soma/main stalk region of AII amacrine cells and only contacted lobular appendages and transversal processes sporadically. In addition to the well-studied contacts between AII cell somata and TH(+) rings in stratum 1 we found that the main stalk region in stratum 3 serves as a secondary major target for TH(+) axons. These data thus clearly show that TH(+) contacts to AII amacrine cells are highly compartment specific. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Genetic fate-mapping of tyrosine hydroxylase-expressing cells in the enteric nervous system.

    Science.gov (United States)

    Obermayr, F; Stamp, L A; Anderson, C R; Young, H M

    2013-04-01

    During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized. We combined in vivo Cre-lox-based genetic fate-mapping with phenotypic analysis to fate-map enteric neuron subtypes arising from tyrosine hydroxylase (TH)-expressing cells. Less than 3% of the total (Hu(+) ) neurons in the myenteric plexus of the small intestine of adult mice are generated from transiently TH-expressing cells. Around 50% of the neurons generated from transiently TH-expressing cells are calbindin neurons, but their progeny also include calretinin, neurofilament-M, and serotonin neurons. However, only 30% of the serotonin neurons and small subpopulations (cells; only 0.2% of nitric oxide synthase neurons arise from TH-expressing cells. Transiently, catecholaminergic cells give rise to subpopulations of multiple enteric neuron subtypes, but the majority of each of the neuron subtypes arises from non-TC cells. © 2013 Blackwell Publishing Ltd.

  7. Tyrosine hydroxylase immunoreactivity is common in the enteric nervous system in teleosts.

    Science.gov (United States)

    Olsson, Catharina

    2016-05-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines and TH immunoreactivity is indicative of cells synthesising either adrenaline/noradrenaline or dopamine. In this study, the distribution of TH immunoreactivity was examined in two distantly related teleost species, zebrafish (Danio rerio) and shorthorn sculpin (Myoxocephalus scorpius). In both species, TH-immunoreactive nerve cell bodies and varicose nerve fibres were common in the myenteric plexus of the intestine. However, no TH-immunoreactive nerve cell bodies were seen in the sculpin stomach. The TH-immunoreactive nerve cell bodies seemed to constitute a larger proportion of the total enteric population in shorthorn sculpin (50 ± 5 %, n = 3067 cells) compared with zebrafish (14 ± 2 %, n = 10,163 cells). In contrast, in sculpin, the TH-immunoreactive cells were smaller than the average enteric nerve cell bodies, whereas in zebrafish, the relationship was the opposite. In developing zebrafish larvae, TH-immunoreactive nerve cell bodies were common (approx. 75 % of the total population) at 3 days post-fertilization (dpf), but decreased in numbers between 3 and 7 dpf. In conclusion, in contrast to previous studies, TH-immunoreactive intrinsic neurons are common in the fish gut. Their role and function need to be further characterized in order to understand the potential importance of this enteric subpopulation in controlling various gut functions.

  8. Peripheral Lipopolysaccharide Challenge Induces Long-Term Changes in Tyrosine Hydroxylase Regulation in the Adrenal Medulla.

    Science.gov (United States)

    Ong, Lin Kooi; Page, Scott; Briggs, Gabrielle D; Guan, Liying; Dun, Matthew D; Verrills, Nicole M; Dunkley, Peter R; Dickson, Phillip W

    2017-08-01

    Immune activation can alter the activity of adrenal chromaffin cells. The effect of immune activation by lipopolysaccharide (LPS) on the regulation of tyrosine hydroxylase (TH) in the adrenal medulla in vivo was determined between 1 day and 6 months after LPS injection. The plasma levels of eleven cytokines were reduced 1 day after LPS injection, whereas the level for interleukin-10 was increased. The levels of all cytokines remained at control levels until 6 months when the levels of interleukin-6 and -4 were increased. One day after LPS injection, there was a decrease in TH-specific activity that may be due to decreased phosphorylation of serine 31 and 40. This decreased phosphorylation of serine 31 and 40 may be due to an increased activation of the protein phosphatase PP2A. One week after LPS injection, there was increased TH protein and increased phosphorylation of serine 40 that this was not accompanied by an increase in TH-specific activity. All TH parameters measured returned to basal levels between 1 month and 3 months. Six months after injection there was an increase in TH protein. This was associated with increased levels of the extracellular regulated kinase isoforms 1 and 2. This work shows that a single inflammatory event has the capacity to generate both short-term and long-term changes in TH regulation in the adrenal medulla of the adult animal. J. Cell. Biochem. 118: 2096-2107, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. The epinephrine increases tyrosine hydroxylase expression through upregulating thioredoxin-1 in PC12 cells.

    Science.gov (United States)

    Jia, Jin-Jing; Zeng, Xian-Si; Yang, Li-Hua; Bai, Jie

    2015-08-01

    Epinephrine is a stress hormone which is sharply increased in response to acute stress and is continuously elevated during persistent stress. Thioredoxin-1 (Trx-1) is a redox regulating protein and is induced under various stresses. Our previous study has shown that epinephrine induces the expression of Trx-1. Tyrosine hydroxylase (TH) is the major rate-limiting enzyme in catecholamine biosynthesis in response to stress. However, how TH is regulated by epinephrine is still unknown. In the present study, we found that epinephrine increased the expression of TH in a dose- and time-dependent manner in PC12 cells, which was inhibited by propranolol (β-adrenergic receptor inhibitor), but not by phenoxybenzamine (α-adrenergic receptor inhibitor). The increase of TH was also inhibited by SQ22536 (adenylyl cyclase inhibitor), H-89(PKA inhibitor) and LY294002 (phosphatidylinositol 3 kinase inhibitor). More importantly, overexpression of Trx-1 significantly enhanced the expression of TH, while Trx-1 siRNA suppressed TH expression induced by epinephrine. These results suggest that Trx-1 is involved in TH expression induced by epinephrine in PC12 cells. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Tyrosine hydroxylase expression in the olfactory/respiratory epithelium in early sheep fetuses (Ovis aries).

    Science.gov (United States)

    Izvolskaia, Marina; Duittoz, Anne H; Ugrumov, Mikhail V; Tillet, Yves

    2006-04-14

    Transient expression of tyrosine hydroxylase (TH, the first enzyme in catecholamine synthesis) has been shown in different brain and peripheral structures of various species. TH-immunoreactive neurons have been reported in the nasal region of human and rat fetuses migrating to the forebrain with GnRH neurons during embryogenesis. In the present study, immunohistochemical analysis and in situ hybridization were performed in fetal sheep and in vitro sheep embryo olfactory placode cultures to confirm this population in this species. On embryonic days 33 to 35, TH-immunoreactive cells as well as TH cDNA-hybridized cells were found in the olfactory and respiratory epithelium and were spatially separated from GnRH-immunoreactive neurons. In days 40 to 44 of gestation, TH-immunoreactive neurons were no longer observed in the olfactory epithelium, and TH-immunoreactive fibers were found on the trajectories of the olfactory nerves. At this stage, some TH-immunoreactive fibers were also labeled for GnRH. TH-immunoreactive cells were also found in primary cultures of olfactory placodes of fetal sheep at 10 to 18 days in vitro. Some of them coexpressed GnRH. These results imply that olfactory epithelium is also able to give rise to TH expressing cells in fetal sheep, but this expression is suppressed earlier in ontogenesis than in humans due to some unidentified factors not present in the primary cultures of olfactory placode. The role of TH expression remains unclear as in other previously described examples.

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

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

    2014-01-24

    Evidence has shown that attempted suicide in psychiatric disorders is a complex interplay of genes and environment. Noradrenergic dysfunction due to abnormalities in the tyrosine hydroxylase (TH) gene has been implicated in the pathogenesis of suicidal behavior in mood disorders. However, suicide is a leading cause of mortality in schizophrenia too. Recent evidence suggests that TH gene variants may also increase the risk of suicide attempts in schizophrenia patients, although the interaction with established clinical risk factors is unclear. This study aimed to identify TH gene variants conferring risk for suicide attempt in schizophrenia while accounting for the interaction between this gene and clinical risk factors. We performed analysis on four TH SNPs (rs11564717, rs11042950, rs2070762, rs689) and the common TCAT repeat (UniSTS:240639) for 234 schizophrenia patients (51 suicide attempters and 183 non-attempters). Clinical risk factors and ethnic stratification were included as covariates. Single marker analysis identified the SNP rs11564717 (p=0.042) and the TCAT(6) (p=0.004) as risk variants for suicide attempt. We also identified the haplotype A-A-A-G as a risk factor for suicide attempt (p=0.0025). In conclusion, our findings suggest that TH polymorphisms may contribute to the risk of attempted suicide in schizophrenia even after accounting for established clinical risk factors and ethnic stratification. Further larger scale studies are needed to confirm these findings and to understand the mechanisms underlying the role of TH gene variants in suicide attempt in schizophrenia. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2011-03-01

    The distribution of tyrosine hydroxylase (TH) in the brainstem of alpaca (Lama pacos) has been analysed using immunohistochemical methods. The following catecholaminergic cell nuclei have been detected: A1, C1, A2, C2 and area postrema in the medulla oblongata; A5, A6d, A7sc and A7d in the pons; as have several mesencephalic groups: A8, A9l, A9m, A9v, A9pc, A10, A10c, A10d and A10dc. This nuclear parcellation differs from that found in rodents, but agrees with the results reported in other members of the Artiodactyla order, such as giraffe or pig, and with the catecholaminergic distribution detected in species of other mammalian orders. Thus, these findings support the hypothesis that the animals included in the same order show the same nuclear complement in the neuromodulatory systems. In addition, it seems that other species share the same catecholaminergic groups as the alpaca, suggesting that a specific nuclear disposition was important and worth maintaining throughout evolution. Moreover, the distribution of TH has been compared with that of CGRP by double immunohistochemistry. Double-labelled neurons were very isolated and observed only in a few catecholaminergic groups: A1 and C2 in the medulla oblongata, A6d, A7sc and A7d in the pons, and A9l in the mesencephalon. However, interaction between TH and CGRP may be possible in more brainstem regions, particularly the area postrema. This interaction may prove important in the regulation of the specific cardiovascular control of alpacas given their morphological characteristics. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Single low doses of MPTP decrease tyrosine hydroxylase expression in the absence of overt neuron loss.

    Science.gov (United States)

    Alam, Gelareh; Edler, Melissa; Burchfield, Shelbie; Richardson, Jason R

    2017-05-01

    Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a prototypical neurotoxicant used in mice to mimic primary features of PD pathology including striatal dopamine depletion and dopamine neuron loss in the substantia nigra pars compacta (SNc). In the literature, there are several experimental paradigms involving multiple doses of MPTP that are used to elicit dopamine neuron loss. However, a recent study reported that a single low dose caused significant loss of dopamine neurons. Here, we determined the effect of a single intraperitoneal injection of one of three doses of MPTP (0.1, 2 and 20mg/kg) on dopamine neurons, labeled by tyrosine hydroxylase (TH + ), and total neuron number (Nissl + ) in the SNc using unbiased stereological counting. Data reveal a significant loss of neurons in the SNc (TH + and Nissl + ) only in the group treated with 20mg/kg MPTP. Groups treated with lower dose of MPTP (0.1 and 2mg/kg) only showed significant loss of TH + neurons rather than TH + and Nissl + neurons. Striatal dopamine levels were decreased in the groups treated with 2 and 20mg/kg MPTP and striatal terminal markers including, TH and the dopamine transporter (DAT), were only decreased in the groups treated with 20mg/kg MPTP. These data demonstrate that lower doses of MPTP likely result in loss of TH expression rather than actual dopamine neuron loss in the SN. This finding reinforces the need to measure both total neuron number along with TH + cells in determining dopamine neuron loss. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Somatic and neuritic spines on tyrosine hydroxylase-immunopositive cells of rat retina.

    Science.gov (United States)

    Fasoli, Anna; Dang, James; Johnson, Jeffrey S; Gouw, Aaron H; Fogli Iseppe, Alex; Ishida, Andrew T

    2017-05-01

    Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABA A R α1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Locomotor response to novelty correlates with the number of midbrain tyrosine hydroxylase positive cells in rats.

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    Jerzemowska, Grażyna; Plucińska, Karolina; Kulikowski, Michał; Trojniar, Weronika; Wrona, Danuta

    2012-01-04

    The present study investigated whether the higher dopaminergic system activation in rats with high (HRs) rather than low (LRs) locomotor activity in response to novelty depend on the number of cells containing the enzyme tyrosine hydroxylase (TH(+)) and/or differences in the morphology of these cells. One week after the novelty test, brains from male Wistar rats (HRs and LRs) were collected and stained for TH expression (immunohistochemistry) and for morphological analysis (immunofluorescent staining). The morphology and total number of TH(+) cells was analyzed for each A9 (substantia nigra) and A10 (ventral tegmental area) group of the midbrain dopaminergic cells. We found that HRs had a higher total number of TH(+) cells in the whole midbrain dopaminergic region (A9-A10) and in the A9 group only than LRs. In particular midbrain dopaminergic groups of neurons, HR/LR differences were regionally specific: HRs had a higher total number of TH(+) cells in the A9, and in the anterior part of the A10. In contrast, the LRs had a higher number of TH(+) cells in the parabrachial pigmented nucleus (A10) and in the posterior part of the A9. There were no significant differences in the morphology of the midbrain dopamine neurons between HRs and LRs. Moreover, there was a positive correlation between the total number of TH(+) neurons and the locomotor activity score in response to novelty in the whole A9-A10 region and in the particular A9 group only. The results obtained indicate that the higher behavioral activation in resting conditions correlates with the higher number rather than changes in the morphology of the midbrain dopaminergic TH(+) cells. It supports findings on the higher level of dopaminergic system activation in high responders to novelty that depends on the number of midbrain dopaminergic TH(+) neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Microglial number is related to the number of tyrosine hydroxylase neurons in SHR and normotensive rats.

    Science.gov (United States)

    Kapoor, Komal; Bhandare, Amol M; Mohammed, Suja; Farnham, Melissa M J; Pilowsky, Paul M

    2016-07-01

    Microglia are ubiquitously distributed throughout the central nervous system (CNS) and play a critical role in the maintenance of neuronal homeostasis. Recent advances have shown that microglia, never resting cells of the CNS, continuously monitor and influence neuronal/synaptic activity levels, by communicating with neurons with the aid of their dynamic processes. The brainstem contains many catecholaminergic nuclei that are key to many aspects of brain function. This includes C1 neurons of the ventrolateral medulla that are thought to play a critical role in control of the circulation. Despite the role of catecholaminergic brainstem neurons in normal physiology, the presence of microglia that surrounds them is poorly understood. Here, we investigate the spatial distribution and morphology of microglia in catecholaminergic nuclei of the brainstem in 3 strains of rat: Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our data reveal that microglia are heterogeneously distributed within and across different strains of rats. Interestingly, intra-strain comparison of tyrosine hydroxylase-immunoreactive (TH-ir) neuronal and microglial number reveals that microglial number varies with the TH-ir neuronal number in the brainstem. Even though microglial spatial distribution varies across brainstem nuclei, microglial morphology (% area covered, number of end point processes and branch length) does not differ significantly. This work provides the first evidence that even though microglia, in their surveilling state, do not vary appreciably in their morphology across brainstem areas, they do have a heterogeneous pattern of distribution that may be influenced by their local environment. Copyright © 2016. Published by Elsevier B.V.

  19. Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms.

    Science.gov (United States)

    Hole, Magnus; Underhaug, Jarl; Diez, Hector; Ying, Ming; Røhr, Åsmund Kjendseth; Jorge-Finnigan, Ana; Fernàndez-Castillo, Noèlia; García-Cazorla, Angels; Andersson, K Kristoffer; Teigen, Knut; Martinez, Aurora

    2015-09-01

    Pharmacological chaperones are small compounds that correct the folding of mutant proteins, and represent a promising therapeutic strategy for misfolding diseases. We have performed a screening of 10,000 compounds searching for pharmacological chaperones of tyrosine hydroxylase (TH), the tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines. A large number of compounds bound to human TH, isoform 1 (hTH1), but only twelve significantly protected wild-type (hTH1-wt) and mutant TH-R233H (hTH1-p.R202H), associated to the rare neurological disorder TH deficiency (THD), from time-dependent loss of activity. Three of them (named compounds 2, 4 and 5) were subjected to detailed characterization of their functional and molecular effects. Whereas compounds 2 and 4 had a characteristic pharmacological chaperone (stabilizing) effect, compound 5 protected the activity in a higher extent than expected from the low conformational stabilization exerted on hTH1. Compounds 4 and 5 were weak competitive inhibitors with respect to the cofactor BH4 and, as seen by electron paramagnetic resonance, they induced small changes to the first coordination sphere of the catalytic iron. Molecular docking also indicated active-site location with coordination to the iron through a pyrimidine nitrogen atom. Interestingly, compound 5 increased TH activity in cells transiently transfected with either hTH1-wt or the THD associated mutants p.L205P, p.R202H and p.Q381K without affecting the steady-state TH protein levels. This work revealed different mechanisms for the action of pharmacological chaperones and identifies a subtype of compounds that preserve TH activity by weak binding to the catalytic iron. This article is part of a Special Issue entitled: Cofactor-dependent proteins: Evolution, chemical diversity and bio-applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The tyrosine hydroxylase 2 (TH2) system in zebrafish brain and stress activation of hypothalamic cells.

    Science.gov (United States)

    Semenova, S A; Chen, Y-C; Zhao, X; Rauvala, H; Panula, P

    2014-12-01

    Two tyrosine hydroxylases (TH1 and TH2) are found in teleost fish, but no antibodies are available for TH2 protein to analyze the detailed structure of the system. We generated antibodies targeting TH2 and used them to characterize the TH2-producing cells in larval and adult zebrafish brain. The rabbit antisera reliably detected two bands corresponding to TH1 and TH2 close to 55 kDa in brain homogenates. The antisera detected neurons in brain nuclei which express th1 and th2 mRNA; knockdown of th2 expression by morpholino oligonucleotide injection abolished both the th2 mRNA signal and immunoreactivity with the rabbit antisera in TH2 cells. Double staining of samples with the rabbit antiserum made against TH2 and a monoclonal antibody which detects only TH1 allowed identification of cell groups expressing either one of the proteins. Cell groups in preoptic area, anterior, intermediate, and posterior part of the paraventricular organ contained neurons stained with the new TH2 antisera but not with the characterized monoclonal TH1 antibody. Neurons immunoreactive for TH2 and 5-HT were distinct. In situ hybridization for the mRNA of the immediate early gene c-fos combined with TH1/TH2 immunohistochemistry was used to characterize the cells of the zebrafish brain reacting to handling stress and a noxious chemical stimulus. Strong upregulation of c-fos expression was detected in hypothalamic nuclei containing TH2 cells, but few of the c-fos-expressing cells were positive for TH2, suggesting that these stressors do not directly activate a large proportion of TH2 cells.

  1. Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

    Science.gov (United States)

    Kiyokage, Emi; Kobayashi, Kazuto; Toida, Kazunori

    2017-04-01

    Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

  2. Distribution of tyrosine hydroxylase-immunoreactive neurons in the brain of the viviparous fish Gambusia affinis.

    Science.gov (United States)

    Bhat, Shilpa K; Ganesh, C B

    2017-11-01

    Tyrosine hydroxylase (TH) is the common precursor enzyme involved in the biosynthetic pathway of the catecholaminergic neurotransmitters, dopamine and norepinephrine. In this investigation, the neuroanatomical distribution of TH-immunoreactivity was studied in the brain of the female mosquitofish Gambusia affinis. Numerous intensely stained TH-immunoreactive (ir) neurons were scattered in the olfactory bulb with their fibres extending towards the medial olfactory tract, whereas few telencephalic TH-ir cells with distinct fibres were observed in the dorsal nucleus of area ventralis telencephali and the posterior nucleus of area ventralis telencephali regions. Large TH-ir cell populations were seen in the suprachiasmatic nucleus and the nucleus dorsomedialis thalami regions of the diencephalon. Distinct TH-ir cells with long fibres were found at the preoptic area and the nucleus preopticus pars magnocellularis as well as the nucleus preopticus pars parvocellularis regions. Numerous intensely stained TH-ir cells were observed in the paraventricular organ and the nucleus posterior tuberis regions, whereas moderately stained cells were present in the nucleus of recessus lateralis medialis. Several TH-ir neurons were detected in medial and lateral subdivisions of the nucleus lateralis tuberis. Furthermore, the projections of the TH-ir fibres were seen in the proximal pars distalis region of the pituitary gland, where gonadotropin-secreting cells are located, suggesting the communication between TH cells and gonadotrope cells. In the rostral spinal cord, dense aggregations of the TH-ir fibres were noticed. Overall, the widespread distribution of the TH-ir neurons throughout the brain and their fibres in the spinal cord and the pituitary gland suggests diverse roles for the catecholaminergic neurons in various physiological functions including reproduction in the mosquitofish. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Effect of tyrosine hydroxylase overexpression in lymphocytes on the differentiation and function of T helper cells.

    Science.gov (United States)

    Huang, Hui-Wei; Zuo, Cong; Chen, Xiao; Peng, Yu-Ping; Qiu, Yi-Hua

    2016-08-01

    The aim of the present study was to examine the effect of the overexpression of tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines (CAs), in lymphocytes on the differentiation and function of T helper (Th) cells. A recombinant TH overexpression plasmid (pEGFP-N1-TH) was constructed and transfected into mesenteric lymphocytes using nucleofection technology. These cells were stimulated with concanavalin A (Con A) for 48 h and then examined for TH expression and CA content, as well as for the percentage of Th1 and Th2 cells, cytokine concentrations and for the levels of signaling molecules. The lymphocytes overexpressing TH also expressed higher mRNA and protein levels of TH, and synthesized more CAs, including norepinephrine (NE), epinephrine (E) and dopamine (DA) than the mock-transfected control cells. TH gene overexpression in the lymphocytes reduced the percentage of interferon-γ (IFN-γ)-producing CD4+ cells and the ratio of CD4+IFN-γ+/CD4+IL-4+ cells, as well as the percentages of CD4+CD26+ and CD4+CD30+ cells and the ratio of CD4+CD26+/CD4+CD30+ cells. TH overexpression also reduced the secretion of IFN-γ and tumor necrosis factor (TNF) from lymphocytes. Moreover, NE inhibited the Con A-induced lymphocyte proliferation and decreased both cyclic adenosine monophosphate (cAMP) levels and p38 mitogen-activated protein kinase (MAPK) expression in the lymphocytes. Our findings thus indicate that TH gene overexpression promotes the polarization and differentiation of CD4+ cells towards Th2 cells, and this effect is mediated by the cAMP and p38 MAPK signaling pathways.

  4. Species differences in the regulation of tyrosine hydroxylase in Cnemidophorus whiptail lizards.

    Science.gov (United States)

    Woolley, Sarah C; Crews, David

    2004-09-05

    Evolution of behavioral phenotype involves changes in the underlying neural substrates. Cnemidophorus whiptail lizards enable the study of behavioral and neural evolution because ancestral species involved in producing unisexual, hybrid species still exist. Catecholaminergic systems modulate the expression of social behaviors in a number of vertebrates, including whiptails, and therefore we investigated how changes in catecholamine production correlated with evolutionary changes in behavioral phenotype by measuring the size and number of catecholamine producing (tyrosine hydroxylase-immunoreactive, or TH-ir) cells across the reproductive cycle in females from two related whiptail species. Cnemidophorusuniparens is a triploid, parthenogenetic species that arose from hybridization events involving the diploid, sexual species C. inornatus. Prior to ovulation, females from both species display femalelike receptive behaviors. However, after ovulation, only parthenogenetic individuals display malelike mounting behavior. In all nuclei measured, we found larger TH-ir cells in the parthenogen, a difference consistent with species differences in ploidy. In contrast, species differences in the number of TH-ir cells were nucleus specific. In the preoptic area and anterior hypothalamus, parthenogens had fewer TH-ir cells than females of the sexual species. Reproductive state only affected TH-ir cell number in the substantia nigra pars compacta (SNpc), and C. uniparens individuals had more TH-ir cells after ovulation than when previtellogenic. Thus, species differences over the reproductive cycle in the SNpc are correlated with species differences in behavior, and it appears that the process of speciation may have produced a novel neural and behavioral phenotype in the parthenogen.

  5. Expression of Aryl Hydrocarbon Hydroxylase Induction and Suppression of Tyrosine Aminotransferase Induction in Somatic-Cell Hybrids

    Science.gov (United States)

    Benedict, W. F.; Nebert, D. W.; Thompson, E. B.

    1972-01-01

    Aryl hydrocarbon hydroxylase activity is inducible in mouse 3T3 fibroblasts by benz[α]anthracene, whereas no detectable basal or inducible levels of this enzyme occur in rat-hepatoma tissue culture cells. Conversely, tyrosine aminotransferase activity is inducible in hepatoma cells by dexamethasone, whereas only low noninducible levels of this enzyme exist in 3T3 cells. In hybrids formed by fusion of these two parent lines, levels of inducible hydroxylase activity range from the same as, to more than 20-fold greater than, that in the 3T3 parent; aminotransferase levels remain very low and noninducible in all of these same hybrids. A majority of the 1S-chromosomal complement from each parent is retained in most of these hybrids. The kinetics of hydroxylase induction and degradation, responses of hydroxylase induction to actinomycin D and cycloheximide, and the relative thermolability of the control and induced activities are similar in the 3T3 parent and in the hybrids. Failure to inactivate any of the aminotransferase activity in the hybrids with antibody specific for the rat enzyme indicates that all of the basal noninducible aminotransferase activity is derived from the mouse 3T3 parent. Images PMID:4403306

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

    Science.gov (United States)

    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 (PSexually inactive rams had fewer (Psexually 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.

  7. Molecular phenotyping of transient postnatal tyrosine hydroxylase neurons in the rat bed nucleus of the stria terminalis.

    Science.gov (United States)

    Carter, David A

    2017-07-01

    The bed nucleus of the stria terminalis (BNST) is a complex integrative centre in the forebrain, composed of multiple sub-nuclei, each with discrete populations of neurons. Progress in understanding BNST function, both in the adult and during postnatal maturation, is dependent upon a more complete characterization of neuronal phenotypes in the BNST. The aim of the current study was to define the molecular phenotype of one postnatal BNST neuronal population, in order to identify molecular factors that may underlie both (protein marker-related) immaturity, and secondly, the transience of this phenotype. This BNST population was originally identified by high, but transient expression of the EGR1 transcription factor (TF) in postnatal rat lateral intermediate BNST (BNSTLI). The current results confirm a high level of Egr1 activation in postnatal day 10 (PN10) male BNSTLI that is lost at PN40, and now demonstrate a similar pattern of transient activation in female brains. Apparent cellular immaturity in this population, as indicated by low levels of the adult neuronal marker NeuN/RBFOX3, was found to be uncorrelated with both key neuronal regulator protein expression (SOX2 and REST), and also RBFOX2 protein levels. The BNSTLI neurons have a partial catecholaminergic phenotype (tyrosine hydroxylase-positive/dopa decarboxylase-negative; TH+ve/DDC-ve) that is lost at PN40. In contrast, the co-expressed neuropeptide, somatostatin, is maintained, albeit at lower levels, at PN40. The transcriptional basis of the transient and partial catecholaminergic phenotype was investigated by analysing TFs known to maintain adult dopaminergic (TH+ve/DDC+ve) neuronal phenotypes. The BNSTLI neurons were shown to lack forkhead TFs including FOXA1, FOXA2 and FOXO1. In addition, the BNSTLI neurons had low, primarily cytoplasmic, expression of NR4A2/NURR1, an orphan nuclear receptor that is critical for adult maintenance of midbrain dopamine neurons. These results detail the molecular features

  8. Effect of runway training on rat brain tyrosine hydroxylase: differential effect of continuous and partial reinforcement schedules.

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    Boarder, M R; Feldon, J; Gray, J A; Fillenz, M

    1979-12-01

    Previous experiments have implicated ascending noradrenergic systems in the development of the behavioural responses to different patterns of reward. In this report food deprived male Sprague--Dawley rats were trained to run a straight alley for good reward on a continuous reinforcement (CRF) or a partial reinforcement (PRF) schedule. Tyrosine hydroxylase measured in a partially solubilized preparation from hippocampus and hypothalamus at the end of acquisition was not different from controls, indicating that enzyme induction does not occur during either training schedules. However, hippocampal synaptosomal tyrosine hydroxylation rates from the CRF group was significantly higher than from either the PRF group or the handled controls. This indicates that at the end of the acquisition schedule the noradrenergic projection to hippocampus was more active in the CRF group than with the PRF group or the handled control.

  9. Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation.

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    Michael F Salvatore

    Full Text Available BACKGROUND: Tyrosine hydroxylase (TH regulates dopamine (DA bioavailability. Its product, L-DOPA, is an established treatment for Parkinson's disease (PD, suggesting that TH regulation influences locomotion. Site-specific phosphorylation of TH at ser31 and ser40 regulates activity. No direct evidence shows that ser40 phosphorylation is the dominating mechanism of regulating TH activity in vivo, and physiologically-relevant stimuli increase L-DOPA biosynthesis independent of ser40 phosphorylation. Significant loss of locomotor activity occurs in aging as in PD, despite less loss of striatal DA or TH in aging compared to the loss associated with symptomatic PD. However, in the substantia nigra (SN, there is equivalent loss of DA or TH in aging and at the onset of PD symptoms. Growth factors increase locomotor activity in both PD and aging models and increase DA bioavailability and ser31 TH phosphorylation in SN, suggesting that ser31 TH phosphorylation status in the SN, not striatum, regulates DA bioavailability necessary for locomotor activity. METHODOLOGY AND PRINCIPAL FINDINGS: We longitudinally characterized locomotor activity in young and older Brown-Norway Fischer 344 F(1 hybrid rats (18 months apart in age at two time periods, eight months apart. The aged group served as an intact and pharmacologically-naïve source of deficient locomotor activity. Following locomotor testing, we analyzed DA tissue content, TH protein, and TH phosphorylation in striatum, SN, nucleus accumbens, and VTA. Levels of TH protein combined with ser31 phosphorylation alone reflected inherent differences in DA levels among the four regions. Measures strictly pertaining to locomotor activity initiation significantly correlated to DA content only in the SN. Nigral TH protein and ser31 phosphorylation together significantly correlated to test subject's maximum movement number, horizontal activity, and duration. CONCLUSIONS/SIGNIFICANCE: Together, these results show ser

  10. Tyrosine hydroxylase positive perisomatic rings are formed around various amacrine cell types in the mammalian retina.

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    Debertin, Gábor; Kántor, Orsolya; Kovács-Öller, Tamás; Balogh, Lajos; Szabó-Meleg, Edina; Orbán, József; Nyitrai, Miklós; Völgyi, Béla

    2015-08-01

    Dopaminergic neurons of the central nervous system are mainly found in nuclei of the midbrain and the hypothalamus that provide subcortical and cortical targets with a rich and divergent innervation. Disturbance of signaling through this system underlies a variety of deteriorating conditions such as Parkinson's disease and schizophrenia. Although retinal dopaminergic signaling is largely independent of the above circuitry, malfunction of the retinal dopaminergic system has been associated with anomalies in visual adaptation and a number of retinal disorders. Dopamine (DA) is released mainly in a paracrine manner by a population of tyrosine hydroxylase expressing (TH(+) ) amacrine cells (AC) of the mammalian retina; thus DA reaches virtually all retinal cell types by diffusion. Despite this paracrine release, however, the so called AII ACs have been considered as the main targets of DA signaling owing to a characteristic and robust ring-like TH(+) innervation to the soma/dendritic-stalk area of AII cells. This apparent selectivity of TH(+) innervation seems to contradict the divergent DAergic signaling scheme of other brain loci. In this study, however, we show evidence for intimate proximity between TH(+) rings and somata of neurochemically identified non-AII cells. We also show that this phenomenon is not species specific, as we observe it in popular mammalian animal models including the rabbit, the rat, and the mouse. Finally, our dataset suggests the existence of further, yet unidentified post-synaptic targets of TH(+) dendritic rings. Therefore, we hypothesize that TH(+) ring-like structures target the majority of ACs non-selectively and that such contacts are wide-spread among mammals. Therefore, this new view of inner retinal TH(+) innervation resembles the divergent DAergic innervation of other brain areas through the mesolimbic, mesocortical, and mesostriatal signaling streams. AII amacrine cells have been considered as the main targets of dopamine

  11. 'Roid rage in rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase.

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    Wood, Ruth I; Armstrong, Abigail; Fridkin, Vlad; Shah, Vivek; Najafi, Allison; Jakowec, Michael

    2013-02-17

    In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight. Alternatively, AAS may increase impulsive behavior, consistent with the popular image of 'roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to respond on a nose-poke on a 10 min fixed-interval schedule for the opportunity to fight in their home cage with an unfamiliar rat. Although testosterone increased aggression (6.3±1.3 fights/5 min vs 2.4±0.8 for controls, ptestosterone, 32.4±7.0 for vehicle). This suggests that testosterone does not enhance motivation for aggression. To test for impulsivity, rats were trained to respond for food in a delay-discounting procedure. In an operant chamber, one lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0, 15, 30 or 45 s). In testosterone- and vehicle-treated rats, body weights and food intake did not differ. However, testosterone-treated rats chose the larger, delayed reward more often (4.5±0.7 times in 10 trials with 45 s delay) than vehicle controls (2.5±0.5 times, ptestosterone enhances aggression, this does not include an increase in impulsive behavior or motivation to fight. This is further supported by measurement of tyrosine hydroxylase (TH) by Western immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and executive function (medial prefrontal cortex, PFC). There were no differences in TH between testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly reduced TH (to 76.9±3.1% of controls, p<0.05) in the caudate-putamen, a brain area important for behavioral inhibition, motor control and habit learning. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

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    E.E.S. Hermel

    2001-09-01

    Full Text Available Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control and handled (pups were handled for 1 min once a day during the first 10 days of life. At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively, paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9 or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7 nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

  13. The effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues.

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    Talas, Zeliha Selamoglu; Yurekli, Muhittin

    2006-01-01

    Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity. Copyright (c) 2005 John Wiley & Sons, Ltd.

  14. Validation of Anti-CSPα, SNAP25, Tyrosine Hydroxylase, Ubiquitin, Cleaved Caspase 3, and pSer PKC Motif Antibodies for Utilization in Western Blotting.

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    Shirafuji, Toshihiko; Ueyama, Takehiko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2017-12-26

    There are many commercial antibodies with little information provided by their suppliers as to their reliability. Accordingly, commercial antibodies require proper validation before being used in scientific research. In this study, we validated several commercial antibodies, including anti-CSPα, SNAP25, tyrosine hydroxylase, ubiquitin, cleaved caspase 3, and pSer PKC motif. Anti-CSPα, SNAP25, and tyrosine hydroxylase antibodies could detect their endogenous target proteins with some degree of cross-reactivity. Furthermore, clear SNAP25 staining was observed with SNAP25 antibody. Antibodies directed against ubiquitin, cleaved caspase 3, and pSer PKC motif could detect poly-ubiquitination, apoptosis, and phosphorylation, respectively.

  15. Stimulatory effect of nobiletin, a citrus polymethoxy flavone, on catecholamine synthesis through Ser19 and Ser40 phosphorylation of tyrosine hydroxylase in cultured bovine adrenal medullary cells.

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    Zhang, Han; Yanagihara, Nobuyuki; Toyohira, Yumiko; Takahashi, Keita; Inagaki, Hirohide; Satoh, Noriaki; Li, Xiaoja; Goa, Xiumei; Tsutsui, Masato; Takahaishi, Kojiro

    2014-01-01

    We previously reported the dual effects of nobiletin, a compound of polymethoxy flavones found in citrus fruits, on catecholamine secretion in cultured bovine adrenal medullary cells. Here, we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine in a time (20-30 min)- and concentration (1.0-100 μM)-dependent manner. Nobiletin (10-100 μM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on (14)C-catecholamine synthesis was not observed when extracellular Ca(2+) was not present in the incubation medium. Protein kinase inhibitors including H-89, an inhibitor of cyclic AMP-dependent protein kinase, and KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. Nobiletin also induced the phosphorylation of tyrosine hydroxylase at Ser(19) and Ser(40). Nobiletin (1.0-100 μM) inhibited (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis through tyrosine hydroxylase phosphorylation at Ser(19) and Ser(40), whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.

  16. Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

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    Cui, X; Pertile, R; Liu, P; Eyles, D W

    2015-09-24

    Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes

  17. Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase.

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    Nandi, Shyam Sundar; Zheng, Hong; Sharma, Neeru M; Shahshahan, Hamid R; Patel, Kaushik P; Mishra, Paras K

    2016-10-01

    MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat's heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function. © 2016 by the American Diabetes Association.

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

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    Pedro Espinosa

    2016-01-01

    Full Text Available 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.

  19. Effects of formaldehyde exposure on anxiety-like and depression-like behavior, cognition, central levels of glucocorticoid receptor and tyrosine hydroxylase in mice.

    Science.gov (United States)

    Li, Yani; Song, Zhuoyi; Ding, Yujuan; Xin, Ye; Wu, Tong; Su, Tao; He, Rongqiao; Tai, Fadao; Lian, Zhenmin

    2016-02-01

    Formaldehyde exposure is toxic to the brains of mammals, but the mechanism remains unclear. We investigated the effects of inhaled formaldehyde on anxiety, depression, cognitive capacity and central levels of glucocorticoid receptor and tyrosine hydroxylase in mice. After exposure to 0, 1 or 2 ppm gaseous formaldehyde for one week, we measured anxiety-like behavior using open field and elevated plus-maze tests, depression-like behavior using a forced swimming test, learning and memory using novel object recognition tests, levels of glucocorticoid receptors in the hippocampus and tyrosine hydroxylase in the Arc, MPOA, ZI and VTA using immuhistochemistry. We found that inhalation of 1 ppm formaldehyde reduced levels of anxiety-like behavior. Inhalation of 2 ppm formaldehyde reduced body weight, but increased levels of depression-like behavior, impaired novel object recognition, and lowered the numbers of glucocorticoid receptor immonureactive neurons in the hippocampus and tyrosine hydroxylase immonureactive neurons in the ventral tegmental area and the zona incerta, medial preoptic area. Different concentrations of gaseous formaldehyde result in different effects on anxiety, depression-like behavior and cognition ability which may be associated with alterations in hippocampal glucocorticoid receptors and brain tyrosine hydroxylase levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Cytoplasmic aggregates of dynactin in iPSC-derived tyrosine hydroxylase-positive neurons from a patient with Perry syndrome.

    Science.gov (United States)

    Mishima, Takayasu; Ishikawa, Taizo; Imamura, Keiko; Kondo, Takayuki; Koshiba, Yasushi; Takahashi, Ryosuke; Takahashi, Jun; Watanabe, Akihiro; Fujii, Naoki; Tsuboi, Yoshio; Inoue, Haruhisa

    2016-09-01

    Perry syndrome is a rare autosomal dominant disorder clinically characterized by parkinsonism with depression/apathy, weight loss, and central hypoventilation. Eight mutations in DCTN1 gene have been reported. A novel disease model is required because the detailed pathogenesis remains unclear. To develop a novel model, we generated induced pluripotent stem cells (iPSCs) from a Perry syndrome patient with F52L mutation in DCTN1, and describe clinical and neuroimaging investigations. We differentiated iPSCs into tyrosine hydroxylase (TH)-positive neurons. Immunocytochemistry analyses of control and mutant were performed. The patient displayed levodopa responsive parkinsonism. Dopamine transporter single photon emission tomography showed markedly decreased uptake in the striatum, and metaiodobenzylguanidine cardiac scintigraphy also showed decreased uptake. Perry syndrome TH-positive neurons showed dynactin aggregates in cytoplasm. TH-positive neurons from Perry syndrome iPSCs recapitulated an aspect of the disease phenotype of Perry syndrome. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Generation of an iPSC line from a patient with tyrosine hydroxylase (TH) deficiency: TH-1 iPSC.

    Science.gov (United States)

    Jung-Klawitter, Sabine; Blau, Nenad; Sebe, Attila; Ebersold, Juliane; Göhring, Gudrun; Opladen, Thomas

    2016-11-01

    Fibroblasts from a male patient with compound heterozygous variants in the tyrosine hydroxylase gene (TH; OMIM: 191290; c.[385-C>T]; [692-G>C]/p.[R129*]; [R231P]), the rate-limiting enzyme for dopamine synthesis, were reprogrammed to iPSCs using episomal reprogramming delivering the reprogramming factors Oct3/4, Sox2, L-Myc, Lin28, Klf4 and p53 shRNA Okita et al. (2011). Pluripotency of TH-1 iPSC was verified by immunohistochemistry and RT-PCR analysis. Cells exhibited a normal karyotype and differentiated spontaneously into the 3 germ layers in vitro. TH-1 iPSC represents the first model system to study the pathomechanism of this rare metabolic disease and provides a useful tool for drug testing. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

  2. Sympathetic Hyperactivity, Increased Tyrosine Hydroxylase and Exaggerated Corpus Cavernosum Relaxations Associated with Oxidative Stress Plays a Major Role in the Penis Dysfunction in Townes Sickle Cell Mouse.

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    Fábio H Silva

    Full Text Available Sickle cell disease patients display priapism that may progress to erectile dysfunction. However, little is known about the pathophysiological alterations of corpus cavernosum in sickle cell disease.Thus, this study aimed to evaluate the functional and molecular alterations of sympathetic machinery and nitric oxide-cyclic guanosine monophosphate signaling pathway in Townes transgenic sickle cell disease mice.Concentration-response curves to contractile (phenylephrine and relaxant agents (acetylcholine and sodium nitroprusside were obtained in corpus cavernosum strips from sickle and C57BL/6 (control mice. Neurogenic contractions and nitrergic relaxations were obtained using electrical-field stimulation. Measurements of endothelial nitric oxide synthase (eNOS, neuronal nitric oxide synthase (nNOS, phosphodiesterase-5 (PDE5 and α1A-, α1B- and α1D-adrenoceptor mRNA expressions and reactive-oxygen species were performed. Tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured.The neurogenic contractions were higher in the sickle cell disease group, in association with elevated tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expression, as well as increased tyrosine hydroxylase mRNA expression. Likewise, phenylephrine-induced contractions were greater in the sickle mice, whereas α1A-, α1B- and α1D-adrenoceptor mRNA expression remained unchanged. Cavernosal relaxations to acetylcholine, sodium nitroprusside and EFS were higher in sickle mice, accompanied by decreased eNOS and nNOS, along with lower PDE5 mRNA expression. An increase of about 40% in reactive-oxygen species generation in corpus cavernosum from sickle mice was also detected.Our study shows that decreased nitric oxide bioavailability in erectile tissue due to increased oxidative stress leads to both sympathetic hyperactivity and dysregulation of nitric oxide

  3. ERK5 activity is required for nerve growth factor-induced neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells.

    Science.gov (United States)

    Obara, Yutaro; Yamauchi, Arata; Takehara, Shin; Nemoto, Wataru; Takahashi, Maho; Stork, Philip J S; Nakahata, Norimichi

    2009-08-28

    Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt(2)cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 mum, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt(2)cAMP, but BIX02188 and BIX02189, at 30 mum, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt(2)cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt(2)cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

  4. The absence of phosphorylated tyrosine hydroxylase expression in the purkinje cells of the ataxic mutant pogo mouse.

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    Lee, N S; Kim, C T; Han, S Y; Kawk, J H; Sawada, K; Fukui, Y; Jeong, Y G

    2006-06-01

    The pogo mouse is a new ataxic autosomal recessive mutant that arose in Korean wild mice (KJR/Mskist). Its ataxic phenotype includes difficulty in maintaining a normal posture and the inability to walk in a straight line. Several studies have reported that tyrosine hydroxylase (TH) is persistently ectopically expressed in particular subsets of Purkinje cells in a parasagittal banding pattern in several ataxic mutant mice, e.g. tottering alleles and pogo mice. In this present study, we examined the expression of an enzymatically active form of TH and phosphorylated TH at Ser(40) (phospho-TH) by using immunohistochemistry and double immunofluorescence in the cerebellum of pogo mice. TH immunostaining appeared in some Purkinje cells in pogo, but in only a few of Purkinje cells of their heterozygous littermate controls. In all groups of mice, no phospho-TH immunoreactive Purkinje cells were observed in the cerebellum, although subsets of TH immunoreactive Purkinje cells were found in adjacent sections. This study suggests that TH expression in the Purkinje cells of pogo abnormally increases without activation of this enzyme by phosphorylation. This may mean that TH in the Purkinje cells of these mutants does not catalyse the conversion of tyrosine to l-DOPA, and is not related to catecholamine synthesis.

  5. Odorant Sensory Input Modulates DNA Secondary Structure Formation and Heterogeneous Ribonucleoprotein Recruitment on the Tyrosine Hydroxylase and Glutamic Acid Decarboxylase 1 Promoters in the Olfactory Bulb.

    Science.gov (United States)

    Wang, Meng; Cai, Elizabeth; Fujiwara, Nana; Fones, Lilah; Brown, Elizabeth; Yanagawa, Yuchio; Cave, John W

    2017-05-03

    Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. For a subset of olfactory bulb interneurons, activity-dependent changes in GABA are reflected by corresponding changes in Glutamate decarboxylase 1 ( Gad1 ) expression levels. Mechanisms regulating Gad1 promoter activity are poorly understood, but here we show that a conserved G:C-rich region in the mouse Gad1 proximal promoter region both recruits heterogeneous nuclear ribonucleoproteins (hnRNPs) that facilitate transcription and forms single-stranded DNA secondary structures associated with transcriptional repression. This promoter architecture and function is shared with Tyrosine hydroxylase ( Th ), which is also modulated by odorant-dependent activity in the olfactory bulb. This study shows that the balance between DNA secondary structure formation and hnRNP binding on the mouse Th and Gad1 promoters in the olfactory bulb is responsive to changes in odorant-dependent sensory input. These findings reveal that Th and Gad1 share a novel transcription regulatory mechanism that facilitates sensory input-dependent regulation of dopamine and GABA expression. SIGNIFICANCE STATEMENT Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. This study shows that transcription of genes encoding rate-limiting enzymes for GABA and dopamine biosynthesis ( Gad1 and Th , respectively) in the mammalian olfactory bulb is regulated by G:C-rich regions that both recruit heterogeneous nuclear ribonucleoproteins (hnRNPs) to facilitate transcription and form single-stranded DNA secondary structures associated with repression. hnRNP binding and formation of DNA secondary structure on the Th and Gad1 promoters are mutually exclusive, and odorant sensory input levels regulate the balance between these regulatory features. These

  6. Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis.

    Science.gov (United States)

    Jenei-Lanzl, Zsuzsa; Capellino, Silvia; Kees, Frieder; Fleck, Martin; Lowin, Torsten; Straub, Rainer H

    2015-02-01

    Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH(+)) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH(+) cells and mode of TH induction are not known. Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH(+) neuronal precursor cells (TH(+) neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe(2+)) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH(+) cells, murine TH(+) neuronal cells were generated from mesenchymal stem cells. TH(+) neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH(+) neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH(+) cells, reversed this effect. The anti-inflammatory effect of TH(+) neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH(+) synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH(+) neuronal cells might open new avenues for cell-based therapy. 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.

  7. Tyrosine hydroxylase expression in CD4(+) T cells is associated with joint inflammatory alleviation in collagen type II-induced arthritis.

    Science.gov (United States)

    Zhao, Xu-Ying; Cui, Shi-Wei; Wang, Xiao-Qin; Peng, Yu-Ping; Qiu, Yi-Hua

    2013-10-01

    We have recently reported that CD4(+) T cells synthesize and secrete catecholamines that facilitate a shift of T helper 1 (Th1)/Th2 balance toward Th2 polarization. In this study, we used an animal model of human rheumatoid arthritis, collagen type II-induced arthritis (CIA), to explore relationship between catecholamine production in CD4(+) T cells and Th1-/Th2-mediated joint inflammation. Histopathological observation of ankle joints of CIA mice displayed an evident inflammatory change on day 35 and a major damage to bones on day 55 post-immunization. Expression of Th1-specific transcription factor, T-bet, and cytokines, IL-2 and IFN-γ, and Th2-specific transcription factor, GATA-3, and cytokines, IL-4 and IL-10, was all upregulated on days 35 and 55 post-immunization, but the elevated Th1 response tended to decrease and the enhanced Th2 response tended to increase with the CIA progression. Expression of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines, dramatically increased in ankle joints of CIA mice, although this increase was reduced on day 55 relative to that on day 35 post-immunization. In synovial tissue of CIA ankle joints but not normal joints, CD4-, T-bet-, GATA-3-, and TH-immunoreactive cells were found. Importantly, co-expressed cells with CD4 and TH, T-bet and TH, and GATA-3 and TH were observed in synovial tissue of CIA ankle joints. These results suggest that an increase in catecholamine production occurs in inflamed joints of CIA. The catecholamines are, at least in part, from Th1 and Th2 cells, and they may be related to joint inflammatory alleviation in CIA progression.

  8. Non-neural tyrosine hydroxylase, via modulation of endocrine pancreatic precursors, is required for normal development of beta cells in the mouse pancreas.

    Science.gov (United States)

    Vázquez, Patricia; Robles, Ana M; de Pablo, Flora; Hernández-Sánchez, Catalina

    2014-11-01

    Apart from transcription factors, little is known about the molecules that modulate the proliferation and differentiation of pancreatic endocrine cells. The early expression of tyrosine hydroxylase (TH) in a subset of glucagon(+) cells led us to investigate whether catecholamines have a role in beta cell development. We studied the immunohistochemical characteristics of TH-expressing cells in wild-type (Th (+/+) ) mice during early pancreas development, and analysed the endocrine pancreas phenotype of TH-deficient (Th (-/-) ) mice. We also studied the effect of dopamine addition and TH-inhibition on insulin-producing cells in explant cultures. In the mouse pancreas at embryonic day (E)12.5-E13.5, the ∼10% of early glucagon(+) cells that co-expressed TH rarely proliferated and did not express the precursor marker neurogenin 3 at E13.5. The number of insulin(+) cells in the Th (-/-) embryonic pancreas was decreased as compared with wild-type embryos at E13.5. While no changes in pancreatic and duodenal homeobox 1 (PDX1)(+)-progenitor cell number were observed between groups at E12.5, the number of neurogenin 3 and NK2 homeobox 2 (NKX2.2)-expressing cells was reduced in Th (-/-) embryonic pancreas, an effect that occurred in parallel with increased expression of the transcriptional repressor Hes1. The potential role of dopamine as a pro-beta cell stimulus was tested by treating pancreas explants with this catecholamine, which resulted in an increase in total insulin content and insulin(+) cells relative to control explants. A non-neural catecholaminergic pathway appears to modulate the pancreatic endocrine precursor and insulin producing cell neogenesis. This finding may have important implications for approaches seeking to promote the generation of beta cells to treat diabetes.

  9. Regulation of Tyrosine Hydroxylase Activity in Cultured Mouse Neuroblastoma Cells: Elevation Induced by Analogs of Adenosine 3′:5′-Cyclic Monophosphate

    Science.gov (United States)

    Waymire, J. C.; Weiner, N.; Prasad, K. N.

    1972-01-01

    Mouse neuroblastoma cells in culture have been used as a model for the study of the mechanism by which activities of tyrosine hydroxylase (EC 1.14.3.a) are regulated in sympathetic tissue. The activity of tyrosine hydroxylase in cultured cells drops to barely detectable activities after 1 week and remains low for months in culture in the uncloned cell line of neuroblastoma. Activity in an adrenergic clone isolated from the uncloned line has about 20% of the activity of the fresh grated tumor cell. N6, O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate causes a concentration and time-dependent increase in enzyme activity in both the cloned and uncloned cell lines. Enzyme activity is elevated by other stable analogs of adenosine 3′:5′-cyclic monophosphate, notably the N6-monobutyryl, 8-aminomethyl, and 8-methylthio derivatives of the cyclic nucleotide; by the inhibitor of cyclic nucleotide phosphodiesterase, papaverine; and by sodium butyrate. Changes in cell morphology and tyrosine hydroxylase activity are shown not to be necessarily related. PMID:4403308

  10. Polymorphism in the tyrosine hydroxylase (TH gene is associated with activity-impulsivity in German Shepherd Dogs.

    Directory of Open Access Journals (Sweden)

    Eniko Kubinyi

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

  11. Locomotor response to novelty correlates with differences in number and morphology of hypothalamic tyrosine hydroxylase positive cells in rats.

    Science.gov (United States)

    Jerzemowska, Grażyna; Plucińska, Karolina; Kuśmierczak, Magda; Myślińska, Dorota; Orzeł-Gryglewska, Jolanta

    2014-02-01

    Individual differences in the intensity of locomotor response to a new environment (exploratory reaction) are generally used as a model to study individual vulnerability to stress and drug addiction. In the present work we studied the number, distribution and morphology of the hypothalamic cells expressing tyrosine hydroxylase (TH+ cells) (immunohistochemical and immunofluorescent staining) in male Wistar rats divided based on high (HR), midline (MR) or low (LR) locomotor activity in response to novelty. Morphology and total number of TH+ cells were analyzed for A11-A15 dopaminergic groups. We found correlation between the total number of hypothalamic TH+ cells in the whole A11-A15 area and the locomotor activity. The differences were most pronounced in some of the hypothalamic nuclei, i.e. in the rostro-caudal extension of the A11, A12 and A14 structures, where the HR rats had a significantly higher number of TH+ cells in comparison to the MR and LR rats. Morphology analysis of TH+ cells showed HR/MR/LR differences in single cell area and perimeter and, to a lesser extent, in the other morphometric parameters such as length of the major and minor axes, or circularity factor. The results suggest that the behavioral traits which characterize the HR animals and are correlated with increased susceptibility to stress and propensity to develop drug addictions can be determined by the number, distribution, activity and perhaps the morphology of the cells in the dopaminergic systems. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Cloning and expression analysis of tyrosine hydroxylase and changes in catecholamine levels in brain during ontogeny and after sex steroid analogues exposure in the catfish, Clarias batrachus.

    Science.gov (United States)

    Mamta, Sajwan Khatri; Raghuveer, Kavarthapu; Sudhakumari, Cheni-Chery; Rajakumar, Anbazhagan; Basavaraju, Yaraguntappa; Senthilkumaran, Balasubramanian

    2014-02-01

    Tyrosine hydroxylase (Th) is the rate-limiting enzyme for catecholamine (CA) biosynthesis and is considered to be a marker for CA-ergic neurons, which regulate the levels of gonadotropin-releasing hormone in brain and gonadotropins in the pituitary. In the present study, we cloned full-length cDNA of Th from the catfish brain and evaluated its expression pattern in the male and female brain during early development and after sex-steroid analogues treatment using quantitative real-time PCR. We measured the CA levels to compare our results on Th. Cloned Th from catfish brain is 1.591 kb, which encodes a putative protein of 458 amino acid residues and showed high homology with other teleosts. The tissue distribution of Th revealed ubiquitous expression in all the tissues analyzed with maximum expression in male and female brain. Copy number analysis showed two-fold more transcript abundance in the female brain when compared with the male brain. A differential expression pattern of Th was observed in which the mRNA levels were significantly higher in females compared with males, during early brain development. CAs, l-3,4-dihydroxyphenylalanine, dopamine, and norepinephrine levels measured using high-performance liquid chromatography with electrochemical detection in the developing male and female brain confirmed the prominence of the CA-ergic system in the female brain. Sex-steroid analogue treatment using methyltestosterone and ethinylestradiol confirmed our findings of the differential expression of Th related to CA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Expression of tyrosine hydroxylase in CD4+T cells contributes to alleviation of Th17/Treg imbalance in collagen-induced arthritis.

    Science.gov (United States)

    Wang, Xiao-Qin; Liu, Yan; Cai, Huan-Huan; Peng, Yu-Ping; Qiu, Yi-Hua

    2016-12-01

    Tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines, is expressed in T lymphocytes. However, the role of T cell-expressed TH in rheumatoid arthritis (RA) is less clear. Herein, we aimed to show the contribution of TH expression by CD4 + T cells to alleviation of helper T (Th)17/regulatory T (Treg) imbalance in collagen-induced arthritis (CIA), a mouse model of RA. CIA was prepared by intradermal injection of collagen type II (CII) at tail base of DBA1/J mice. Expression of TH in the spleen and the ankle joints was measured by real-time polymerase chain reaction and Western blot analysis. Percentages of TH-expressing Th17 and Treg cells in splenic CD4 + T cells were determined by flow cytometry. Overexpression and knockdown of TH gene in CD4 + T cells were taken to evaluate effects of TH on Th17 and Treg cells in CIA. TH expression was upregulated in both the inflamed tissues (spleen and ankle joints) and the CD4 + T cells of CIA mice. In splenic CD4 + T cells, the cells expressing TH were increased during CIA. These cells that expressed more TH in CIA were mainly Th17 cells rather than Treg cells. TH gene overexpression in CD4 + T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion, whereas TH gene knockdown enhanced the Th17 cell activity. In contrast, TH gene overexpression increased Treg-related cytokine expression and secretion in CD4 + T cells of CIA mice, while TH gene knockdown decreased the Treg cell changes. Collectively, these findings show that CIA induces TH expression in CD4 + T cells, particularly in Th17 cells, and suggest that the increased TH expression during CIA represents an anti-inflammatory mechanism.

  14. Squamosamide derivative FLZ protected tyrosine hydroxylase function in a chronic MPTP/probenecid mouse model of Parkinson's disease.

    Science.gov (United States)

    Bao, Xiu-Qi; Wu, Liang-Yu; Wang, Xiao-Liang; Sun, Hua; Zhang, Dan

    2015-05-01

    Parkinson's disease (PD) is a chronic, progressive neurodegenerative disorder characterized by motor impairments and loss of dopaminergic neurons in the substantia nigra. FLZ (formulated as: N-2-(4-hydroxy-phenyl)-ethyl]-2-(2, 5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) is a novel synthetic derivative of squamosamide from a Chinese herb and has been proven to protect dopaminergic neurons in subacute PD models. However, whether FLZ has a neuroprotective effect on chronic PD model is still unknown. The present study was designed to verify the neuroprotection of FLZ on chronic PD mouse model induced by MPTP combined with probenecid (MPTP/p). The results showed that treatment of mice with FLZ for 9 weeks significantly improved motor behavior and dopaminergic neuronal function of mice injected with MPTP/p. The beneficial effects of FLZ attributed to the elevation of dopaminergic neuron number, dopamine level, and tyrosine hydroxylase (TH) activity, as well as decrease of α-synuclein (α-Syn) expression, α-Syn phosphorylation, nitration, and aggregation. Moreover, FLZ decreased the interaction between α-Syn and TH, which eventually improved dopaminergic neuronal function. Mechanistic study demonstrated that FLZ increased Akt and mTOR phosphorylation, suggesting that FLZ activated Akt/mTOR signaling pathway and this might be involved in the neuroprotection of FLZ. The present results provided more elaborate in vivo evidences to support the neuroprotective effect of FLZ on dopaminergic neurons of chronic PD mouse model and the potential of FLZ to be developed as new drug to treat PD.

  15. The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

    Science.gov (United States)

    Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

    2016-07-01

    In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

  16. Striatal tyrosine hydroxylase-positive neurons are associated with L-DOPA-induced dyskinesia in hemiparkinsonian mice.

    Science.gov (United States)

    Keber, U; Klietz, M; Carlsson, T; Oertel, W H; Weihe, E; Schäfer, M K-H; Höglinger, G U; Depboylu, C

    2015-07-09

    L-3,4-Dihydroxyphenylalanine (L-DOPA) is the therapeutic gold standard in Parkinson's disease. However, long-term treatment is complicated by the induction of debilitating abnormal involuntary movements termed L-DOPA-induced dyskinesias (LIDs). Until today the underlying mechanisms of LID pathogenesis are not fully understood. The aim of this study was to reveal new factors, which may be involved in the induction of LID. We have focused on the expression of striatal tyrosine hydroxylase-positive (TH+) neurons, which are capable of producing either L-DOPA or dopamine (DA) in target areas of ventral midbrain DAergic neurons. To address this issue, a daily L-DOPA dose was administered over the course of 15 days to mice with unilateral 6-hydroxydopamine-induced lesions of the medial forebrain bundle and LIDs were evaluated. Remarkably, the number of striatal TH+ neurons strongly correlated with both induction and severity of LID as well as ΔFosB expression as an established molecular marker for LID. Furthermore, dyskinetic mice showed a marked augmentation of serotonergic fiber innervation in the striatum, enabling the decarboxylation of L-DOPA to DA. Axial, limb and orolingual dyskinesias were predominantly associated with TH+ neurons in the lateral striatum, whereas medially located TH+ neurons triggered locomotive rotations. In contrast, identified accumbal and cortical TH+ cells did not contribute to the generation of LID. Thus, striatal TH+ cells and serotonergic terminals may cooperatively synthesize DA and subsequently contribute to supraphysiological synaptic DA concentrations, an accepted cause in LID pathogenesis. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Manganese tissue accumulation and tyrosine hydroxylase immunostaining response in the Neotropical freshwater crab, Dilocarcinus pagei, exposed to manganese.

    Science.gov (United States)

    Ponzoni, Silvia

    2017-06-01

    Manganese (Mn) is an essential metal for the development and function of the mammalian brain; however, excess Mn accumulation may cause neurological abnormalities resembling Parkinson's disease due to reductions in brain dopamine levels. Because dopamine also regulates many functions in crustaceans, this study examined the effects of Mn accumulation in Dilocarcinus pagei, a Neotropical freshwater crab. Following a 72-h exposure to graded concentrations of MnCl 2 , Mn accumulation was assessed in several tissues. Glycaemia and the tyrosine hydroxylase (TH) immunostaining response were also examined as indicators of catecholaminergic function and catecholaminergic cell integrity, respectively. Tissue Mn accumulation was variable and occurred in the following order: gills > hepatopancreas > claw muscle > haemolymph. Exposure to 2 mM Mn reduced the gill levels of calcium, copper and iron, whereas Mn at all concentrations decreased zinc levels. All Mn-exposed animals showed lower copper levels in the hepatopancreas and haemolymph. Exposure to 2.0 mM Mn increased the haemolymph calcium. Mn exposure had no effect on glycaemia, whereas exposure to low Mn concentrations reduced the TH immunostaining response. Analysis of the central nervous system revealed the greatest Mn effect in the cerebral ganglion and the least effect in the abdominal ganglia. These results suggest the operation of an adaptive mechanism for tissue accumulation that could be responsible for the lack of an association between Mn concentrations and metal accumulation. The findings also suggest that Mn, calcium, iron and zinc share a transporter in gill cells and that Mn resistance is greater in the TH-positive cells of this crustacean than in mammalian cells.

  18. Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Li-Juan Zhang

    Full Text Available Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH expression of dopaminergic (DA neurons induced by 6-hydroxydopamine (6-OHDA toxicity that is most commonly used to create a rat model of Parkinson's disease (PD. In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

  19. Effects of Selective Deletion of Tyrosine Hydroxylase from Kisspeptin Cells on Puberty and Reproduction in Male and Female Mice.

    Science.gov (United States)

    Stephens, Shannon B Z; Rouse, Melvin L; Tolson, Kristen P; Liaw, Reanna B; Parra, Ruby A; Chahal, Navi; Kauffman, Alexander S

    2017-01-01

    The neuropeptide kisspeptin, encoded by Kiss1 , regulates reproduction by stimulating GnRH secretion. Kiss1- syntheizing neurons reside primarily in the hypothalamic anteroventral periventricular (AVPV/PeN) and arcuate (ARC) nuclei. AVPV/PeN Kiss1 neurons are sexually dimorphic, with females expressing more Kiss1 than males, and participate in estradiol (E 2 )-induced positive feedback control of GnRH secretion. In mice, most AVPV/PeN Kiss1 cells coexpress tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis (in this case, dopamine). Dopamine treatment can inhibit GnRH neurons, but the function of dopamine signaling arising specifically from AVPV/PeN Kiss1 cells is unknown. We generated a novel TH flox mouse and used Cre-Lox technology to selectively ablate TH specifically from Kiss1 cells. We then examined the effects of selective TH knock-out on puberty and reproduction in both sexes. In control mice, 90% of AVPV/PeN Kiss1 neurons coexpressed TH , whereas in mice lacking TH exclusively in Kiss1 cells (termed Kiss THKOs), TH was successfully absent from virtually all Kiss1 cells. Despite this absence of TH , both female and male Kiss THKOs displayed normal body weights, puberty onset, and basal gonadotropin levels in adulthood, although testosterone (T) was significantly elevated in adult male Kiss THKOs. The E 2 -induced LH surge was unaffected in Kiss THKO females, and neuronal activation status of kisspeptin and GnRH cells was also normal. Supporting this, fertility and fecundity were normal in Kiss THKOs of both sexes. Thus, despite high colocalization of TH and Kiss1 in the AVPV/PeN, dopamine produced in these cells is not required for puberty or reproduction, and its function remains unknown.

  20. Three-dimensional distribution of tyrosine hydroxylase, vasopressin and oxytocin neurones in the transparent postnatal mouse brain.

    Science.gov (United States)

    Godefroy, D; Dominici, C; Hardin-Pouzet, H; Anouar, Y; Melik-Parsadaniantz, S; Rostène, W; Reaux-Le Goazigo, A

    2017-12-01

    Over the years, advances in immunohistochemistry techniques have been a critical step in detecting and mapping neuromodulatory substances in the central nervous system. The better quality and specificity of primary antibodies, new staining procedures and the spectacular development of imaging technologies have allowed such progress. Very recently, new methods permitting tissue transparency have been successfully used on brain tissues. In the present study, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with the iDISCO+ clearing method, light-sheet microscopy and semi-automated counting of three-dimensionally-labelled neurones to obtain a (3D) distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us, with high resolution, to map TH staining of the various catecholaminergic cell groups and their ascending and descending fibre pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to that observed in the adult rat brain. We also provide new information on the postnatal distribution of OXT and AVP immunoreactive cells in the mouse hypothalamus, and show that, compared to AVP neurones, OXT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei are not yet mature in the early postnatal period. 3D semi-automatic quantitative analysis of the PVN reveals that OXT cell bodies are more numerous than AVP neurones, although their immunoreactive soma have a volume half smaller. More AVP nerve fibres compared to OXT were observed in the PVN and the retrochiasmatic area. In conclusion, the results of the present study demonstrate the utility and the potency of imaging large brain tissues with clearing procedures coupled to novel 3D imaging technologies to study, localise and quantify neurotransmitter substances involved in brain and neuroendocrine functions. © 2017 British Society for

  1. Localization of choline acetyltransferase and tyrosine hydroxylase immunoreactivities in the superior colliculus of the microbat,Rhinolophus ferrumequinum.

    Science.gov (United States)

    Jeong, Se-Jin; Jeon, Chang-Jin

    2017-06-01

    The purpose of this study was to determine whether the superior colliculus (SC) of the microbat has the same neurochemical makeup as that of other mammals. We examined the organization of choline acetyltransferase (ChAT)- and tyrosine hydroxylase-immunoreactive (TH-IR) fibers/cells using standard immunohistochemistry with antibodies against ChAT and TH. ChAT-IR fibers observed in the superficial layers were denser than those in the deeper layers, and these fibers were classified into two types: small varicose fibers and large varicose fibers. ChAT-IR cells were predominantly located in the superficial layers with diverse morphologies. Among the well-known sources of cholinergic fibers in the mammalian SC, pedunculopontine tegmental nucleus (PPTN) and laterodorsal tegmental nucleus (LDTN) contained strongly labeled ChAT-IR cells, while no cholinergic structures were found in the parabigeminal nucleus (PBG) in the microbat brain. TH-immunoreactivity was found within fibers but not within cells. The density of TH-IR fibers was high in the zonal layer, moderate in the superficial gray and optic layers, and low in the deeper layers. Well-labeled TH-IR cells were also observed within area 13 and the locus coeruleus, known as the sources of catecholaminergic fibers in other mammalian SC. Although there are some cytoarchitectural variations among species, our results clearly showed elaborately organized ChAT-IR and TH-IR fibers/cells in the microbat SC. Our findings will contribute significantly to the understanding of actively constructed microbat visual systems.

  2. Effect of tyrosine hydroxylase gene silencing in CD4+ T lymphocytes on differentiation and function of helper T cells.

    Science.gov (United States)

    Liu, Yan; Huang, Yan; Wang, Xiao-Qin; Peng, Yu-Ping; Qiu, Yi-Hua

    2012-01-01

    We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs. CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis. CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA. TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.

  3. The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization.

    Science.gov (United States)

    Parra, Leonardo A; Baust, Tracy B; Smith, Amanda D; Jaumotte, Juliann D; Zigmond, Michael J; Torres, Soledad; Leak, Rehana K; Pino, Jose A; Torres, Gonzalo E

    2016-08-19

    We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells.

    Science.gov (United States)

    Nakashima, Akira; Ohnuma, Syuhei; Kodani, Yu; Kaneko, Yoko S; Nagasaki, Hiroshi; Nagatsu, Toshiharu; Ota, Akira

    2016-04-15

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Rotenone induces KATP channel opening in PC12 cells in association with the expression of tyrosine hydroxylase.

    Science.gov (United States)

    Bai, Qunhua; He, Junlin; Qiu, Jingfu; Wang, Yang; Wang, Shibo; Xiu, Yun; Yu, Chao

    2012-10-01

    The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibitor glibenclamide were used to treat cells for 24 h, and the expression of TH was detected. Our results showed that treatment of PC12 cells with glibenclamide for 24 h can notably promote TH expression and can also enhance the expression of TH which were reduced by rotenone. These data indicate that the energy states in PC12 induced by various doses of rotenone could significantly influence the opening states of KATP channels. However long-term energy stress may raise the opening rate and opening sensitivity of this channel. In addition, our results demonstrate for the first time that activation of plasma membrane KATP channels induced by rotenone inhibits TH expression which influences DA synthesis in PC12 cells.

  6. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity.

    Science.gov (United States)

    Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

    2016-01-01

    Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥ 0.80 m in women and ≥ 0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome.

  7. Parents induced- conditioned place preference and the neuronal expression of oxytocin and tyrosine hydroxylase in preweanling female pups.

    Science.gov (United States)

    Wang, Jianli; Liu, Chaobao; Ma, Yongping

    2017-01-15

    Parents-offspring bonding is critical for development of offspring in mammals. While it is known that pups stimuli provide rewarding effects on their parents, few studies have assessed whether parental stimuli serve as a reinforcing agent to their pups, and what the neural mechanisms underlying this reward process may be. In addition to maternal care, male ICR mice display pairmate-dependent parental behavior. Using the conditioned place preference (CPP) paradigm, we examined the effects of maternal and paternal conditioning on the postnatal day 17-21 female ICR mice pups, and compared the expression of oxytocin (OT)- and tyrosine hydroxylase (TH)- immunoreactive (IR) neurons. We found that the pups established dam- or sire- induced CPP when using mother conditioning (MC) or father conditioning (FC) alone. However, the pups failed to show any preference when using mother versus father conditioning (MFC). Compared to the control group, the MC and MFC groups displayed more OT-IR neurons in the supraoptic nucleus and more TH-IR neurons in the ventral tegmental area (VTA). The FC group showed more TH-IR neurons in the VTA compared to the control group, but there were no significant differences in OT-IR neurons. These findings indicate that female ICR mice pups may establish mother- or father- induced CPP. The underpinnings of preference for parents are associated with the activity of VTA dopaminergic neurons, and the preference of pups for mother in particular appears to be associated with OT levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. TH01, a Tetrameric Short Tandem Repeat Locus in the Tyrosine Hydroxylase Gene: Association with Myocardial Hypertrophy and Death from Myocardial Infarction?

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    Michael Klintschar

    2005-01-01

    Full Text Available TH01 is a tetrameric short tandem repeat locus located in intron 01 of the tyrosine hydroxylase gene. The tyrosine hydroxylase catalyzes the hydroxylation of L-tyrosine to L-DOPA and is the rate limiting enzyme in the synthesis of catecholamines like noradrenaline or adrenaline, which are pivotal in the regulation of blood pressure. In a clinical study a strong correlation between alleles *9.3 and *10 and essential hypertension was observed ([2] Hypertension 32: 676–682. To further investigate this association, we typed TH01 in 296 autopsy cases and correlated the genotypes to the heart weight as parameter for myocardial hypertrophy. No significant correlation was observed. Moreover, dividing the studied cases into 2 groups, one including 172 casualties from hypertension-associated diseases (myocardial infarction, left heart failure, aortic aneurysm, spontaneous intracerebral bleeding and cerebral infarction and one consisting of 124 cases of death unrelated to hypertension, revealed similar allelic frequencies for both groups. Our data thus suggest that TH01 long alleles appear not to lead to a significant increase in the incidence of myocardial hypertrophy or other hypertension associated diseases. This could be explained by a relatively small impact of the TH01 genotype on the blood pressure or by counteraction of another mechanism related to catecholamines and their effect on the human body.

  9. Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Jung-Hyuck Ahn

    Full Text Available Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A. A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

  10. The alpha(2)-adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons ion the Long Evans and Brattleboro rats

    DEFF Research Database (Denmark)

    Bundzikova, J; Pirnik, Z; Zelena, D

    2010-01-01

    The hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei are activated by body salt-fluid variations. Stimulation of alpha(2)-adrenoceptors by an agonist-xylazine (XYL) activates oxytocinergic but not vasopressinergic magnocellular neurons. In this study, tyrosine hydroxylase (TH...... sections of 30 mum thickness double immunolabeled with Fos/neuropeptide were evaluated under light microscope. Under basal conditions, di/di in comparison with control Long Evans rats, displayed significantly higher number of TH, CRH, and NPY immunoreactive neurons in the SON and PVN (except NPY cells...

  11. Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

    NARCIS (Netherlands)

    Molenaar, W M; Lee, V M; Trojanowski, J Q

    1990-01-01

    The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of

  12. Effect of ghrelin receptor agonist and antagonist on the activity of arcuate nucleus tyrosine hydroxylase containing neurons in C57BL/6 male mice exposed to normal or high fat diet

    Czech Academy of Sciences Publication Activity Database

    Pirník, Z.; Majerčíková, Z.; Holubová, Martina; Pirník, R.; Železná, Blanka; Maletínská, Lenka; Kiss, A.

    2014-01-01

    Roč. 65, č. 4 (2014), s. 477-486 ISSN 0867-5910 Institutional support: RVO:61388963 Keywords : growth hormone secretagogue receptor * ghrelin receptor agonist * ghrelin receptor antagonist * high fat diet * tyrosine hydroxylase * arcuate nucleus * food intake Subject RIV: CE - Biochemistry Impact factor: 2.386, year: 2014

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

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available 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 SNCAA53T 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.

  14. Gene therapy in hemiparkinsonian rhesus monkeys: long-term survival and behavioral recovery by transplantation of autologous human tyrosine hydroxylase-expressing neural stem cells.

    Science.gov (United States)

    Xu, Qiang; Jiang, Xiaodan; Ke, Yiquan; Zhang, Shizhong; Xu, Ruxiang; Zeng, Yanjun

    2010-04-01

    Neural stem cells (NSC) derived from bone marrow stromal cells (BMSC) (BMSC-D-NSC) are remarkably versatile in response to environmental signals, which render them useful in the search for neurodegenerative disease treatments. We isolated NSC from rhesus monkey bone marrow (BM), transfected them with the human tyrosine hydroxylase (hTH) gene, and transplanted them into 1-methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned hemiparkinsonian rhesus monkeys to determine changes in neural transmitter production and alterations in behavior. hTH-expressing cells produced monoamine agents in vitro, such as noradrenalin and dopamine. After cell transplantation in the caudate nucleus and substantia nigra of the experimental monkeys, their disease symptoms and dysfunctional glucose metabolism and dopamine transport were ameliorated. hTH-expressing BMSC-D-NSC survived in transplantation sites and assumed normal dopaminergic neuronal properties, playing an instrumental role in functional restoration.

  15. Pirenzepine Inhibits Myopia in Guinea Pig Model by Regulating the Balance of MMP-2 and TIMP-2 Expression and Increased Tyrosine Hydroxylase Levels.

    Science.gov (United States)

    Qian, Lifeng; Zhao, Hong; Li, Xiaoxia; Yin, Juanjuan; Tang, Wenjian; Chen, Peng; Wang, Qian; Zhang, Jinsong

    2015-04-01

    In this study, we investigated the effects and mechanism of action of pirenzepine in a guinea pig model of myopia induced by exposure to monochromatic light. It was observed that pirenzepine inhibited the increase of diopter and extension of ocular axial length. Immunohistochemistry staining showed that the number of tyrosine hydroxylase (TH)-positive cells in pirenzepine group was significantly higher compared to the other treatment groups pointing to a highly positive correlation between TH expression levels and the diopter and axial length change. RT-PCR analysis further showed that pirenzepine treatment reduced the expression of matrix metalloproteinase (MMP-2) and enhanced the expression of tissue inhibitors of metalloproteinase (TIMP-2) compared to the other treatment and control groups. To conclude, we demonstrate that pirenzepine may improve the prognosis of monochromatic light-induced myopia in guinea pigs, possibly by both regulating the balance of MMP-2 and TIMP-2 in sclera and increasing the TH expression in retina.

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

    Science.gov (United States)

    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.

  17. Tyrosine hydroxylase immunoreactivity and [3H]WIN 35,428 binding to the dopamine transporter in a hamster model of idiopathic paroxysmal dystonia

    International Nuclear Information System (INIS)

    Nobrega, J.N.; Gernert, M.; Loescher, W.; Raymond, R.; Belej, T.; Richter, A.

    1999-01-01

    Recent pharmacological studies and receptor analyses have suggested that dopamine neurotransmission is enhanced in mutant dystonic hamsters (dt sz ), a model of idiopathic paroxysmal dystonia which displays attacks of generalized dystonia in response to mild stress. In order to further characterize the nature of dopamine alterations, the present study investigated possible changes in the number of dopaminergic neurons, as defined by tyrosine hydroxylase immunohistochemistry, as well as binding to the dopamine transporter labelled with [ 3 H]WIN 35,428 in dystonic hamsters. No differences in the number of tyrosine hydroxylase-immunoreactive neurons were found within the substantia nigra and ventral tegmental area of mutant hamsters compared to non-dystonic control hamsters. Similarly, under basal conditions, i.e. in the absence of a dystonic episode, no significant changes in [ 3 H]WIN 35,428 binding were detected in dystonic brains. However, in animals killed during the expression of severe dystonia, significant decreases in dopamine transporter binding became evident in the nucleus accumbens and ventral tegmental area in comparison to controls exposed to the same external stimulation. Since stimulation tended to increase [ 3 H]WIN 35,428 binding in control brains, the observed decrease in the ventral tegmental area appeared to be due primarily to the fact that binding was increased less in dystonic brains than in similarly stimulated control animals.This finding could reflect a diminished ability of the dopamine transporter to undergo adaptive changes in response to external stressful stimulation in mutant hamsters. The selective dopamine uptake inhibitor GBR 12909 (20 mg/kg) aggravated dystonia in mutant hamsters, further suggesting that acute alterations in dopamine transporter function during stimulation may be an important component of dystonia in this model. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  18. NF-kappaB regulates the transcription of protein tyrosine kinase Tec.

    Science.gov (United States)

    Yu, Liang; Simonson, Oscar E; Mohamed, Abdalla J; Smith, C I Edvard

    2009-11-01

    The tyrosine kinase expressed in hepatocellular carcinoma (Tec) is a non-receptor protein tyrosine kinase (PTK) that is expressed in hematopoietic cells, such as B and T lymphocytes, myeloid lineage cells and neutrophils. Mutations in the human Btk gene cause X-linked agammaglobulinemia (XLA), but the corresponding mutation in mice results in a much milder defect. However, the combined inactivation of Btk and Tec genes in mice cause a severe phenotype resembling XLA. Tec is involved in the regulation of both B and T lymphocytes, fine-tuning of TCR/BCR signaling, and also activation of the nuclear factor of activated T cells. Previous work has shown that the transcription factors Sp1 and PU.1 can bind and regulate the Tec promoter. In this study, we demonstrate that NF-kappaB is an essential transcription factor for optimal expression of the Tec gene, and identify a unique functionally active NF-kappaB binding site in its promoter. The NF-kappaB subunit p65/RelA directly induced transcriptional activity of the Tec promoter. Moreover, we also found that proteasome inhibitors, including Bortezomib, repress Tec transcription through inactivation of the NF-kappaB signaling pathway. This study, together with our previous findings on the transcriptional regulation of Btk (Bruton's tyrosine kinase) by proteasome inhibitors, provides important insight into the molecular mechanism(s) underlying the role of NF-kappaB in Tec family kinase signaling and lymphocyte development.

  19. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    Science.gov (United States)

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients.

  20. Elevated blood plasma levels of epinephrine, norepinephrine, tyrosine hydroxylase, TGFβ1, and TNFα associated with high-altitude pulmonary edema in Indian population

    Directory of Open Access Journals (Sweden)

    Pandey P

    2016-08-01

    Full Text Available Priyanka Pandey,1,2 Zahara Ali,1,2 Ghulam Mohammad,3 MA Qadar Pasha1,2 1Functional Genomics Unit, CSIR-Institute of Genomics and Integrative Biology, Delhi, 2Department of Biotechnology, Savitribai Phule Pune University, Pune, 3Department of Medicine, SNM Hospital, Ladakh, Jammu and Kashmir, India Abstract: Biomarkers are essential to unravel the locked pathophysiology of any disease. This study investigated the role of biomarkers and their interactions with each other and with the clinical parameters to study the physiology of high-altitude pulmonary edema (HAPE in HAPE-patients (HAPE-p against adapted highlanders (HLs and healthy sojourners, HAPE-controls (HAPE-c. For this, seven circulatory biomarkers, namely, epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor beta 1, tumor necrosis factor alpha (TNFα, platelet-derived growth factor beta beta, and C-reactive protein (CRP, were measured in blood plasma of the three study groups. All the subjects were recruited at ~3,500 m, and clinical features such as arterial oxygen saturation (SaO2, body mass index, and mean arterial pressure were measured. Increased levels of epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor-beta 1, and TNFα were observed in HAPE-p against the healthy groups, HAPE-c, and HLs (P<0.0001. CRP levels were decreased in HAPE-p against HAPE-c and HLs (P<0.0001. There was no significant difference or very marginal difference in the levels of these biomarkers in HAPE-c and HLs (P>0.01. Correlation analysis revealed a negative correlation between epinephrine and norepinephrine (P=4.6E-06 in HAPE-p and positive correlation in HAPE-c (P=0.004 and HLs (P=9.78E-07. A positive correlation was observed between TNFα and CRP (P=0.004 in HAPE-p and a negative correlation in HAPE-c (P=4.6E-06. SaO2 correlated negatively with platelet-derived growth factor beta beta (HAPE-p; P=0.05, norepinephrine (P=0.01, and TNFα (P=0.005 and

  1. A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase

    International Nuclear Information System (INIS)

    Wu Shaoping; Fu Ailing; Wang Yuxia; Yu Leiping; Jia Peiyuan; Li Qian; Jin Guozhang; Sun Manji

    2006-01-01

    The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 μM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD

  2. Tyrosine Hydroxylase, Vesicular Monoamine Transporter and Dopamine Transporter mRNA Expression in Nigrostriatal Tissue of Rats with Pedunculopontine Neurotoxic Lesion

    Directory of Open Access Journals (Sweden)

    Lisette Blanco-Lezcano

    2018-02-01

    Full Text Available Background: The degeneration of the pedunculopontine nucleus (PPN precedes the degeneration of the nigral cells in the pre-symptomatic stages of Parkinson’s disease (PD. Although the literature recognizes that a lesion of the PPN increases the vulnerability of dopaminergic cells, it is unknown if this risk is associated with the loss of capability of handling the dopaminergic function. Methods: In this paper, the effects of a unilateral neurotoxic lesion of the PPN in tyrosine hydroxylase (TH, vesicular monoamine transporter 2 (VMAT2 and dopamine transporter (DAT mRNA expression in nigrostriatal tissue were evaluated. Three experimental groups were organized: non-treated rats, NMDA-lesioned rats and Sham-operated rats. Results: Seven days after the PPN lesion, in nigral tissue, TH mRNA expression was higher in comparison with control groups (p < 0.05; in contrast, VMAT2 mRNA expression showed a significant decrease (p < 0.01. DAT mRNA expression showed a significant decrease (p < 0.001 in the striatal tissue. Comparing nigral neuronal density of injured and control rats revealed no significant difference seven days post-PPN injury. Conclusions: Findings suggest that the PPN lesion modifies the mRNA expression of the proteins associated with dopaminergic homeostasis at nigrostriatal level. It could represent vulnerability signals for nigral dopaminergic cells and further increase the risk of degeneration of these cells.

  3. Photobiomodulation-induced changes in a monkey model of Parkinson's disease: changes in tyrosine hydroxylase cells and GDNF expression in the striatum.

    Science.gov (United States)

    El Massri, Nabil; Lemgruber, Ana P; Rowe, Isobel J; Moro, Cécile; Torres, Napoleon; Reinhart, Florian; Chabrol, Claude; Benabid, Alim-Louis; Mitrofanis, John

    2017-06-01

    Intracranial application of red to infrared light, known also as photobiomodulation (PBM), has been shown to improve locomotor activity and to neuroprotect midbrain dopaminergic cells in rodent and monkey models of Parkinson's disease. In this study, we explored whether PBM has any influence on the number of tyrosine hydroxylase (TH) + cells and the expression of GDNF (glial-derived neurotrophic factor) in the striatum. Striatal sections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice and monkeys and 6-hydroxydopamine (6OHDA)-lesioned rats that had PBM optical fibres implanted intracranially (or not) were processed for immunohistochemistry (all species) or western blot analysis (monkeys). In our MPTP monkey model, which showed a clear loss in striatal dopaminergic terminations, PBM generated a striking increase in striatal TH + cell number, 60% higher compared to MPTP monkeys not treated with PBM and 80% higher than controls. This increase was not evident in our MPTP mouse and 6OHDA rat models, both of which showed minimal loss in striatal terminations. In monkeys, the increase in striatal TH + cell number in MPTP-PBM cases was accompanied by similar increases in GDNF expression, as determined from western blots, from MPTP and control cases. In summary, these results offer insights into the mechanisms by which PBM generates its beneficial effects, potentially with the use of trophic factors, such as GDNF.

  4. Tyrosine hydroxylase-producing neurons in the human cerebral cortex do not colocalize with calcium-binding proteins or the serotonin 3A receptor.

    Science.gov (United States)

    Asmus, Stephen E; Raghanti, Mary Ann; Beyerle, Eric R; Fleming-Beattie, Julia C; Hawkins, Sarah M; McKernan, Courtney M; Rauh, Nicholas A

    2016-12-01

    Interneurons of the cerebral cortex play a significant role in cortical information processing and are of clinical interest due to their involvement in neurological disorders. In the human neocortex, three subsets of interneurons can be identified based on the production of the calcium-binding proteins parvalbumin, calretinin or calbindin. A subset of interneurons in the mouse cortex expresses the serotonin 3A receptor (5-HT 3A R). Previous work in humans has also demonstrated the presence of a subgroup of cortical neurons that produces the catecholaminergic enzyme tyrosine hydroxylase (TH). Many TH-producing cells in the rat cortex coexpress calretinin and are adjacent to blood vessels. However, little is known about the phenotype of these TH interneurons in humans. Here we immunohistochemically examined the coexpression of TH with parvalbumin, calretinin, calbindin or 5-HT 3A R in human Brodmann's areas 10 and 24, cortical regions with high densities of TH-containing neurons. Colocalization of TH with these calcium-binding proteins and with 5-HT 3A R was not detected in either area. Cortical TH cells were rarely apposed to blood vessels, denoted by immunolabeling for the gliovascular marker aquaporin-4. Our results suggest that the TH-immunoreactive cells in the human cortex do not overlap with any known neurochemically-defined subsets of interneurons and provide further evidence of differences in the phenotype of these cells across species. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Sesamin Modulates Tyrosine Hydroxylase, Superoxide Dismutase, Catalase, Inducible No Synthase and Interleukin-6 Expression in Dopaminergic Cells Under Mpp+-Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Vicky Lahaie-Collins

    2008-01-01

    Full Text Available Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP+ ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP+-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP+-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP+ stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP+-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

  6. Exercise-Mediated Increase in Nigral Tyrosine Hydroxylase Is Accompanied by Increased Nigral GFR-α1 and EAAC1 Expression in Aging Rats.

    Science.gov (United States)

    Arnold, Jennifer C; Salvatore, Michael F

    2016-02-17

    Exercise may alleviate locomotor impairment in Parkinson's disease (PD) or aging. Identifying molecular responses immediately engaged by exercise in the nigrostriatal pathway and allied tissue may reveal critical targets associated with its long-term benefits. In aging, there is loss of tyrosine hydroxylase (TH) and the glial cell line-derived neurotrophic factor (GDNF) receptor, GFR-α1, in the substantia nigra (SN). Exercise can increase GDNF expression, but its effect on GFR-α1 expression is unknown. Infusion of GDNF into striatum or GFR-α1 in SN, respectively, can increase locomotor activity and TH function in SN but not striatum in aged rats. GDNF may also increase glutamate transporter expression, which attenuates TH loss in PD models. We utilized a footshock-free treadmill exercise regimen to determine the immediate impact of short-term exercise on GFR-α1 expression, dopamine regulation, glutamate transporter expression, and glutamate uptake in 18 month old male Brown-Norway/Fischer 344 F1 hybrid rats. GFR-α1 and TH expression significantly increased in SN but not striatum. This exercise regimen did not affect glutamate uptake or glutamate transporter expression in striatum. However, EAAC1 expression increased in SN. These results indicate that nigral GFR-α1 and EAAC1 expression increased in conjunction with increased nigral TH expression following short-term exercise.

  7. Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment.

    Science.gov (United States)

    Taufique, S K Tahajjul; Kumar, Vinod

    2016-11-01

    Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) interacts with the phytanoyl-CoA alpha-hydroxylase associated protein 1 (PAHX-AP1), a brain specific protein.

    Science.gov (United States)

    Bescond, Marilyne; Rahmani, Zohra

    2005-04-01

    Down syndrome (DS) is the most common genetic defect correlated with mental retardation and delayed development. The specific genes responsible for these phenotypic alterations have not yet been defined. Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A), the human ortholog of the Drosophila minibrain gene (mnb), maps to the Down syndrome critical region of human chromosome 21 and is overexpressed in Down syndrome fetal brain. In Drosophila, minibrain is involved in postembryonic neurogenesis. In human, DYRK1A encodes a serine-threonine kinase but despite its potential involvement in the neurobiological alterations associated with Down syndrome, its physiological function has not yet been defined. To gain some insight into its biological function, we used the yeast two-hybrid approach to identify binding partners of DYRK1A. We found that the C-terminal region of DYRK1A interacts with a brain specific protein, phytanoyl-CoA alpha-hydroxylase-associated protein 1 (PAHX-AP1, also named PHYHIP) which was previously shown to interact with phytanoyl-CoA alpha-hydroxylase (PAHX, also named PHYH), a Refsum disease gene product. This interaction was confirmed by co-immunoprecipitation of PC12 cells co-transfected with DYRK1A and PAHX-AP1. Furthermore, immunofluorescence analysis of PC12 cells co-transfected with both plasmids showed a re-distribution of DYRK1A from the nucleus to the cytoplasm where it co-localized with PAHX-AP1. Finally, in PC12 cells co-transfected with both plasmids, DYRK1A was no longer able to interact with the nuclear transcription factor CREB, thereby confirming that the intracellular localization of DYRK1A was changed from the nucleus to the cytoplasm in the presence of PAHX-AP1. Therefore, these data indicate that by inducing a re-localization of DYRK1A into the cytoplasm, PAHX-AP1 may contribute to new cellular functions of DYRK1A and suggest that PAHX-AP1 may be involved in the development of neurological abnormalities

  9. Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene

    Directory of Open Access Journals (Sweden)

    Jeong-Woong Park

    2017-10-01

    Full Text Available Objective Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form in equine skeletal muscle to gain insight(s into the role of each alternative transcript during exercise. Methods We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR, and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR. Results Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3 and immunoglobin (Ig domain was different between two alternative isoforms. Conclusion It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s and significance of the alternative splicing isoforms in race horse skeletal muscle.

  10. A dopaminergic projection to the rat mammillary nuclei demonstrated by retrograde transport of wheat germ agglutinin-horseradish peroxidase and tyrosine hydroxylase immunohistochemistry

    Science.gov (United States)

    Gonzalo-Ruiz, A.; Alonso, A.; Sanz, J. M.; Llinas, R. R.

    1992-01-01

    The presence and distribution of dopaminergic neurons and terminals in the hypothalamus of the rat were studied by tyrosine hydroxylase (TH) immunohistochemistry. Strongly labelled TH-immunoreactive neurons were seen in the dorsomedial hypothalamic nucleus, periventricular region, zona incerta, arcuate nucleus, and supramammillary nucleus. A few TH-positive neurons were also identified in the dorsal and ventral premammillary nucleus, as well as the lateral hypothalamic area. TH-immunoreactive fibres and terminals were unevenly distributed in the mammillary nuclei; small, weakly labelled terminals were scattered in the medial mammillary nucleus, while large, strongly labelled, varicose terminals were densely concentrated in the internal part of the lateral mammillary nucleus. A few dorsoventrally oriented TH-positive axon bundles were also identified in the lateral mammillary nucleus. A dopaminergic projection to the mammillary nuclei from the supramammillary nucleus and lateral hypothalamic area was identified by double labelling with retrograde transport of wheat germ agglutinin-horseradish peroxidase and TH-immunohistochemistry. The lateral mammillary nucleus receives a weak dopaminergic projection from the medial, and stronger projections from the lateral, caudal supramammillary nucleus. The double-labelled neurons in the lateral supramammillary nucleus appear to encapsulate the caudal end of the mammillary nuclei. The medial mammillary nucleus receives a very light dopaminergic projection from the caudal lateral hypothalamic area. These results suggest that the supramammillary nucleus is the principal source of the dopaminergic input to the mammillary nuclei, establishing a local TH-pathway in the mammillary complex. The supramammillary cell groups are able to modulate the limbic system through its dopaminergic input to the mammillary nuclei as well as through its extensive dopaminergic projection to the lateral septal nucleus.

  11. Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

    Science.gov (United States)

    Kawahata, Ichiro; Ohtaku, Shiori; Tomioka, Yoshihisa; Ichinose, Hiroshi; Yamakuni, Tohru

    2015-09-11

    The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice.

    Science.gov (United States)

    Smith, Jeremy T; Reichenbach, Alex; Lemus, Moyra; Mani, Bharath K; Zigman, Jeffrey M; Andrews, Zane B

    2013-09-01

    Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals.

    Science.gov (United States)

    Massari, V J; Dickerson, L W; Gray, A L; Lauenstein, J M; Blinder, K J; Newsome, J T; Rodak, D J; Fleming, T J; Gatti, P J; Gillis, R A

    1998-08-17

    Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by

  14. Expression of the μ, κ, and δ-opioid receptors and tyrosine hydroxylase in MN9D cells.

    Science.gov (United States)

    Tian, Pengxiang; Shi, Weibo; Liu, Jie; Wang, Jie; Ma, Chunling; Qi, Qian; Cong, Bin; Li, Yingmin

    2015-01-01

    Dopaminergic neurons are suggested to be a critical physiopathology substrate for addiction disorders. It is not well known whether the clonal mesencephalic dopaminergic cell line MN9D cells can be applied to study morphine addiction. Immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect protein and mRNA expression of the μ, κ, and δ-opioid receptors in MN9D cells. Immunofluorescence staining of TH was applied to quantify the number of dopaminergic neurons. The results showed that the μ, κ, and δ-receptors were all expressed in MN9D cells, and the number of TH-positive cells was significantly greater in the MN9D cells than SH-SY5Y cells. The data suggest that MN9D cells can be used as an in vitro models in future studies to explore the mechanisms of morphine addiction related to dopaminergic neurons.

  15. From the Cover: Prenatal Nicotinic Exposure Attenuates Respiratory Chemoreflexes Associated With Downregulation of Tyrosine Hydroxylase and Neurokinin 1 Receptor in Rat Pup Carotid Body.

    Science.gov (United States)

    Zhao, Lei; Zhuang, Jianguo; Gao, Xiuping; Ye, Chunyan; Lee, Lu-Yuan; Xu, Fadi

    2016-09-01

    Maternal cigarette smoke is the major risk of sudden infant death syndrome (SIDS). A depressed ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) is thought to be responsible for the pathogenesis of SIDS and the carotid body is critically involved in these responses. We have recently reported that prenatal nicotinic exposure (PNE) over the full gestation induces depressed HVR in rat pups. Here, we asked whether PNE (1) depressed not only HVR but also HCVR that were dependent on the carotid body, (2) affected some important receptors and neurochemicals expressed in the carotid body, such as tyrosine hydroxylase (TH), neurokinin-1 receptor (NK1R), and α7 nicotinic acetylcholine receptor (α7nAChR), and (3) blunted the ventilatory responses to activation of these receptors. To this end, HVR and HCVR in Ctrl and PNE pups were measured with plethysmography before and after carotid body ablation (Series I), mRNA expression and/or immunoreactivity (IR) of TH, NK1R, and α7nAChR in the carotid body were examined by RT-PCR and immunohistochemistry (Series II), and the ventilatory responses were tested before and after intracarotid injection of substance P (NK1R agonist) and AR-R17779 (α7nAChR agonist) (Series III). Our results showed that PNE (1) significantly depressed both HVR and HCVR and these depressions were abolished by carotid body ablation, (2) reduced the relative population of glomus cells, mRNA NK1R, and α7nAChR and IR of NK1R and TH in the carotid body, and (3) decreased ventilatory responses to intracarotid injection of substance P or AR-R17779. These results suggest that PNE acting via the carotid body could strikingly blunt HVR and HCVR, likely through downregulating TH and NK1R. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

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    Hei, Ming-Yan; Luo, Ya-Li; Zhang, Xiao-Chun; Liu, Hong; Gao, Ru; Wu, Jing-Jiang [Department of Pediatrics, the Third Xiangya Hospital, Central South University, Changsha, Hunan (China)

    2011-12-09

    Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O{sub 2}/92% N{sub 2}) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

  17. Intraventricular injection of 6-hydroxydopamine results in an increased number of tyrosine hydroxylase immune-positive cells in the rat cortex.

    Science.gov (United States)

    Wachter, B; Caradonna, S; Gittinger, K; Schläger, A; Küppers, E

    2014-11-07

    Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells 4 days after lesion (Wachter et al., 2010). To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH(+)) somata in the cortices of 6-OHDA-lesioned animals. The number of TH(+) cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham-operated animals, where virtually no TH(+) somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼45% of the TH(+) cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as glial fibrillary acidic protein (GFAP) or the neural stem/progenitor marker Nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100β) and with polysialylated-neural cell adhesion molecule (PSA-NCAM), which has been shown to be expressed in immature, but not recently generated cortical neurons. Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH(+) cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010). The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine-(DA)-depleted system. Future studies are needed to determine

  18. Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

    Directory of Open Access Journals (Sweden)

    Hei Ming-Yan

    2012-01-01

    Full Text Available Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI, and severe HI groups (N = 10 in each group at each time on postnatal day 7 (P7 to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH in the substantia nigra (SN. The mild and severe HI groups were exposed to hypoxia (8% O2/92% N2 for 90 and 150 min, respectively. The elevated plus-maze (EPM test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05. The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2% and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05. The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

  19. Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

    Science.gov (United States)

    Barroso-Chinea, Pedro; Cruz-Muros, Ignacio; Afonso-Oramas, Domingo; Castro-Hernández, Javier; Salas-Hernández, Josmar; Chtarto, Abdelwahed; Luis-Ravelo, Diego; Humbert-Claude, Marie; Tenenbaum, Liliane; González-Hernández, Tomás

    2016-04-01

    The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Early life stress and post-weaning high fat diet alter tyrosine hydroxylase regulation and AT1 receptor expression in the adrenal gland in a sex dependent manner.

    Science.gov (United States)

    Bobrovskaya, Larisa; Maniam, Jayanthi; Ong, Lin Kooi; Dunkley, Peter R; Morris, Margaret J

    2013-04-01

    Previous studies have shown that early life stress induced by maternal separation or non-handling can lead to behavioural deficits in rats and that these deficits can be alleviated by providing palatable cafeteria high-fat diet (HFD). In these studies we investigated the effects of maternal separation or non-handling and HFD on tyrosine hydroxylase (TH) protein and TH phosphorylation at Ser40 (pSer40TH) and the expression of angiotensin II receptor type 1 (AT1R) protein in the adrenal gland as markers of sympatho-adrenomedullary activation. After littering, Sprague-Dawley rats were assigned to short maternal separation, S15 (15 min), prolonged maternal separation, S180 (180 min) daily from postnatal days 2-14 or were non-handled (NH) until weaning. Siblings were exposed to HFD or chow from day 21 until 19 weeks when adrenals were harvested. Maternal separation and non-handling had no effects on adrenal TH protein in both sexes. We found an effect of HFD only in the females; HFD significantly increased TH levels in NH rats and pSer40TH in S180 rats (relative to corresponding chow-fed groups), but had no effect on AT1R expression in any group. In contrast, in male rats HFD had no effect on TH protein levels, but significantly increased pSer40TH across all treatment groups. There was no effect of HFD on AT1R expression in male rats; however, maternal separation (for 15 or 180 min) caused significant increases in AT1R expression (relative to NH group regardless of diet). This is the first study to report that early life stress and diet modulate TH protein, pSer40TH and AT1R protein levels in the adrenal gland in a sex dependent manner. These results are interpreted in respect to the potential adverse effects that these changes in the adrenal gland may have in males and females in adult life.

  1. Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

    Science.gov (United States)

    Bashyam, Murali D; Chaudhary, Ajay K; Kiran, Manjari; Nagarajaram, Hampapathalu A; Devi, Radha Rama; Ranganath, Prajnya; Dalal, Ashwin; Bashyam, Leena; Gupta, Neerja; Kabra, Madhulika; Muranjan, Mamta; Puri, Ratna D; Verma, Ishwar C; Nampoothiri, Sheela; Kadandale, Jayarama S

    2014-03-01

    Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients. © 2013 Wiley Periodicals, Inc.

  2. KIT(D816V) Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma

    DEFF Research Database (Denmark)

    Phung, Bengt; Kazi, Julhash U; Lundby, Alicia

    2017-01-01

    The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KIT(D816V) has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KIT(D816V) induces tyrosine phosphorylat.......Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KIT(D816V), can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265-74. ©2017 AACR....... complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KIT(D816V) in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KIT(D816V) transformed cells...

  3. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2...

  4. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

  5. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein...

  6. Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations.

    Science.gov (United States)

    Hedlund, Eva; Pruszak, Jan; Ferree, Andrew; Viñuela, Angel; Hong, Sunghoi; Isacson, Ole; Kim, Kwang-Soo

    2007-05-01

    Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression. Disclosure of potential conflicts of interest is found at the end of this article.

  7. Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

    Directory of Open Access Journals (Sweden)

    F. Rigon

    Full Text Available Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG cells, in the present study, 18 adult male frogs (Rana catesbeiana were divided into three experimental groups: naive (frogs not subjected to surgical manipulation, sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve, and SNT (frogs in which the sciatic nerve was exposed and transected. After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase. SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

  8. Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

    Directory of Open Access Journals (Sweden)

    F. Rigon

    2013-06-01

    Full Text Available Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG cells, in the present study, 18 adult male frogs (Rana catesbeiana were divided into three experimental groups: naive (frogs not subjected to surgical manipulation, sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve, and SNT (frogs in which the sciatic nerve was exposed and transected. After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase. SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

  9. 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.

    Science.gov (United States)

    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.

  10. Dataset of mRNA levels for dopaminergic receptors, adrenoceptors and tyrosine hydroxylase in lymphocytes from subjects with clinically isolated syndromes

    Directory of Open Access Journals (Sweden)

    Marco Cosentino

    2016-12-01

    Full Text Available This data article presents a dataset of mRNA levels for dopaminergic receptors, adrenoceptors and for tyrosine hydoxylase, the rate-limiting enzyme in the synthesis of catecholamines, in peripheral blood mononuclear cells as well as in CD4+ T effector and regulatory cells from subjects with clinically isolated syndromes (CIS, which is a first episode of neurological disturbance(s suggestive of multiple sclerosis. CIS subjects are divided into two groups according to their eventual progression, after 12 months from CIS, to clinically established multiple sclerosis. The data reported are related to the article entitled "Dopaminergic receptors and adrenoceptors in circulating lymphocytes as putative biomarkers for the early onset and progression of multiple sclerosis" (M. Cosentino, M. Zaffaroni, M. Legnaro, R. Bombelli, L. Schembri, D. Baroncini, A. Bianchi, R. Clerici, M. Guidotti, P. Banfi, G. Bono, F. Marino, 2016 [1].

  11. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

    Science.gov (United States)

    Congo Carbajosa, Nadia A; Carbajosa, Nadia A Longo; Corradi, Gerardo; Verrilli, María A Lopez; Guil, María J; Vatta, Marcelo S; Gironacci, Mariela M

    2015-01-01

    Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

  12. Pre- and postnatal bisphenol A treatment does not alter the number of tyrosine hydroxylase-positive cells in the anteroventral periventricular nucleus (AVPV) of weanling male and female rats.

    Science.gov (United States)

    Ferguson, Sherry A; Paule, Merle G; He, Zhen

    2015-10-22

    Exposure to Bisphenol A (BPA) may interfere with brain sexual differentiation. Altered numbers of tyrosine hydroxylase (TH) cells in the rodent anteroventral periventricular nucleus (AVPV) after developmental BPA treatment have been reported; however, definitive conclusions are lacking. The current study incorporated many of the guidelines suggested for endocrine disrupter research. Specifically, ethinyl estradiol (EE2) served as a reference estrogen, exogenous environmental estrogen exposure was controlled, BPA was administered orally, and subjects consumed a low phytoestrogen diet. Here, on gestational days 6-21, Sprague-Dawley rats (10-15/group) were gavaged with 2.5 or 25.0 µg BPA/kg/day or 5.0 or 10.0 µg EE2/kg/day or the vehicle (5 ml of 0.3% aqueous carboxymethylcellulose/kg/day). A naïve control group was weighed and restrained, but not gavaged. Beginning on postnatal day (PND) 1 and continuing until PND 21, the 4 pups/sex/litter were orally treated with the same dose their dam had received. On PND 21, 1/sex/litter was perfused and the brain removed. TH immunoreactivity (TH-ir) was counted in 8 images/pup by a technician blind to treatment status. ANOVA results indicated significantly higher TH-ir cells/mm(2) in females (main effect of sex: p<0.01); however, there was no significant effect of treatment or a significant interaction of treatment with sex. In a separate untreated group of PND 21 Sprague-Dawley pups, AVPV volume was quantified and no significant sexual dimorphism was apparent. Similar to our reported results of behavioral assessments, the BPA treatment paradigm used here (2.5 or 25.0 µg BPA/kg/day administered orally) does not appear to cause significant alterations in AVPV TH-ir. Published by Elsevier B.V.

  13. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

    Directory of Open Access Journals (Sweden)

    Nadia A Congo Carbajosa

    Full Text Available Aberrations in the ubiquitin-proteasome system (UPS are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

  14. Distribution of orexin-1 receptor-green fluorescent protein- (OX1-GFP) expressing neurons in the mouse brain stem and pons: Co-localization with tyrosine hydroxylase and neuronal nitric oxide synthase.

    Science.gov (United States)

    Darwinkel, A; Stanić, D; Booth, L C; May, C N; Lawrence, A J; Yao, S T

    2014-10-10

    We used a reporter mouse line in which green fluorescent protein (GFP) was inserted into the orexin-1 receptor (OX1) locus to systematically map the neuroanatomical distribution of the OX1 receptor in the mouse brainstem and pons. Here, we show that the OX1 receptor is expressed in a select subset of medullary and pontine nuclei. In the medulla, we observed OX1-GFP expression in the cuneate, gracile, dorsal motor nucleus of the vagus (10N), nucleus of the solitary tract and medullary raphe areas. In the pons, the greatest expression was found in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). High to moderate expression was found in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus, A5 noradrenergic cell group (A5) and the periaqueductal gray. Double-labeling with neuronal nitric oxide synthase (NOS1) revealed extensive co-localization in cell bodies and fibers of the 10N, A5 cell group and the PPTg. Double-staining with tyrosine hydroxylase revealed extensive co-expression in the LC, DRN and the lateral paragigantocellularis cell group in the ventral medulla. Our findings faithfully recapitulate the findings of OX1 mRNA expression previously reported. This is the first study to systematically map the neuroanatomical distribution of OX1 receptors within the mouse hindbrain and suggest that this OX1-GFP transgenic reporter mouse line might be a useful tool with which to study the neuroanatomy and physiology of OX1 receptor-expressing cells. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  15. Species differences in the immunoreactive expression of oxytocin, vasopressin, tyrosine hydroxylase and estrogen receptor alpha in the brain of Mongolian gerbils (Meriones unguiculatus and Chinese striped hamsters (Cricetulus barabensis.

    Directory of Open Access Journals (Sweden)

    Yu Wang

    Full Text Available Species differences in neurochemical expression and activity in the brain may play an important role in species-specific patterns of social behavior. In the present study, we used immunoreactive (ir labeling to compare the regional density of cells containing oxytocin (OT, vasopressin (AVP, tyrosine hydroxylase (TH, or estrogen receptor alpha (ERα staining in the brains of social Mongolian gerbils (Meriones unguiculatus and solitary Chinese striped hamsters (Cricetulus barabensis. Multiple region- and neurochemical-specific species differences were found. In the anterior hypothalamus (AH, Mongolian gerbils had higher densities of AVP-ir and ERα-ir cells than Chinese striped hamsters. In the lateral hypothalamus (LH, Mongolian gerbils also had higher densities of AVP-ir and TH-ir cells, but a lower density of OT-ir cells, than Chinese striped hamsters. Furthermore, in the anterior nucleus of the medial preoptic area (MPOAa, Mongolian gerbils had higher densities of OT-ir and AVP-ir cells than Chinese striped hamsters, and an opposite pattern was found in the posterior nucleus of the MPOA (MPOAp. Some sex differences were also observed. Females of both species had higher densities of TH-ir cells in the MPOAa and of OT-ir cells in the intermediate nucleus of the MPOA (MPOAi than males. Given the role of these neurochemicals in social behaviors, our data provide additional evidence to support the notion that species-specific patterns of neurochemical expression in the brain may be involved in species differences in social behaviors associated with different life strategies.

  16. De-repression of PDGFRβ transcription promotes acquired resistance to EGFR tyrosine kinase inhibitors in glioblastoma patients

    Science.gov (United States)

    Akhavan, David; Pourzia, Alexandra L.; Nourian, Alex A.; Williams, Kevin J.; Nathanson, David; Babic, Ivan; Villa, Genaro R.; Tanaka, Kazuhiro; Nael, Ali; Yang, Huijun; Dang, Julie; Vinters, Harry V.; Yong, William H.; Flagg, Mitchell; Tamanoi, Fuyuhiko; Sasayama, Takashi; James, C. David; Kornblum, Harley I.; Cloughesy, Tim F.; Cavenee, Webster K.; Bensinger, Steven J.; Mischel, Paul S.

    2013-01-01

    Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here we demonstrate the first example of this alternate mechanism in brain tumors by showing that EGFR-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing PDGFRβ. Mechanistic studies demonstrate that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1 and ERK-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, non-genetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy. PMID:23533263

  17. PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina PBMC

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2005-01-01

    Full Text Available Mechanisms underlying in vitro immunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs and polychlorinated biphenyls (PCBs were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs Fyn and Itk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP, 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169, a model immunotoxic PCB, or DMSO (vehicle control. Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKs Fyn and Itk were both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part by disruption of T cell receptor (TCR signaling and cytokine production.

  18. morphological features of tyrosine hydroxylase immunoreactive cells ...

    African Journals Online (AJOL)

    Mgina

    glandular cells. TH immunoreactive fibers are considered to be extrinsic in origin, arising from the cell bodies in the celiac ganglia (Beckman 1986). Observations made in the rat have showed that neurones in the celiac ganglia that innervate the pancreas are immunopositive for TH (Hökfelt et al. 1977; Schultzberg et a l .

  19. Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Lehmann, E.M.; Princen, H.M.G.

    1993-01-01

    We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most

  20. A unique dual activity amino acid hydroxylase in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Gaskell

    Full Text Available The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s of these bi-functional enzymes during host infection are discussed.

  1. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  2. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    Science.gov (United States)

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  3. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  4. Protonation Effect of Tyrosine in a Segment of the SRF Transcription Factor: A Combined Optical Spectroscopy, Molecular Dynamics, and Density Functional Theory Calculation Study

    Czech Academy of Sciences Publication Activity Database

    Profantová, B.; Profant, V.; Zíma, V.; Kopecký, V. Jr.; Bednárová, Lucie; Zentz, Ch.; Baumruk, V.; Turpin, P. Y.; Štěpánek, J.

    2013-01-01

    Roč. 117, č. 50 (2013), s. 16086-16095 ISSN 1520-6106 R&D Projects: GA ČR GA202/09/0193 Institutional support: RVO:61388963 Keywords : MADS box * protein secondary structure * tyrosine * pHtransition * Raman spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

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

    Energy Technology Data Exchange (ETDEWEB)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

    1988-11-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  7. Serum Inter-α-inhibitor activates the Yes tyrosine kinase and YAP/TEAD transcriptional complex in mouse embryonic stem cells.

    Science.gov (United States)

    Pijuan-Galitó, Sara; Tamm, Christoffer; Annerén, Cecilia

    2014-11-28

    We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Serum Inter-α-inhibitor Activates the Yes Tyrosine Kinase and YAP/TEAD Transcriptional Complex in Mouse Embryonic Stem Cells*

    Science.gov (United States)

    Pijuan-Galitó, Sara; Tamm, Christoffer; Annerén, Cecilia

    2014-01-01

    We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal. PMID:25301940

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

    Directory of Open Access Journals (Sweden)

    Mike Petrassi

    2017-05-01

    Full Text Available 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.

  10. Phenylalanine hydroxylase from Legionella pneumophila is a thermostable enzyme with a major functional role in pyomelanin synthesis.

    Directory of Open Access Journals (Sweden)

    Marte I Flydal

    Full Text Available Legionella pneumophila is a pathogenic bacterium that can cause Legionnaires' disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from L. pneumophila (lpPAH, the product of the phhA gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.Comparative studies of wild-type and phhA mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. phhA and letA (gacA appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. phhA is expressed in L. pneumophila growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (T(m = 79 ± 0.5°C, a high specific activity at 37°C (10.2 ± 0.3 µmol L-Tyr/mg/min and low affinity for the substrate (K(m (L-Phe = 735 ± 50 µM.lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.

  11. EB-1, a tyrosine kinase signal transduction gene, is transcriptionally activated in the t(1;19) subset of pre-B ALL, which express oncoprotein E2a-Pbx1.

    Science.gov (United States)

    Fu, X; McGrath, S; Pasillas, M; Nakazawa, S; Kamps, M P

    1999-09-02

    The t(1;19) translocation of pre-B cell acute lymphocytic leukemia (ALL) produces E2a-Pbx1, a chimeric oncoprotein containing the transactivation domains of E2a joined to the homeodomain protein, Pbx1. E2a-Pbx1 causes T cell and myeloid leukemia in mice, blocks differentiation of cultured myeloid progenitors, and transforms fibroblasts through a mechanism accompanied by aberrant expression of tissue-specific and developmentally-regulated genes. Here we investigate whether aberrant gene expression also occurs specifically in the t(1;19)-containing subset of pre-B cell ALL in man. Two new genes, EB-1 and EB-2, as well as Caldesmon were transcriptionally activated in each of seven t(1;19) cell lines. EB-1 expression was extremely low in marrow from patients having pre-B ALL not associated with the t(1;19), and elevated more than 100-fold in marrow from patients with pre-B ALL associated with the t(1;19). Normal EB-1 expression was strong in brain and testis, the same tissues exhibiting the highest levels of PBX1 expression. EB-1 encodes a signaling protein containing a phosphotyrosine binding domain homologous to that of dNumb developmental regulators and two SAM domains homologous to those in the C-terminal tail of Eph receptor tyrosine kinases. We conclude that aberrant expression of tissue-specific genes is a characteristic of t(1;19) pre-B ALL, as was previously found in fibroblasts transformed by E2a-Pbx1. Potentially, EB-1 overexpression could interfere with normal signaling controlling proliferation or differentiation.

  12. Genetics Home Reference: dopamine beta-hydroxylase deficiency

    Science.gov (United States)

    ... Twitter Home Health Conditions Dopamine beta-hydroxylase deficiency Dopamine beta-hydroxylase deficiency Printable PDF Open All Close ... Javascript to view the expand/collapse boxes. Description Dopamine beta (β)-hydroxylase deficiency is a condition that ...

  13. Seventeen Alpha-hydroxylase Deficiency

    Directory of Open Access Journals (Sweden)

    Siew-Lee Wong

    2006-01-01

    Full Text Available Seventeen a-hydroxylase deficiency (17OHD is a rare form of congenital adrenal hyperplasia in which defects in the biosynthesis of cortisol and sex steroid result in mineralocorticoid excess, hypokalemic hypertension and sexual abnormalities such as pseudohermaphroditism in males, and sexual infantilism in females. The disease is inherited in an autosomal recessive pattern, and is caused by mutations in the gene encoding cytochrome P450c17 (CYP17, which is the single polypeptide that mediates both 17α-hydroxylase and 17,20-lyase activities. We report the case of a 15-year-old patient with 17OHD who had a female phenotype but male karyotype (46,XY. The diagnosis was made based on classical clinical features, biochemical data and molecular genetic study. Two mutations were identified by polymerase chain reaction amplification and sequencing, including a S106P point mutation in exon 2 and a 9-bp (GACTCTTTC deletion from nucleotide position 1519 in exon 8 of CYP17. The first of these mutations was found in the father and the second in the mother, and both have been previously reported in Asia. The patient's hypertension and hypokalemia resolved after glucocorticoid replacement and treatment with potassium-sparing diuretics. Sex hormone replacement was prescribed for induction of sexual development and reduction of the final height. Prophylactic gonadectomy was scheduled. In summary, 17OHD should be suspected in patients with hypokalemic hypertension and lack of secondary sexual development so that appropriate therapy can be implemented.

  14. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

    Science.gov (United States)

    2012-01-01

    Background Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant

  15. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

    Directory of Open Access Journals (Sweden)

    Lin Yuheng

    2012-04-01

    Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

  16. Dopamine beta-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Senard Jean-Michel

    2006-03-01

    Full Text Available Abstract Dopamine beta-hydroxylase (DβH deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS. Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

  17. Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions.

    Science.gov (United States)

    Marino, Nicole D; Boothroyd, John C

    2017-05-01

    Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Exploring oxidative modifications of tyrosine

    DEFF Research Database (Denmark)

    Houée-Lévin, C; Bobrowski, K; Horakova, L

    2015-01-01

    residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... processes are important in vivo and can contribute to cellular pathology....

  19. Characterization of two carnation petal prolyl 4 hydroxylases.

    Science.gov (United States)

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis

    2010-10-01

    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers. Copyright © Physiologia Plantarum 2010.

  20. Tyrosine supplementation for phenylketonuria.

    Science.gov (United States)

    Webster, Diana; Wildgoose, Joanne

    2013-06-05

    Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap. However, the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in phenylketonuria. Therefore, this review aims to assess the efficacy of tyrosine supplementation for phenylketonuria. To assess the effects of tyrosine supplementation alongside or instead of a phenylalanine-restricted diet for people with phenylketonuria, who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of a phenylalanine-restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register which is comprised of references identified from comprehensive electronic database searches, handsearches of relevant journals and abstract books of conference proceedings. Additional studies were identified from handsearches of the Journal of Inherited Metabolic Disease (from inception in 1978 to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references.Date of the most recent search of the Group's Inborn Errors of Metabolism Trials Register: 28 June 2012. All randomised or quasi-randomised trials investigating the use of tyrosine supplementation versus placebo in people with phenylketonuria in addition to, or instead of, a phenylalanine-restricted diet. People treated for maternal phenylketonuria were excluded. Two authors independently assessed the trial eligibility, methodological quality

  1. Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

    Science.gov (United States)

    Huang, Jin; Lin, Yuheng; Yuan, Qipeng; Yan, Yajun

    2015-04-01

    Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

  2. Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

    Directory of Open Access Journals (Sweden)

    Yang M

    2014-08-01

    Full Text Available Ming Yang,1 Huizhong Su,1 Tomoyoshi Soga,2 Kamil R Kranc,3 Patrick J Pollard1 1Cancer Biology and Metabolism Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 2Institute for Advanced Biosciences, Keio University, Mizukami, Tsuruoka, Yamagata, Japan; 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Abstract: The hypoxia-inducible factor (HIF prolyl hydroxylase domain enzymes (PHDs regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2 availability enables its association with the von Hippel-Lindau (VHL tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. Keywords: prolyl hydroxylase domain (PHD

  3. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

    Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

    2008-11-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

  4. Tyrosine Modifications in Aging

    OpenAIRE

    Feeney, Maria B.; Schöneich, Christian

    2012-01-01

    Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may cont...

  5. Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

    Science.gov (United States)

    Baptista, Melisa J; O'Farrell, Casey; Daya, Sneha; Ahmad, Rili; Miller, David W; Hardy, John; Farrer, Matthew J; Cookson, Mark R

    2003-05-01

    Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

  6. Biodegradation of the allelopathic chemical m-tyrosine by Bacillus aquimaris SSC5 involves the homogentisate central pathway.

    Science.gov (United States)

    Khan, Fazlurrahman; Kumari, Munesh; Cameotra, Swaranjit Singh

    2013-01-01

    m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and (1)H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms.

  7. The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

    Directory of Open Access Journals (Sweden)

    Marta Perez

    2017-11-01

    Full Text Available Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi was implicated in interaction among the two clusters.

  8. Regulation of ex vivo tyrosine hydroxylase (TH) activity is not altered by chronic lead (Pb) exposure

    International Nuclear Information System (INIS)

    Lasley, S.M.; Green, M.C.

    1991-01-01

    Previous studies have suggested that chronic Pb exposure results in impaired regulation of CNS dopamine (DA) synthesis in rats. The present study was designed to directly assess TH activity in exposed animals compared to controls, employing a pharmacological model that assesses the functional status of dopaminergic synthesis-modulating autoreceptors. At birth dams received 0.2% Pb acetate in drinking water. Offspring were weaned to and maintained on the same solution until termination at 60 or 120 days. Rats were given saline or a DA agonist (EMD 23448 or CGS 15855A) 45 min before sacrifice followed 15 min later by gamma-butyrolactone (GBL). Regional TH activity was measured by a modification of the tritium release method. DA content was determined by liquid chromatography. The ability of EMD 23448 to prevent the GBL-induced increase in DA content was significantly diminished in caudate-putamen (C-P) of exposed rats compared to controls, similar to previous observations. However, an analogous effect of Pb on TH activity in this drug model was not observed using CGS 15855A in rats either 60 or 120 days of age. These findings suggest that chronic Pb exposure has no effect on autoreceptor-mediated regulation of TH in DA neurons when TH activity is measured ex vivo

  9. A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio.

    Science.gov (United States)

    Hernández-Romero, Diana; Sanchez-Amat, Antonio; Solano, Francisco

    2006-01-01

    The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature 415, 497-502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil-borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.

  10. Effects of repetitive hypoglycemia on neuroendocrine response and brain tyrosine hydroxylase activity in the rat.

    NARCIS (Netherlands)

    Figlewicz, D. P.; Van Dijk, G.; Wilkinson, C. W.; Gronbeck, P.; Higgins, M.; Zavosh, A.

    2002-01-01

    Hypoglycemia-associated autonomic failure (HAAF) is a syndrome of acute adaptation to a metabolic stressor, in which neuroendocrine responses to repetitive hypoglycemic bouts are blunted. The CNS mechanisms that contribute to HAAF are unknown. In the present study, we modeled HAAF in the rat and

  11. Novel mechanism for Fc epsilon RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production

    Czech Academy of Sciences Publication Activity Database

    Pullen, N.A.; Barnstein, B.O.; Falanga, Y.T.; Wang, Z.Q.; Suzuki, R.; Tamang, T.D.L.; Khurana, M.C.; Harry, E.A.; Dráber, Petr; Bunting, K.D.; Mizuno, K.; Wilson, B.S.; Ryan, J.J.

    2012-01-01

    Roč. 287, č. 3 (2012), s. 2045-2054 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA ČR GAP302/10/1759; GA ČR GA301/09/1826 Grant - others:NIH(US) 1R01AI59638; NIH(US) R01DK059380; NIH(US) AI051575; NIH(US) GM065794; VCU(US) U19A1077435 Institutional support: RVO:68378050 Keywords : cytokine induction * STAT transcription factor * mast cell * Fyn kinase * STAT5 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.651, year: 2012

  12. Hypoxia Inducible Factor (HIF Hydroxylases as Regulators of Intestinal Epithelial Barrier FunctionSummary

    Directory of Open Access Journals (Sweden)

    Mario C. Manresa

    2017-05-01

    Full Text Available Human health is dependent on the ability of the body to extract nutrients, fluids, and oxygen from the external environment while at the same time maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin, and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients, fluid, and waste products, while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease often is associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease, chronic obstructive pulmonary disease, and atopic dermatitis. These chronic inflammatory disorders often are characterized by diminished tissue oxygen levels (hypoxia. Hypoxia triggers an adaptive transcriptional response governed by hypoxia-inducible factors (HIFs, which are repressed by a family of oxygen-sensing HIF hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms. Keywords: Epithelial Barrier, Inflammatory Bowel Disease, Hypoxia, Hypoxia-Inducible Factor (HIF Hydroxylases

  13. CRYSTAL STRUCTURE OF HUMAN DOPAMINE BETA-HYDROXYLASE

    DEFF Research Database (Denmark)

    2017-01-01

    A crystalline form of dopamine β-hydroxylase is provided. X-ray crystallography reveals the space group and cell dimensions, as well as the atomic coordinates. The information can be used for identifying one or more modulators of dopamine β-hydroxylase, which can then be chemically synthesised...

  14. Structural and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase

    NARCIS (Netherlands)

    Montersino, S.

    2012-01-01

    The thesis deals with the characterization of a new flavoprotein hydroxylase 3 hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1. 3HB6H is able to insert exclusively oxygen in para-position and the enzyme has been chosen to study the structural basis of such regioselectivity. As

  15. Catabolism of L-tyrosine in Trichosporon cutaneum.

    Science.gov (United States)

    Sparnins, V L; Burbee, D G; Dagley, S

    1979-01-01

    Protocatechuic acid was a catabolite in the degradation of L-tyrosine by Trichosporon cutaneum. Intact cells oxidized to completion various compounds proposed as intermediates in this conversion, but they did not readily oxidize catabolites of the homogentisate and homoprotocatechuate metabolic pathways, which are known to function in other organisms. Cell extracts converted tyrosine first to 4-hydroxycinnamic acid and then to 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. The proposed hydration product of 4-hydroxycinnamic acid, namely, beta-(4-hydroxyphenyl)-hydracrylic acid, was synthesized chemically, and its enzymatic degradation to 4-hydroxybenzaldehyde was shown to be dependent upon additions of adenosine triphosphate and coenzyme A. The hydroxylase that attacked 4-hydroxybenzoate showed a specific requirement for reduced nicotinamide adenine dinucleotide phosphate. Protocatechuate, the product of this reaction, was oxidized by cell extracts supplemented with reduced nicotinamide adenine dinucleotide or, less effectively, with reduced nicotinamide adenine dinucleotide phosphate, but these extracts contained no ring fission dioxygenase for protocatechuate. Evidence is presented that the principal hydroxylation product of protocatechuate was hydroxyquinol, the benzene nucleus of which was cleaved oxidatively to give maleylacetic acid. PMID:571434

  16. Protein-tyrosine phosphatases in zebrafish gastrulation

    NARCIS (Netherlands)

    van Eekelen, M.J.L.

    2011-01-01

    Protein tyrosine phosphorylation plays a key role in relaying external stimuli and signals into the cell towards the appropriate responses. This process is mediated by protein-tyrosine kinases adding a phosphor group to a tyrosine residue and protein-tyrosine phosphatases removing a phosphor group

  17. Tyrosine modifications in aging.

    Science.gov (United States)

    Feeney, Maria B; Schöneich, Christian

    2012-12-01

    The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3'-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind.

  18. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  19. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    Energy Technology Data Exchange (ETDEWEB)

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  20. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

    Science.gov (United States)

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Katz, Maximiliano Javier; Gándara, Lautaro; Misra, Tvisha; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-05-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

  1. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU).

    Science.gov (United States)

    Harding, Cary O; Winn, Shelley R; Gibson, K Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

    2014-09-01

    Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU.

  2. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU)

    Science.gov (United States)

    Harding, Cary O.; Winn, Shelley R.; Gibson, K. Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

    2014-01-01

    Summary Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU. PMID:24487571

  3. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes...

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

    Science.gov (United States)

    ... fatty acid 2-hydroxylase adds a single oxygen atom to a hydrogen atom at a particular point on a fatty acid ... direct-to-consumer genetic testing? What is precision medicine? What is newborn screening? New Pages Obstructive sleep ...

  5. Treatment of Nonclassic 11-Hydroxylase Deficiency with Ashwagandha Root

    Directory of Open Access Journals (Sweden)

    Daniel Powell

    2017-01-01

    Full Text Available An elderly woman presented with acne and male pattern alopecia, which upon diagnostic evaluation was found to be due to nonclassic 11-hydroxylase deficiency. We previously reported that Ashwagandha root ameliorates nonclassic 3-β-ol dehydrogenase and aldosterone synthase deficiencies. This is the first report of its use being associated with amelioration of nonclassic 11-hydroxylase deficiency, where its apparent effects appear to be dose-related.

  6. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

    2002-01-01

    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

  7. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

    Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

    1988-01-01

    Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

  8. Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

    2016-06-03

    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 of 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.

  9. Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression.

    Science.gov (United States)

    Nishimura, Kaneyasu; Kitamura, Yoshihisa; Inoue, Takeshi; Umesono, Yoshihiko; Yoshimoto, Kanji; Taniguchi, Takashi; Agata, Kiyokazu

    2008-12-01

    The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that Dj

  10. Reciprocal regulation of C-Maf tyrosine phosphorylation by Tec and Ptpn22.

    Science.gov (United States)

    Liu, Chih-Chun; Lai, Chen-Yen; Yen, Wei-Feng; Lin, Yu-Hsien; Chang, Hui-Hsin; Tai, Tzong-Shyuan; Lu, Yu-Jung; Tsao, Hsiao-Wei; Ho, I-Cheng; Miaw, Shi-Chuen

    2015-01-01

    C-Maf plays an important role in regulating cytokine production in TH cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

  11. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    Science.gov (United States)

    Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

    1992-01-01

    The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

  12. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  13. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  14. Expression of the vitamin D receptor, 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Andersen, Claus B.; Nielsen, John E

    2010-01-01

    The vitamin D receptor (VDR), CYP27B1 and CYP24A1 are expressed in the human kidney, but the segmental expression of the 25-hydroxylases is unknown. A comprehensive analysis of CYP2R1, CYP27A1, CYP27B1, VDR and CYP24A1 expression in normal kidney and renal clear cell cancer (CCc) would reveal...

  15. Age-related changes in immunoreactivity for dopamine β-hydroxylase in carotid body glomus cells in spontaneously hypertensive rats.

    Science.gov (United States)

    Kato, Kouki; Fushuku, Seigo; Yamamoto, Yoshio

    2017-07-01

    The purpose of this study was to investigate immunoreactivity for dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in carotid body (CB) glomus cells in spontaneously hypertensive rats (SHR/Izm) at 4 (prehypertensive stage), 8 (early stage of developmental hypertension), 12 (later stage of developmental hypertension), and 16weeks of age (established hypertensive stage). Age-matched Wistar Kyoto rats (WKY/Izm) were used as controls. Staining properties for TH were similar between both strains at each age. Regarding DBH immunostaining, although some glomus cells showed intense DBH immunoreactivity at 4weeks of age, these cells were rarely observed at 8, 12, and 16weeks of age in WKY/Izm. In SHR/Izm, intense DBH immunoreactivity was observed in some glomus cells at 4weeks of age, these cells were also observed at 8 and 12weeks of age, and their number increased at 16weeks of age. An image analysis showed that the percentage of DBH-immunopositive glomus cells in WKY/Izm was approximately 30% at 4weeks of age and significantly decreased to approximately 10% at 8, 12, and 16weeks of age (pcells was similar in both strains at 4weeks of age, but became significantly lower in WKY/Izm and higher in SHR/Izm with increase in age (pcells plays an important role in the regulation of neurotransmission between CB and afferent nerves during developmental hypertension. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X

    1997-01-01

    . Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA...

  17. Use of deuterated tyrosine and phenylalanine in the study of catecholamine and aromatic acid metabolism

    International Nuclear Information System (INIS)

    Curtius, H.C.; Redweik, U.; Steinmann, B.; Leimbacher, W.; Wegmann, H.

    1975-01-01

    Deuterated tyrosine and phenylalanine have been used for the study of their respective metabolism in patients with phenylketonuria (PKU) and in healthy persons. Urinary excretion of dopamine and its metabolites was studied by GC-MS after oral administration of deuterated L-tyrosine in 2 patients with PKU and in normal controls at low and high plasma phenylalanine levels. From these studies it seemed that the in vivo tyrosine 3-hydroxylase activity and thus the formation of L-dopa depend on the phenylalanine concentration in plasma and also in tissues. After loading 3 mentally retarded patients with 3,5-[ 2 H 2 ]-4-hydroxyphenylalanine, we found, among others, excretion of deuterated m-hydroxyphenyl-hydracrylic acid, p-hydroxymandelic acid, p-hydroxybenzoic acid, p-hydroxyhippuric acid, benzoic acid and hippuric acid. An intramolecular rearrangement is postulated. Deuterated phenylalanine was used to investigate phenylalanine and dopa metabolism in PKU. In addition, one untreated person with PKU of normal intelligence and normal excretion of catecholamines at high plasma phenylalanine concentration was investigated in order to see whether there exists an alternative metabolic pathway from phenylalanine to dopa formation

  18. Functional Identification of Putrescine C- and N-Hydroxylases.

    Science.gov (United States)

    Li, Bin; Lowe-Power, Tiffany; Kurihara, Shin; Gonzales, Stephen; Naidoo, Jacinth; MacMillan, John B; Allen, Caitilyn; Michael, Anthony J

    2016-10-21

    The small polyamine putrescine (1,4-diaminobutane) is ubiquitously and abundantly found in all three domains of life. It is a precursor, through N-aminopropylation or N-aminobutylation, for biosynthesis of the longer polyamines spermidine, sym-homospermidine, spermine, and thermospermine and longer and branched chain polyamines. Putrescine is also biochemically modified for purposes of metabolic regulation and catabolism, e.g. N-acetylation and N-glutamylation, and for incorporation into specialized metabolites, e.g. N-methylation, N-citrylation, N-palmitoylation, N-hydroxylation, and N-hydroxycinnamoylation. Only one example is known where putrescine is modified on a methylene carbon: the formation of 2-hydroxyputrescine by an unknown C-hydroxylase. Here, we report the functional identification of a previously undescribed putrescine 2-hydroxylase, a Rieske-type nonheme iron sulfur protein from the β-proteobacteria Bordetella bronchiseptica and Ralstonia solanacearum. Identification of the putrescine 2-hydroxylase will facilitate investigation of the physiological functions of 2-hydroxyputrescine. One known role of 2-hydroxyputrescine has direct biomedical relevance: its role in the biosynthesis of the cyclic hydroxamate siderophore alcaligin, a potential virulence factor of the causative agent of whooping cough, Bordetella pertussis. We also report the functional identification of a putrescine N-hydroxylase from the γ-proteobacterium Shewanella oneidensis, which is homologous to FAD- and NADPH-dependent ornithine and lysine N-monooxygenases involved in siderophore biosynthesis. Heterologous expression of the putrescine N-hydroxylase in E. coli produced free N-hydroxyputrescine, never detected previously in a biological system. Furthermore, the putrescine C- and N-hydroxylases identified here could contribute new functionality to polyamine structural scaffolds, including C-H bond functionalization in synthetic biology strategies.

  19. Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

    Science.gov (United States)

    Radich, Jerald P; Mauro, Michael J

    2017-08-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

    Energy Technology Data Exchange (ETDEWEB)

    Mai, Sanyue [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Qu, Xiuhua [General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037 (China); Li, Ping; Ma, Qingjun [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Liu, Xuan, E-mail: liux931932@163.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Cao, Cheng, E-mail: cao_c@sohu.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China)

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

  1. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

    International Nuclear Information System (INIS)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-01-01

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

  2. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specifi city and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology....... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  3. Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.

    Directory of Open Access Journals (Sweden)

    Jonggeun Kim

    2013-01-01

    Full Text Available The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.. Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84, is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF encoding 518 amino acids (GenBank Accession number JX524278. The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78% with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h, wounding (24 h, cold (24 h, abscisic acid (24 h, and methyl jasmonate (24 h.

  4. Antisense-induced suppression of taxoid 14β- hydroxylase gene ...

    African Journals Online (AJOL)

    Jane

    2011-08-15

    Aug 15, 2011 ... the 11(12)-diene might be directed to the production of useful C-13 oxygenated taxoids such as Taxol. Here, we reported a general method for adjusting regulation of the taxoid pathway and provide evidence for the suppression of taxoid 14β-hydroxylase gene expression in transgenic Taxus × media cell ...

  5. Sleep patterns in congenital dopamine beta-hydroxylase deficiency

    NARCIS (Netherlands)

    J.H.M. Tulen (Joke); A.J. Man in't Veld (A.); K. Mechelse (Karel); F. Boomsma (Frans)

    1990-01-01

    textabstractSleep patterns of two young female patients with congenital dopamine beta-hydroxylase deficiency are described. In this orthostatic syndrome central and peripheral noradrenergic failure occurs as a result of impaired beta-hydroxylation of dopamine. Consequently, the levels of dopamine

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

    Science.gov (United States)

    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…

  7. Modelling the active site properties of dopamine b-hydroxylase

    Indian Academy of Sciences (India)

    Administrator

    Dopamine b-hydroxylase (DbH) is a copper-containing glycoprotein that hydroxylates dopamine to norepinephrine 1,2. Based on spectroscopic studies the active site of the metalloenzyme is proposed to have two copper centres. The enzyme in the oxidized dicopper(II) form gets reduced to the dicopper(I) unit by ascorbate ...

  8. Nonreceptor Tyrosine Kinases in Prostate

    Directory of Open Access Journals (Sweden)

    Cancer Yu-Ming Chang

    2007-02-01

    Full Text Available BACKGROUND: Carcinoma of the prostate (CaP is the most commonly diagnosed cancer in men in the United States. Signal transduction molecules such as tyrosine kinases play important roles in CaP. Src, a nonreceptor tyrosine kinase (NRTK and the first proto-oncogene discovered is shown to participate in processes such as cell proliferation and migration in CaP. Underscoring NRTK's and, specifically, Src's importance in cancer is the recent approval by the US Food and Drug Administration of dasatinib, the first commercial Src inhibitor for clinical use in chronic myelogenous leukemia (CML. In this review we will focus on NRTKs and their roles in the biology of CaP. MATERIALS AND METHODS: Publicly available literature from PubMed regarding the topic of members of NRTKs in CaP was searched and reviewed. RESULTS: Src, FAK, JaK1/2, and ETK are involved in processes indispensable to the biology of CaP: cell growth, migration, invasion, angiogenesis, and apoptosis. CONCLUSIONS: Src emerges as a common signaling and regulatory molecule in multiple biological processes in CaP. Src's relative importance in particular stages of CaP, however, required further definition. Continued investigation of NRTKs will increase our understanding of their biological function and potential role as new therapeutic targets.

  9. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    Supplemental tyrosine is effective at limiting cold-induced decreases in working memory, presumably by augmenting brain catecholamine levels, since tyrosine is a precursor for catecholamine synthesis...

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

    DEFF Research Database (Denmark)

    Hundahl, C A; Fahrenkrug, J; Luuk, H

    2012-01-01

    Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen...... reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression...

  11. Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

    Directory of Open Access Journals (Sweden)

    Akahoshi Eiichi

    2009-06-01

    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene transactivation via an AhR-AHRE-III-mediated pathway. The AhR- mediated pathway could have a particular AhR-mediated genomic control pathway transmitting the effects of TCDD action to target cells in the development of dopaminergic disabilities.

  12. Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

    Energy Technology Data Exchange (ETDEWEB)

    Schnicker, Nicholas J. [Department; Razzaghi, Mortezaali [Department; Guha Thakurta, Sanjukta [Department; Chakravarthy, Srinivas [Biophysics; Dey, Mishtu [Department

    2017-10-17

    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, 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.

  13. Expression of the vitamin D receptor, 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Andersen, Claus B.; Nielsen, John E

    2010-01-01

    The vitamin D receptor (VDR), CYP27B1 and CYP24A1 are expressed in the human kidney, but the segmental expression of the 25-hydroxylases is unknown. A comprehensive analysis of CYP2R1, CYP27A1, CYP27B1, VDR and CYP24A1 expression in normal kidney and renal clear cell cancer (CCc) would reveal...... the segmental location of expression, and clarify whether the reported loss of VDR in CCc is coincident with alterations of vitamin D metabolism....

  14. Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-02-01

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

  16. The carboxyl terminal tyrosine 417 residue of NOK has an autoinhibitory effect on NOK-mediated signaling transductions

    International Nuclear Information System (INIS)

    Li Yinghua; Zhong Shan; Rong Zhili; Ren Yongming; Li Zhiyong; Zhang Shuping; Chang Zhijie; Liu Li

    2007-01-01

    Receptor protein tyrosine kinases (RPTKs) are essential mediators of cell growth, differentiation, migration, and metabolism. Recently, a novel RPTK named NOK has been cloned and characterized. In current study, we investigated the role of the carboxyl terminal tyrosine 417 residue of NOK in the activations of different signaling pathways. A single tyrosine to phenylalanine point mutation at Y417 site (Y417 F) not only dramatically enhanced the NOK-induced activation of extracellular signal-regulated kinase (ERK), but also markedly promoted the NOK-mediated activation of both signal transducer and activator of transcription 1 and 3 (STAT1 and 3). Moreover, the proliferation potential of NIH3T3-NOK (Y417F) stable cells were significantly elevated as compared with that of NIH3T3-NOK. Overall, our results demonstrate that the tyrosine Y417 residue at the carboxyl tail of NOK exhibits an autoinhibitory role in NOK-mediated signaling transductions

  17. Sleep patterns in congenital dopamine beta-hydroxylase deficiency

    OpenAIRE

    Tulen, Joke; Man in't Veld, A.; Mechelse, Karel; Boomsma, Frans

    1990-01-01

    textabstractSleep patterns of two young female patients with congenital dopamine beta-hydroxylase deficiency are described. In this orthostatic syndrome central and peripheral noradrenergic failure occurs as a result of impaired beta-hydroxylation of dopamine. Consequently, the levels of dopamine and its metabolites are elevated. The relative importance of noradrenaline deficit in the face of dopamine excess for sleep-regulatory mechanisms can be inferred from the sleep pattern of these patie...

  18. Tryptophan hydroxylase-1 regulates immune tolerance and inflammation

    OpenAIRE

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

    2012-01-01

    Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 defic...

  19. Dityrosine formation is impaired by tyrosine phosphorylation.

    Science.gov (United States)

    Christian, S; Bernhard, G; Patrizia, R; Brigitte, M

    1992-10-15

    Using pure tyrosine and phosphotyrosine we have recently shown that phosphotyrosine is unable to form peroxidase catalyzed dimers (1989, FEBS Lett. 255, 395-397). In the present report, the effect of phosphotyrosine residues within a protein structure on dityrosine formation was studied using casein as a model protein. Dephosphorylation of casein resulted in a dose and time dependent increased synthesis of dityrosines following treatment with peroxidase/H2O2. The extent of crosslink formation was inversely related to the amount of phosphorylated tyrosine residues as quantitated by immunoblotting. Thus, phosphorylation of tyrosine residues could play a regulatory role in protein-crosslinking where dityrosine bonds are involved.

  20. Restraint stress increases prolactin-mediated phosphorylation of signal transducer and activator of transcription 5 in the hypothalamus and adrenal cortex in the male mouse.

    Science.gov (United States)

    Kirk, S E; Xie, T Y; Steyn, F J; Grattan, D R; Bunn, S J

    2017-06-01

    Prolactin is a pleiotropic peptide hormone produced by the lactotrophs in the anterior pituitary. Its rate of secretion is primarily regulated by a negative-feedback mechanism where prolactin stimulates the activity of the tuberoinfundibular dopaminergic (TIDA) neurones, increasing their release of dopamine, which accesses the pituitary via the median eminence to suppress further prolactin secretion. In addition to its well established role in lactation, circulating prolactin is secreted in response to stress, although the mechanism by which this is achieved or its cellular targets remains unknown. In the present study, we show that 15 minutes of restraint stress causes an approximately seven-fold increase in circulating prolactin concentration in male mice. Monitoring prolactin receptor activation, using immunohistochemistry to determine the level and distribution of tyrosine phosphorylated signal transducer and activator of transcription 5 (pSTAT5), we show that this stress-induced increase in prolactin interacts with both central and peripheral targets. Restraint stress for 15 minutes significantly increased pSTAT5 staining in the arcuate nucleus, median eminence and the zona fasciculata of the adrenal cortex. In each case, this response was prevented by pretreating the animals with bromocriptine to block prolactin secretion from the pituitary. Interestingly, in contrast to many cells in the arcuate nucleus, stress reduced pSTAT5 staining of the TIDA neurones (identified by dual-labelling for tyrosine hydroxylase). This suggests that there is reduced prolactin signalling in these cells and thus potentially a decline in their inhibitory influence on prolactin secretion. These results provide evidence that prolactin secreted in response to acute stress is sufficient to activate prolactin receptors in selected target tissues known to be involved in the physiological adaptation to stress. © 2017 British Society for Neuroendocrinology.

  1. The transcription factor Uncx4.1 acts in a short window of midbrain dopaminergic neuron differentiation

    Directory of Open Access Journals (Sweden)

    Rabe Tamara I

    2012-12-01

    Full Text Available Abstract Background The homeobox containing transcription factor Uncx4.1 is, amongst others, expressed in the mouse midbrain. The early expression of this transcription factor in the mouse, as well as in the chick midbrain, points to a conserved function of Uncx4.1, but so far a functional analysis in this brain territory is missing. The goal of the current study was to analyze in which midbrain neuronal subgroups Uncx4.1 is expressed and to examine whether this factor plays a role in the early development of these neuronal subgroups. Results We have shown that Uncx4.1 is expressed in GABAergic, glutamatergic and dopaminergic neurons in the mouse midbrain. In midbrain dopaminergic (mDA neurons Uncx4.1 expression is particularly high around E11.5 and strongly diminished already at E17.5. The analysis of knockout mice revealed that the loss of Uncx4.1 is accompanied with a 25% decrease in the population of mDA neurons, as marked by tyrosine hydroxylase (TH, dopamine transporter (DAT, Pitx3 and Ngn2. In contrast, the number of glutamatergic Pax6-positive cells was augmented, while the GABAergic neuron population appears not affected in Uncx4.1-deficient embryos. Conclusion We conclude that Uncx4.1 is implicated in the development of mDA neurons where it displays a unique temporal expression profile in the early postmitotic stage. Our data indicate that the mechanism underlying the role of Uncx4.1 in mDA development is likely related to differentiation processes in postmitotic stages, and where Ngn2 is engaged. Moreover, Uncx4.1 might play an important role during glutamatergic neuronal differentiation in the mouse midbrain.

  2. Adaptation of phenylalanine and tyrosine catabolic pathway to hibernation in bats.

    Science.gov (United States)

    Pan, Yi-Hsuan; Zhang, Yijian; Cui, Jie; Liu, Yang; McAllan, Bronwyn M; Liao, Chen-Chung; Zhang, Shuyi

    2013-01-01

    Some mammals hibernate in response to harsh environments. Although hibernating mammals may metabolize proteins, the nitrogen metabolic pathways commonly activated during hibernation are not fully characterized. In contrast to the hypothesis of amino acid preservation, we found evidence of amino acid metabolism as three of five key enzymes, including phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase (HGD), fumarylacetoacetase (FAH), involved in phenylalanine and tyrosine catabolism were co-upregulated during hibernation in two distantly related species of bats, Myotis ricketti and Rhinolophus ferrumequinum. In addition, the levels of phenylalanine in the livers of these bats were significantly decreased during hibernation. Because phenylalanine and tyrosine are both glucogenic and ketogenic, these results indicate the role of this catabolic pathway in energy supply. Since any deficiency in the catabolism of these two amino acids can cause accumulations of toxic metabolites, these results also suggest the detoxification role of these enzymes during hibernation. A higher selective constraint on PAH, HPD, and HGD in hibernators than in non-hibernators was observed, and hibernators had more conserved amino acid residues in each of these enzymes than non-hibernators. These conserved amino acid residues are mostly located in positions critical for the structure and activity of the enzymes. Taken together, results of this work provide novel insights in nitrogen metabolism and removal of harmful metabolites during bat hibernation.

  3. Regulation of rabbit lung cytochrome P-450 prostaglandin omega-hydroxylase (P-450/sub PG-omega/) during pregnancy

    International Nuclear Information System (INIS)

    Muerhoff, A.S.; Williams, D.E.; Jackson, V.; Leithauser, M.T.; Waterman, M.R.; Johnson, E.F.; Masters, B.S.S.

    1987-01-01

    The mechanism of induction during pregnancy of a rabbit lung prostaglandin omega-hydroxylase cytochrome P-450 has been investigated. This activity has been demonstrated to be induced over 100-fold in 28-day pregnant rabbits, as compared to nonpregnant rabbits. The induction is reflected by an increase in the amount of P-450/sub PG-omega/ protein as measured by Western blotting. P-450/sub PG-omega/ microsomal protein increases throughout gestation concomitant with an increase in PGE 1 omega-hydroxylase activity. Elucidation of the level of induction involved extraction of RNA from rabbit lungs obtained at various days of gestation followed by in vitro translation of the RNA in the presence of 35 S-methionine. Immunoprecipitation of newly synthesized P-450 and analysis of the immunoisolates by SDS-PAGE, autoradiography and densitometry of the P-450/sub PG-omega/ band revealed that the P-450/sub PG-omega/ mRNA levels followed the gestational time-dependent increase observed for both PGE 1 omega-hydroxylase activity and P-450/sub PG-omega/ protein, i.e., a gradual increase peaking at 28-days, dropping precipitously to near control levels following parturition. These data suggest that control of P-450/sub PG-omega expression occurs at the transcriptional level. Western blots of human lung bronchioloalveolar-carcinoma cell lines NCL-H322 and NCL-H358 utilizing a guinea pig IgG to P-450/sub PG-omega/ detect a cross-reactive species

  4. Genomic organization of Bruton`s tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Rohrer, J.; Conley, M.E. [Univ. of Tennessee, Memphis, TN (United States)

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

  5. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  6. 25-Hydroxyvitamin D3 1-Alpha-Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-11-01

    Full Text Available Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1 by FGF23. Conversely, 1,25(OH2D3 stimulates, by activating the vitamin D3 receptor (Vdr, the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293 or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR or of mineralocorticoid receptor (NR3C2. Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours and mice (8 hours or 5 days. Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours. Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of

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

    Science.gov (United States)

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

    2015-12-01

    Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  8. No effect of oral tyrosine on total tyrosine levels in breast milk: implications for dietary supplementation in early postpartum.

    Science.gov (United States)

    Dowlati, Yekta; Ravindran, Arun V; Maheux, Maxim; Steiner, Meir; Stewart, Donna E; Meyer, Jeffrey H

    2014-12-01

    Postpartum depression (PPD) is the most common complication of childbearing with a 13 % prevalence rate, and there is no widespread approach for prevention. There is an appealing theoretical rationale for oral tyrosine to help prevent PPD. However, the effect of oral tyrosine on its total and free concentrations in breast milk and plasma of breastfeeding mothers is not known. Twenty-four healthy breastfeeding women were randomly assigned to 0, 2, 5, or 10 g of oral tyrosine. Free and total tyrosine in breast milk and free tyrosine in plasma were measured. Free tyrosine was also measured in 12 different infant formulas. Total tyrosine in breast milk did not rise, but there was a slight tendency towards a reduction (up to −12 %; repeated measures ANOVA (RMANOVA): p = 0.074). Maternal plasma tyrosine rose (RMANOVA: p oral tyrosine on its concentration in breast milk supports further development of oral tyrosine as part of a prevention strategy for PPD.

  9. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  10. Regulation of human sterol 27-hydroxylase gene (CYP27A1) by bile acids and hepatocyte nuclear factor 4alpha (HNF4alpha).

    Science.gov (United States)

    Chen, Wenling; Chiang, John Y L

    2003-08-14

    Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt -147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4alpha, is identified. HNF4alpha strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4alpha plays a pivotal role in transcriptional regulation of this gene.

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

    Science.gov (United States)

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

    2011-03-24

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

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

    Directory of Open Access Journals (Sweden)

    Haiying Yu

    2011-03-01

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

  13. Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARγ ligands

    OpenAIRE

    Quinn, Carmel M.; Jessup, Wendy; Wong, Jenny; Kritharides, Leonard; Brown, Andrew J.

    2005-01-01

    CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However...

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

    Science.gov (United States)

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

    2014-09-01

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

  15. Expression of ovine ubiquitin C-terminal hydroxylase 1, pH and ...

    African Journals Online (AJOL)

    Ubiquitin C-terminal hydroxylase (UCH-L1) has been identified in few transcriptome studies as a biomarker coding for trauma and perception of pain in non-meat species. For the first time, real-time polymerase chain reaction (qPCR) assay was used to quantitate the expression of ovine ubiquitin C-terminal hydroxylase 1 ...

  16. 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.

    Science.gov (United States)

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

    2006-01-24

    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. 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. 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

  17. 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

    Directory of Open Access Journals (Sweden)

    Paillard Sophie

    2006-01-01

    Full Text Available Abstract 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

  18. Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives

    Science.gov (United States)

    Bishop, Tammie; Ratcliffe, Peter J

    2014-01-01

    By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. PMID:27774477

  19. Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

    International Nuclear Information System (INIS)

    Wang, J.-H.; Tuohimaa, Pentti

    2006-01-01

    Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

  20. Mechanisms of Peroxynitrite Mediated Nitration of Tyrosine

    Science.gov (United States)

    Gunaydin, Hakan; Houk, K. N.

    2009-01-01

    The mechanisms of tyrosine nitration by peroxynitrous acid or nitrosoperoxycarbonate were investigated with the CBS-QB3 method. Either the protonation of peroxynitrite, or a reaction with carbon dioxide gives a reactive peroxide intermediate. Peroxynitrous acid mediated nitration of phenol occurs via the unimolecular decomposition to give nitrogen dioxide and hydroxyl radicals. Nitrosoperoxycarbonate also undergoes unimolecular decomposition to give carbonate and nitrogen dioxide radicals. The reactions of tyrosine with the hydroxyl or carbonate radicals give a phenoxy radical intermediate. The reaction of the nitrogen dioxide with this radical intermediate followed by tautomerization gives nitrated tyrosine in both cases. According to CBS-QB3 calculations, the rate-limiting step for the nitration of phenol is the decomposition of peroxynitrous acid or of nitrosoperoxycarbonate. PMID:19374346

  1. Rat-liver cholesterol 7α-hydroxylase. Pt. 1

    International Nuclear Information System (INIS)

    Cantfort, J. van; Renson, J.; Gielen, J.

    1975-01-01

    A new assay is described to measure the activity of cholesterol 7α-hydroxylase and compared to the conventional 14 C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7α-hydrogen by a hydroxyl group. [7α- 3 H]cholesterol is incubated at 37 0 C and in the presence of molecular O 2 , in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9,000 x g supernatant), NADPH, MgCl 2 and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 μM) in the incubation mixture to saturate the ezyme (K(m) = 100 μM). Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4- 14 C]cholesterol technique (r = 0.96; P 3 H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7α-hydroxycholesterol, the tritiated water representing the entire cholesterol 7α-hydroxylase activity. (orig.) [de

  2. Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

    Science.gov (United States)

    Dekanty, Andrés; Wappner, Pablo

    2010-01-01

    Background The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain. PMID:20811646

  3. Oxygen sensing in Drosophila: multiple isoforms of the prolyl hydroxylase fatiga have different capacity to regulate HIFalpha/Sima.

    Science.gov (United States)

    Acevedo, Julieta M; Centanin, Lazaro; Dekanty, Andrés; Wappner, Pablo

    2010-08-25

    The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.

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

    Science.gov (United States)

    Schlangen, Karin; Miosic, Silvija; Halbwirth, Heidi

    2010-02-01

    In the petals of Dahlia variabilis, hydroxylation of chalcones at position 3 can be detected, except the well-known flavonoid 3'-hydroxylation. Although the reaction is well characterized at the enzymatic level, it remained unclear whether it is catalyzed by a flavonoid 3'-hydroxylase (F3'H, EC1.14.13.21, CYP75B) with broad substrate specificity. Two novel allelic variants of F3'H were cloned from D. variabilis, which differ only in three amino acids within their 508 residues. The corresponding recombinant enzymes show significant differences in their chalcone 3-hydroxylase (CH3H) activity. A substitution of alanine at position 425 with valine enables CH3H activity, whereas the reciprocal substitution leads to a loss of CH3H activity. Interaction of the valine at position 425 with not yet identified structural properties seems to be decisive for chalcone acceptance. This is the first identification of an F3'H which is able to catalyze chalcone 3-hydroxylation to a physiologically relevant extent from any plant species. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  5. Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

    NARCIS (Netherlands)

    van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

    Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some

  6. Differential evolutionary wiring of the tyrosine kinase Btk.

    Directory of Open Access Journals (Sweden)

    Hossain M Nawaz

    Full Text Available BACKGROUND: A central question within biology is how intracellular signaling pathways are maintained throughout evolution. Btk29A is considered to be the fly-homolog of the mammalian Bruton's tyrosine kinase (Btk, which is a non-receptor tyrosine-kinase of the Tec-family. In mammalian cells, there is a single transcript splice-form and the corresponding Btk-protein plays an important role for B-lymphocyte development with alterations within the human BTK gene causing the immunodeficiency disease X-linked agammaglobulinemia in man and a related disorder in mice. In contrast, the Drosophila Btk29A locus encodes two splice-variants, where the type 2-form is the more related to the mammalian Btk gene product displaying more than 80% homology. In Drosophila, Btk29A displays a dynamic pattern of expression through the embryonic to adult stages. Complete loss-of-function of both splice-forms is lethal, whereas selective absence of the type 2-form reduces the adult lifespan of the fly and causes developmental abnormalities in male genitalia. METHODOLOGY/PRINCIPAL FINDINGS: Out of 7004-7979 transcripts expressed in the four sample groups, 5587 (70-79% were found in all four tissues and strains. Here, we investigated the role of Btk29A type 2 on a transcriptomic level in larval CNS and adult heads. We used samples either selectively defective in Btk29A type 2 (Btk29A(ficP or revertant flies with restored Btk29A type 2-function (Btk29A(fic Exc1-16. The whole transcriptomic profile for the different sample groups revealed Gene Ontology patterns reflecting lifespan abnormalities in adult head neuronal tissue, but not in larvae. CONCLUSIONS: In the Btk29A type 2-deficient strains there was no significant overlap between transcriptomic alterations in adult heads and larvae neuronal tissue, respectively. Moreover, there was no significant overlap of the transcriptomic changes between flies and mammals, suggesting that the evolutionary conservation is confined

  7. Chlorinated tyrosine derivatives in insect cuticle

    DEFF Research Database (Denmark)

    Andersen, Svend Olav

    2004-01-01

    , not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed...

  8. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  9. Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

    Science.gov (United States)

    Santos, Christine Nicole S; Xiao, Wenhai; Stephanopoulos, Gregory

    2012-08-21

    Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.

  10. Phenylketonuria : Tyrosine beyond the phenylalanine-restricted diet

    NARCIS (Netherlands)

    van Spronsen, FJ; Smit, PGA; Koch, R

    Controversies exist on the role of tyrosine in the pathogenesis of phenylketonuria (PKU) and, consequently, on the therapeutic role of tyrosine. This review examines data and theoretical considerations on the role of tyrosine in the pathogenesis and treatment of PKU. It is concluded that treatment

  11. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

    DEFF Research Database (Denmark)

    Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

    2004-01-01

    requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...

  12. Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function

    DEFF Research Database (Denmark)

    Lobie, P E; Allevato, G; Norstedt, G

    1995-01-01

    We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine...... (CHO-GHR1-638 Y333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also...... similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol...

  13. Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

    DEFF Research Database (Denmark)

    Tidemand, Kasper Damgaard; Peters, Günther H.J.; Harris, Pernille

    2017-01-01

    Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis...... of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH4) results in a hybrid Ping Pong-ordered mechanism...... in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been...

  14. Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors.

    Science.gov (United States)

    Aprelikova, Olga; Chandramouli, Gadisetti V R; Wood, Matthew; Vasselli, James R; Riss, Joseph; Maranchie, Jodi K; Linehan, W Marston; Barrett, J Carl

    2004-06-01

    Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Copyright 2004 Wiley-Liss, Inc.

  15. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

    International Nuclear Information System (INIS)

    Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto

    2007-01-01

    The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein

  16. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phos...

  17. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

    Science.gov (United States)

    Lee, Eun-Jeong; Facchini, Peter J

    2011-11-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

  18. Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

    International Nuclear Information System (INIS)

    Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

    1988-01-01

    A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM - phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level

  19. Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

    Energy Technology Data Exchange (ETDEWEB)

    Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

    1988-04-19

    A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM/sup -/ phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.

  20. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

  1. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein......Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

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

    Science.gov (United States)

    ... breasts and pubic hair, and do not menstruate (amenorrhea). Women with partial 17α-hydroxylase/17,20-lyase ... have female internal reproductive organs, these individuals have amenorrhea and do not develop female secondary sex characteristics. ...

  3. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  4. Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-05

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

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

    Science.gov (United States)

    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.

  6. Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine beta-hydroxylase

    DEFF Research Database (Denmark)

    Bjerrum, Ole Jannik; Helle, K B; Bock, Elisabeth Marianne

    1979-01-01

    . The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic...... interaction immunoelectrophoresis with phenyl-Sepharose. Furthermore, immunoelectrophoretic analysis in the presence of Triton X-100 plus the cationic detergent cetyltrimethylammonium bromide indicates additional heterogeneity of the membrane-bound dopamine-beta-hydroxylase. By limited proteolysis...

  7. Studies on estradiol-2/4-hydroxylase activity in rat brain and liver

    International Nuclear Information System (INIS)

    Theron, C.N.

    1985-03-01

    A sensitive and specific radio-enzymatic assay was used to study estradiol-2/4-hydroxylase activity in rat liver microsomes and in microsomes obtained from 6 discrete brain areas of the rat. Kinetic parameters were determined for these enzyme activities. The effects of different P-450 inhibitors on estradiol-2/4-hydroxylase activity in brain and liver microsomes were also studied. In both organs these enzyme activities were found to be located mainly in the microsomal fraction and were inhibited by the 3 P-450 inhibitors tested. The hepatic estradiol-2/4-hydroxylase activity in adult male rats was significantly higher than that of females, but the enzyme activity in the brain did not exhibit a similar sex difference. Furthermore, estradiol-2/4-hydroxylase activity in rat liver was strongly induced by phenobarbitone treatment, but not in the brain. The phenobarbitone-induced activity in male and female rats exhibited significant kinetic differences. In female rats sexual maturation was associated with significant changes in the apparent Km of estradiol-2/4-hydroxylases in the liver and hypothalamus. Evidence was found that the in vitro estradiol-2/4-hydroxylase activity in rat brain and liver is due to more than one form of microsomal P-450. Kinetic studies showed important differences between the estradiol-2/4-hydroxylase activities in the hippocampus and hypothalamus. Significant differences in estradiol-2/4-hydroxylase activities were observed in the 6 brain areas studied, with the hippocampus showing the highest, and the hypothalamus the lowest activity at all developmental stages in both male and female rats

  8. CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

    Science.gov (United States)

    Teerawatanasuk, N; Skalnik, D G; Carr, L G

    1999-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene.

  9. [Properties of unrelated salicylate hydroxylases in bacteria of the genus pseudomon].

    Science.gov (United States)

    Puntus, T F; Vlasova, E P; Sokolov, A P; Zaharchenko, N S; Funtikova, T V

    2015-01-01

    The unrelated salicylate hydroxylases NahG and NahU of the strains Pseudomonasfluorescens 142 NF and P. Putida BS3701 were extracted and purified by ion-exchange and hydrophobic and gel permeation chromatography. The extracted enzymes differed in kinetic and catalyst performance during salicylate hydrolysis. For NahU salicylate hydroxylase, Km and Vmax were found to be higher (3.1 +/- 0.6 microM and 7.7 +/- 0.4 microM/min, respectively) than for NahG salicylate hydroxylase (1.3 +/- 0.1 microM and 4.7 +/- 0.1 microM/min, respectively). The activity of both enzymes toward substituted salicylates was higher in cases where the substituent groups were in para position than in cases with those in meta position. The activity toward substituted salicylates with substituent groups in meta position was different. The activity of salicylate hydroxylase NahG was higher toward salicylates with substituent groups in position 3; salicylate hydroxylase NahU activity was higher toward those with substituent groups in position 5. This suggests a difference in the spatial configuration of active sites in extracted unrelated salicylate hydroxylases.

  10. Characterization of Phenyalanine Hydroxylase Gene Mutations in Chilean PKU Patients.

    Science.gov (United States)

    Hamilton, V; Santa María, L; Fuenzalida, K; Morales, P; Desviat, L R; Ugarte, M; Pérez, B; Cabello, J F; Cornejo, V

    2017-12-30

    Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive disease, caused by mutations in the Phenylalanine Hydroxylase (PAH) gene situated in chromosome 12q22-q24.2. This gene has 13 exons. To date, 991 mutations have been described. The genotype is one of the main factors that determine the phenotype of this disease. Characterize PKU genotype and phenotype seen in Chilean PKU patients. We studied the PAH gene by restriction fragment length polymorphism (RFLP) and/or sequencing techniques to identify pathogenic mutations in 71 PKU subjects. We classified the phenotype according to Guldberg predicted value. We identified 26 different mutations in 134 of the 142 alleles studied (94.4%), 88.7% of the subjects had biallelic pathogenic mutations while 11.3% had only one pathogenic mutation identified. Compound heterozygous represented 85.9% of the cases. Exon 7 included the majority of mutations (26.9%) and 50% of mutations were missense. The most frequent mutations were c.1066-11G > A, c.442-?_509+?del and p.Val388Met. The majority of subjects (52.3%) had the classic phenotype. The most frequent mutations in our Chilean PKU population were p.Val388Met, c.442?_509+?del and c.1066-11G > A. It is possible to predict phenotype by detecting the genotype, and use this information to determine disease prognosis and adjust patient's medical and nutritional management accordingly.

  11. Updates on the biology of serotonin and tryptophan hydroxylase.

    Science.gov (United States)

    Swami, Tara; Weber, H Christian

    2018-02-01

    To summarize the most recent findings relevant to the biology of serotonin (5-hydroxytryptamine; 5-HT) and the enzyme tryptophan hydroxylase (TPH) in human gastrointestinal disease. Serotonin is synthesized in the central nervous system (CNS) and the gastrointestinal tract where it is secreted from enteroendocrine cells. Its biosynthesis is regulated by two isoforms of the enzyme TPH of which TPH1 is localized predominantly in gastrointestinal enteroendocrine cells. Serotonin activates the peristaltic reflexes, regulates gastrointestinal motility, and has a role in intestinal inflammation. Inhibition of TPH with novel molecules represents a new pharmacological tool in the successful management of carcinoid syndrome in patients with gastrointestinal neuroendocrine tumors (GI-NETs). Certain 5-HT receptor subtype agonists and antagonists are useful in the treatment of functional gastrointestinal disorders. The gastrointestinal tract is the largest storage organ for serotonin where its biosynthesis is regulated by TPH1. It has several important functions in gastrointestinal motility, secretion, and inflammation. Furthermore, TPH represents a target for inhibitory pharmacological therapy of serotonin access states such as the carcinoid syndrome.

  12. Characterization of mutations at the mouse phenylalanine hydroxylase locus

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, J.D.; Charlton, C.K. [Wichita State Univ., KS (United States)

    1997-02-01

    Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAH{sup ENU1}, the phenotype is mild. The Pah{sup enu1} mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAH{sup ENU2} the phenotype is severe. The Pah{sup enu2} mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAH{sup ENU2}, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site. 26 refs., 2 figs., 1 tab.

  13. Dopamine-beta hydroxylase polymorphism and cocaine addiction

    Directory of Open Access Journals (Sweden)

    Collier David

    2008-01-01

    Full Text Available Abstract Cocaine addiction involves a number of medical, psychological and social problems. Understanding the genetic aetiology of this disorder will be essential for design of effective treatments. Dopamine-beta hydroxylase (DbH catalyzes the conversion of dopamine to norepinephrine and could, therefore, have an influence on both cocaine action and the basal sensitivity of neurotransmitter systems to cocaine. Recently, the -1021C>T polymorphism have been found to strongly correlated with individual variation in plasma DbH activity. To test the influence of this polymorphism on the susceptibility of cocaine addiction, we decided to genotype it in a sample of 689 cocaine addicts and 832 healthy individuals. Genotypic and allelic analyses did not show any evidence of association with cocaine addiction, even after correcting for the effect of population stratification and other possible confounders. Our results do not support a major role of the -1021C>T polymorphism or the gene itself in the development of cocaine addiction but further examination of other variants within this gene will be necessary to completely rule out an effect.

  14. Tryptophan Hydroxylase 2 Gene and Alcohol Use among College Students

    Science.gov (United States)

    Gacek, Paul; Conner, Tamlin S.; Tennen, Howard; Kranzler, Henry R.; Covault, Jonathan

    2009-01-01

    Objective Genes that regulate serotonin activity are regarded as promising predictors of heavy alcohol use. Tryptophan Hydroxylase (TPH2) plays an important role in serotonergic neurotransmission by serving as the rate-limiting enzyme for serotonin biosynthesis in the midbrain and serotonergic neurons. Despite the link between TPH2 and serotonergic function, TPH2’s role in the pathogenesis of alcohol use disorders remains unclear. The goal of this study was to examine whether variation in the TPH2 gene is associated with risky alcohol consumption. Specifically, this study examined whether the TPH2 G-703T polymorphism predicted alcohol consumption among college students. Methods In two successive years, 351 undergraduates were asked to record their alcohol use each day for 30 days using an internet-based electronic diary. Participants’ DNA was collected and polymerase chain reaction genotyping was performed. Results Alcohol consumption was not associated with the TPH2 G-703T polymorphism alone, or the interaction of TPH2 with two other candidate polymorphisms (TPH1 C218A, and the SLC6A4 tri-allelic 5-HTTLPR) or negative life events. Conclusions This study supports recent null findings relating TPH2 to drinking outcomes. It also extends these findings by showing null interactions with the TPH1 C218A polymorphism, the SLC6A4 tri-allelic 5-HTTLPR polymorphism, and environmental stressors in predicting sub-clinical alcohol use among Caucasian American young adults. PMID:18782386

  15. Identification of a variant form of tyrosine phosphatase LYP

    Directory of Open Access Journals (Sweden)

    Ho Wanting T

    2010-11-01

    Full Text Available Abstract Background Protein tyrosine phosphatases (PTPs are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22 is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease. Results In this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals. Conclusions We thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.

  16. Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

    Science.gov (United States)

    Si, Yuan; Ji, Xinyan; Cao, Xiaolei; Dai, Xiaoming; Xu, Lingyi; Zhao, Hongxia; Guo, Xiaocan; Yan, Huan; Zhang, Haitao; Zhu, Chu; Zhou, Qi; Tang, Mei; Xia, Zongping; Li, Li; Cong, Yu-Sheng; Ye, Sheng; Liang, Tingbo; Feng, Xin-Hua; Zhao, Bin

    2017-09-15

    The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. A novel mutation in the lysyl hydroxylase 1 gene causes decreased lysyl hydroxylase activity in an ehlers-danlos VIA patient

    NARCIS (Netherlands)

    Walker, L.C.; Overstreet, M.A.; Siddiqui, A.; Paepe, A. de; Ceylaner, G.; Malfait, F.; Symoens, S.; Atsawasuwan, P.; Yamauchi, M.; Ceylaner, S.; Bank, R.A.; Yeowell, H.N.

    2005-01-01

    The clinical diagnosis of a patient with the phenotype of Ehlers-Danlos syndrome type VI was confirmed biochemically by the severely diminished level of lysyl hydroxylase (LH) activity in the patient's skin fibroblasts. A novel homozygous mutation, a single base change of T1360 → G in exon 13 of the

  18. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  19. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. (Brandeis Univ., Waltham, MA (USA))

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  20. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    Science.gov (United States)

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  1. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    ... examined. This study evaluated the effect of tyrosine supplementation on cognitive, psychomotor, and physical performance following a cold water immersion protocol that lowered body core temperature...

  2. SH3 domain tyrosine phosphorylation--sites, role and evolution.

    Directory of Open Access Journals (Sweden)

    Zuzana Tatárová

    Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

  3. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

    Science.gov (United States)

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

    1996-09-01

    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  4. Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria

    NARCIS (Netherlands)

    vanSpronsen, FJ; vanDijk, T; Smit, GPA; vanRijn, M; Reijngoud, DJ; Berger, Ruud; Heymans, HSA

    1996-01-01

    In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were

  5. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  6. Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

    Science.gov (United States)

    Beaudoin, Guillaume A W; Facchini, Peter J

    2013-02-15

    Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively. Copyright © 2013. Published by Elsevier Inc.

  7. Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase.

    Science.gov (United States)

    Tidemand, Kasper D; Peters, Günther H; Harris, Pernille; Stensgaard, Eva; Christensen, Hans E M

    2017-11-21

    Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH 4 ) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, K i , and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH 4 , binding pocket, which is induced by Trp binding.

  8. Molecular characterization and functional expression of flavonol 6-hydroxylase

    Directory of Open Access Journals (Sweden)

    Ibrahim Ragaï K

    2004-12-01

    Full Text Available Abstract Background Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD, rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis. Results A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H, was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome. Conclusions Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.

  9. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-06-09

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

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

    Science.gov (United States)

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

    2015-01-01

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

  12. CYP94A1, a plant cytochrome P450-catalyzing fatty acid omega-hydroxylase, is selectively induced by chemical stress in Vicia sativa seedlings.

    Science.gov (United States)

    Benveniste, Irène; Bronner, Roberte; Wang, Yong; Compagnon, Vincent; Michler, Pierre; Schreiber, Lukas; Salaün, Jean-Pierre; Durst, Francis; Pinot, Franck

    2005-08-01

    CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) omega-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 microM range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.

  13. Deuterium Labelling of L-Tyrosine with Raney Alloys in Alkaline Deuterium Oxide Solutions

    OpenAIRE

    Tsuzuki, Hirohisa; Mukumoto, Mamoru; Udagawa, Jun; Mataka, Shuntaro; Tashiro, Masashi

    1997-01-01

    The synthesis of deuteriated L-tyrosines with Raney alloys in alkaline deuterium oxide solutions, involving reductive debromination of brominated L-tyrosines and hydrogen-deuterium (H-D) exchange of L-tyrosines, without causing racemization, is presented.

  14. Determination of o-tyrosine in irradiated chicken

    International Nuclear Information System (INIS)

    Zoller, O.; Schoeni, D.; Zimmerli, B.

    1991-01-01

    The author explains his method to determine O-Tyrosine in irradiated chickens with a high-performance liquid chromatography. The method is simple and fast, but a proper chromatographic separation is difficult. The detection limit with a high sensitive detector is about 0.05-0.1 mg O-Tyrosine/kg meat (9 refs)

  15. Tyrosine improves working memory in a multitasking environment.

    Science.gov (United States)

    Thomas, J R; Lockwood, P A; Singh, A; Deuster, P A

    1999-11-01

    Previous studies indicate that tyrosine may prove useful in promoting improved performance in situations in which performance is compromised by stress. To extend the generality of previous tyrosine findings, the present study examined the effects of tyrosine ingestion on performance during both a Multiple Task and a Simple Task battery. The multiple task battery was designed to measure working memory, arithmetic skills, and visual and auditory monitoring simultaneously, whereas the simple task battery measured only working memory and visual monitoring. Ten men and 10 women subjects underwent these batteries 1 h after ingesting 150 mg/kg of l-tyrosine or placebo. Administration of tyrosine significantly enhanced accuracy and decreased frequency of list retrieval on the working memory task during the multiple task battery compared with placebo. However, tyrosine induced no significant changes in performance on the arithmetic, visual, or auditory tasks during the Multiple Task, or modified any performance measures during the Simple Task battery. Blood levels of ACTH and cortisol were not, but heart rate and blood pressure were significantly increased during the performance tasks. The present results indicate that tyrosine may sustain working memory when competing requirements to perform other tasks simultaneously degrade performance, and that supplemental tyrosine may be appropriate for maintaining performance when mild to severe decrements are anticipated.

  16. [scpA the new salicylate hydroxylase gene localized on salicylate/caprolactam degradation plasmids].

    Science.gov (United States)

    Panov, A V; Volkova, O V; Puntus, I F; Esikova, T Z; Kosheleva, I A; Boronin, A M

    2013-01-01

    Both caprolactam and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders is controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids determined to be the members of IncP-7 group. The new salicylate 1-hydroxylase gene (scpA) on SAL/CAP-plasmids has been detected and partially sequenced. Gene scpA was equally related to closest homologs nahG (NAH7), salA (P. reinekei MT1) and nahU (pND6-1), but identity of scpA to these genes did not exceed 72-74%. Synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited highest specific activity with 4-methylsalicylate and nonsubstituted salicylate. Besides pseudomonad's salicylate degradative conjugative plasmids without "classical" nah2-operon and harboring only salicylate 1-hydroxylase gene nahU have been firstly described.

  17. Measurement of optical purity of p-BPA-Tyrosine dipeptide

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, K.; Sato, N.; Kitta, K.; Saitake, Y. [Shinshu Univ., Faculty of Science, Matsumoto, Nagano (Japan); Hiratsuka, J. [Kawasaki Medical School, Dept. of Radiation Oncology, Kurashiki, Okayama (Japan); Ichihashi, M. [Kobe Univ. (Japan). School of Medicine

    2000-10-01

    Melanin biosynthesis is very active in melanoma cells, and tyrosine is one of the substrates of the melanin biosynthesis. Tyrosine is oxidized to dopa by tyrosinase at the beginning of melanin biosynthesis process. Therefore, p-boronophenylalanine (BPA)-tyrosine dipeptide is expected to be a substrate of melanin biosynthesis process, and the peptide will be incorporated in melanoma cells, and then tumor boron concentration lasts in their cells for long time. Since p-BPA tyrosine are amino acids, they have D, L isomers. Therefore, we have tried to synthesize four isomers (L-L, L-D, D-L, D-D) of p-BPA-Tyrosine dipeptide, and have measured their optical purity with HPLC. (author)

  18. The Sulfinator: predicting tyrosine sulfation sites in protein sequences.

    Science.gov (United States)

    Monigatti, Flavio; Gasteiger, Elisabeth; Bairoch, Amos; Jung, Eva

    2002-05-01

    Protein tyrosine sulfation is an important post-translational modification of proteins that go through the secretory pathway. No clear-cut acceptor motif can be defined that allows the prediction of tyrosine sulfation sites in polypeptide chains. The Sulfinator is a software tool that can be used to predict tyrosine sulfation sites in protein sequences with an overall accuracy of 98%. Four different Hidden Markov Models were constructed, each of them specialized to recognize sulfated tyrosine residues depending on their location within the sequence: near the N-terminus, near the C-terminus, in the center of a window with a size of at least 25 amino acids, as well as in windows containing several tyrosine residues. The Sulfinator is accessible at (http://www.expasy.org/tools/sulfinator/). Sulfinator documentation is accessible at (http://www.expasy.org/tools/sulfinator/sulfinator-doc.html).

  19. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

    Directory of Open Access Journals (Sweden)

    Mun Chiang Chan

    Full Text Available As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs. Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs. Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4 induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

  20. Protein Tyrosine Nitration: Selectivity, physicochemical and biological consequences, denitration and proteomics methods for the identification of tyrosine-nitrated proteins

    NARCIS (Netherlands)

    Abello, N.; Kerstjens, H.A.M.; Postma, D.S; Bischoff, Rainer

    2009-01-01

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate

  1. Protein Tyrosine Nitration : Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins

    NARCIS (Netherlands)

    Abello, Nicolas; Kerstjens, Huib A. M.; Postma, Dirkje S.; Bischoff, Rainer

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO(2)). In the present article, we review the main nitration reactions and

  2. Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing.

    Science.gov (United States)

    Duscher, Dominik; Januszyk, Michael; Maan, Zeshaan N; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Dong, Yixiao; Khong, Sacha M; Longaker, Michael T; Gurtner, Geoffrey C

    2017-03-01

    A hallmark of diabetes mellitus is the breakdown of almost every reparative process in the human body, leading to critical impairments of wound healing. Stabilization and activity of the transcription factor hypoxia-inducible factor (HIF)-1α is impaired in diabetes, leading to deficits in new blood vessel formation in response to injury. In this article, the authors compare the effectiveness of two promising small-molecule therapeutics, the hydroxylase inhibitor dimethyloxalylglycine and the iron chelator deferoxamine, for attenuating diabetes-associated deficits in cutaneous wound healing by enhancing HIF-1α activation. HIF-1α stabilization, phosphorylation, and transactivation were measured in murine fibroblasts cultured under normoxic or hypoxic and low-glucose or high-glucose conditions following treatment with deferoxamine or dimethyloxalylglycine. In addition, diabetic wound healing and neovascularization were evaluated in db/db mice treated with topical solutions of either deferoxamine or dimethyloxalylglycine, and the efficacy of these molecules was also compared in aged mice. The authors show that deferoxamine stabilizes HIF-1α expression and improves HIF-1α transactivity in hypoxic and hyperglycemic states in vitro, whereas the effects of dimethyloxalylglycine are significantly blunted under hyperglycemic hypoxic conditions. In vivo, both dimethyloxalylglycine and deferoxamine enhance wound healing and vascularity in aged mice, but only deferoxamine universally augmented wound healing and neovascularization in the setting of both advanced age and diabetes. This first direct comparison of deferoxamine and dimethyloxalylglycine in the treatment of impaired wound healing suggests significant therapeutic potential for topical deferoxamine treatment in ischemic and diabetic disease.

  3. The Hypoxia-Inducible Factor Pathway, Prolyl Hydroxylase Domain Protein Inhibitors, and Their Roles in Bone Repair and Regeneration

    Directory of Open Access Journals (Sweden)

    Lihong Fan

    2014-01-01

    Full Text Available Hypoxia-inducible factors (HIFs are oxygen-dependent transcriptional activators that play crucial roles in angiogenesis, erythropoiesis, energy metabolism, and cell fate decisions. The group of enzymes that can catalyse the hydroxylation reaction of HIF-1 is prolyl hydroxylase domain proteins (PHDs. PHD inhibitors (PHIs activate the HIF pathway by preventing degradation of HIF-α via inhibiting PHDs. Osteogenesis and angiogenesis are tightly coupled during bone repair and regeneration. Numerous studies suggest that HIFs and their target gene, vascular endothelial growth factor (VEGF, are critical regulators of angiogenic-osteogenic coupling. In this brief perspective, we review current studies about the HIF pathway and its role in bone repair and regeneration, as well as the cellular and molecular mechanisms involved. Additionally, we briefly discuss the therapeutic manipulation of HIFs and VEGF in bone repair and bone tumours. This review will expand our knowledge of biology of HIFs, PHDs, PHD inhibitors, and bone regeneration, and it may also aid the design of novel therapies for accelerating bone repair and regeneration or inhibiting bone tumours.

  4. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

    Science.gov (United States)

    2013-01-01

    Background Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Results Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Conclusions Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of

  5. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  6. Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy.

    Science.gov (United States)

    Cackowski, Frank C; Eber, Matthew R; Rhee, James; Decker, Ann M; Yumoto, Kenji; Berry, Janice E; Lee, Eunsohl; Shiozawa, Yusuke; Jung, Younghun; Aguirre-Ghiso, Julio A; Taichman, Russell S

    2017-04-01

    Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling inDrosophilaaxon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

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

    Science.gov (United States)

    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

  9. Wood chemistry analysis and expression profiling of a poplar clone expressing a tyrosine-rich peptide.

    Science.gov (United States)

    Xu, Yi; Chen, Chin-Fu; Thomas, Tina P; Azadi, Parastoo; Diehl, Brett; Tsai, Chung-Jui; Brown, Nicole; Carlson, John E; Tien, Ming; Liang, Haiying

    2013-12-01

    Our study has identified pathways and gene candidates that may be associated with the greater flexibility and digestibility of the poplar cell walls. With the goal of facilitating lignin removal during the utilization of woody biomass as a biofuel feedstock, we previously transformed a hybrid poplar clone with a partial cDNA sequence encoding a tyrosine- and hydroxyproline-rich glycoprotein from parsley. A number of the transgenic lines released more polysaccharides following protease digestion and were more flexible than wild-type plants, but otherwise normal in phenotype. Here, we report that overexpression of the tyrosine-rich peptide encoding sequence in these transgenic poplar plants did not significantly alter total lignin quantity or quality (S/G lignin ratio), five- and six-carbon sugar contents, growth rate, or susceptibility to a major poplar fungal pathogen, Septoria musiva. Whole-genome microarray analysis revealed a total of 411 differentially expressed transcripts in transgenic lines, all with decreased transcript abundance relative to wild-type plants. Their corresponding genes were overrepresented in functional categories such as secondary metabolism, amino acid metabolism, and energy metabolism. Transcript abundance was decreased primarily for five types of genes encoding proteins involved in cell-wall organization and in lignin biosynthesis. The expression of a subset of 19 of the differentially regulated genes by qRT-PCR validated the microarray results. Our study has identified pathways and gene candidates that may be the underlying cause for the enhanced flexibility and digestibility of the stems of poplar plants expressing the TYR transgene.

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

    Science.gov (United States)

    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 ...

  11. Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase.

    Science.gov (United States)

    Edgar, Steven; Zhou, Kang; Qiao, Kangjian; King, Jason R; Simpson, Jeffrey H; Stephanopoulos, Gregory

    2016-02-19

    The anticancer molecule taxol (Paclitaxel) stands as one of the most medically and economically important natural products. However, despite decades of extensive study, its biosynthesis remains poorly understood. Unpredictable behavior of the first oxygenation enzyme, taxadiene-5α-hydroxylase, which produces a range of undesired products, currently stands as a key bottleneck to improved taxol production. We herein present chemical and biological evidence of an unreported epoxidase activity of taxadiene-5α-hydroxylase that puts into question the previously proposed radical-rebound mechanism. We demonstrate that the poor selectivity of taxadiene-5α-hydroxylase arises from nonselective degradation of an epoxide intermediate produced via a selective oxidation step, rather than from promiscuous oxidation, as previously proposed. We support these conclusions by demonstrating variable enzyme behavior in differing hosts and conditions, similarity of products and product ratios generated from chemical epoxidation, and taxadiene-5α-hydroxylase, and differing enzymatic activity on alternative taxadiene isomers. Additionally, we use directed mutagenesis to describe the oxidizing species of the P450, demonstrate that further in vivo functionalization of oxidized taxadiene is unable to improve selectivity of the oxidation, and show that multiple products are produced in the Taxus cuspidata and are not simply an artifact of heterologous expression. Our results highlight an important, and previously unknown, obstacle to improved taxol production. We further offer insights to overcome the challenges posed by an epoxide-mediated reaction, which sets the basis for further engineering of taxol biosynthesis.

  12. Mechanism-based inactivation of benzo[a]pyrene hydroxylase by aryl acetylenes and aryl olefins

    International Nuclear Information System (INIS)

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

    1986-01-01

    A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo[a]pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo[a]pyrene hydroxylase. The mechanism-based loss of benzo[a]pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenes therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, 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

  13. 3-Ketosteroid 9 alpha-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Hu, Yanmin; van der Geize, Robert; Besra, Gurdyal S.; Gurcha, Sudagar S.; Liu, Alexander; Rohde, Manfred; Singh, Mahavir; Coates, Anthony

    Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3-ketosteroid 9a-hydroxylase (KSH). Consistent with their predicted roles, the Delta kshA and Delta kshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione

  14. Longitudinal analysis of growth and puberty in 21-hydroxylase deficiency patients

    NARCIS (Netherlands)

    van der Kamp, H. J.; Otten, B. J.; Buitenweg, N.; de Muinck Keizer-Schrama, S. M. P. F.; Oostdijk, W.; Jansen, M.; Delemarre-de Waal, H. A.; Vulsma, T.; Wit, J. M.

    2002-01-01

    To evaluate growth from diagnosis until final height (FH) in 21-hydroxylase deficiency patients. A retrospective longitudinal study was performed. Only patients treated with hydrocortisone and fludrocortisone (in case of salt wasting) were evaluated. This resulted in a sample of 34 (21 male, 13

  15. Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization

    DEFF Research Database (Denmark)

    Tidemand, Kasper Damgaard; Christensen, Hans Erik Mølager; Hoeck, Niclas

    2016-01-01

    Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full length TPH2 is poorly characterized due to low purification...

  16. Functional expression of the ectoine hydroxylase gene (thpD) from Streptomyces chrysomallus in Halomonas elongata.

    Science.gov (United States)

    Prabhu, Julia; Schauwecker, Florian; Grammel, Nicolas; Keller, Ullrich; Bernhard, Michael

    2004-05-01

    The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

  17. Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

    OpenAIRE

    Prabhu, Julia; Schauwecker, Florian; Grammel, Nicolas; Keller, Ullrich; Bernhard, Michael

    2004-01-01

    The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

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

    International Nuclear Information System (INIS)

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

    1981-01-01

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

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

    Science.gov (United States)

    ... of the kidneys and produce a variety of hormones that regulate many essential functions in the body. In people with CAH due to 11-beta-hydroxylase deficiency, the adrenal glands produce excess androgens, which are male sex hormones. There are two types of CAH ...

  20. Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1

    NARCIS (Netherlands)

    Sucharitakul, J.; Wongnate, T.; Montersino, S.; Berkel, van W.J.H.; Chaiyen, P.

    2012-01-01

    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate

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

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen; Harris, Pernille; Zhao, Y.

    2016-01-01

    , Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600...... into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system....

  2. Novel vitamin D 1α-hydroxylase gene mutations in a Chinese ...

    Indian Academy of Sciences (India)

    hydroxylase gene mutations in a Chinese vitamin-D-dependent rickets type I patient. Lihua Cao Fang Liu Yu Wang Jian Ma Shusen Wang Libo Wang Yang Zhang Chen Chen Yang Luo Hongwei Ma. Research Note Volume 90 Issue 2 August ...

  3. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2011-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  4. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2012-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  5. Recent advances in biochemical and molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Jin-Ho Choi

    2016-03-01

    Full Text Available The term congenital adrenal hyperplasia (CAH covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency.

  6. Novel vitamin D 1α-hydroxylase gene mutations in a Chinese ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... 25-Hydroxyvitamin D 1α- hydroxylase is a typical mitochondrial (type I) cytochrome. P450 enzyme that functions as an oxidase, using electrons from reduced nicotinamide adenine dinucleotide phosphate and molecular oxygen. To date, 39 different mutations in CYP27B1 gene asso- ciated with VDDR-I ...

  7. Carnitine biosynthesis. Purification of gamma-butyrobetaine hydroxylase from rat liver

    NARCIS (Netherlands)

    Vaz, F. M.; van Gool, S.; Ofman, R.; IJlst, L.; Wanders, R. J.

    1999-01-01

    gamma-Butyrobetaine hydroxylase catalyse the last step in carnitine biosynthesis, the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on Fe2+, alpha-ketoglutarate, ascorbate and oxygen. Initial attempts to purify the protein from rat liver showed that gamma-butyrobetaine

  8. Crosstalk between G protein-coupled receptors (GPCRs and tyrosine kinase receptor (TXR in the heart after morphine withdrawal

    Directory of Open Access Journals (Sweden)

    Pilar eAlmela

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signalling, resulting in high expression of protein kinase (PK A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK, one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl-5-isoquinolinesulfonamide (HA-1004, a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

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

    Science.gov (United States)

    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.

  10. 1α-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines

    Science.gov (United States)

    Lagishetty, Venu; Chun, Rene F.; Liu, Nancy Q.; Lisse, Thomas S.; Adams, John S.; Hewison, Martin

    2010-01-01

    Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). In cells such as macrophages, expression of 1α-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and β-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1α-hydroxylase. Alternative activators of 1α-hydroxylase such as transforming growth factor β were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. PMID:20152900

  11. 1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines.

    Science.gov (United States)

    Lagishetty, Venu; Chun, Rene F; Liu, Nancy Q; Lisse, Thomas S; Adams, John S; Hewison, Martin

    2010-07-01

    Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  12. Activity of 25-hydroxylase in human gingival fibroblasts and periodontal ligament cells.

    Science.gov (United States)

    Liu, Kaining; Meng, Huanxin; Hou, Jianxia

    2012-01-01

    We previously demonstrated that 25-hydroxyvitamin D(3) concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D(3) can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells. Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D(3) that resulted in the production of 1α,25-dihydroxyvitamin D(3). Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D(3) and 1α,25-dihydroxyvitamin D(3) production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D(3) synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells. The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

  13. Activity of 25-hydroxylase in human gingival fibroblasts and periodontal ligament cells.

    Directory of Open Access Journals (Sweden)

    Kaining Liu

    Full Text Available We previously demonstrated that 25-hydroxyvitamin D(3 concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D(3 can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D(3, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D(3 that resulted in the production of 1α,25-dihydroxyvitamin D(3. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D(3 and 1α,25-dihydroxyvitamin D(3 production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D(3 synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.; Croteau, R. (Washington State Univ., Pullman (United States))

    1993-04-01

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

  15. Direct observation of spin-injection in tyrosinate-functionalized single-wall carbon nanotubes

    NARCIS (Netherlands)

    Tsoufis, Theodoros; Ampoumogli, Asem; Gournis, Dimitrios; Georgakilas, Vasilios; Jankovic, Lubos; Christoforidis, Konstantinos C.; Deligiannakis, Yiannis; Mavrandonakis, Andreas; Froudakis, George E.; Maccallini, Enrico; Rudolf, Petra; Mateo-Alonso, Aurelio; Prato, Maurizio

    In this work, we report on the interaction of a tyrosinate radical with single wall carbon nanotubes (CNT). The tyrosinate radical was formed from tyrosine (ester) by Fenton's reagent and, reacted in situ with carbon nanotubes resulting in novel tyrosinated carbon nanotube derivatives. The covalent

  16. Tyrosine phosphorylation of Rab7 by Src kinase.

    Science.gov (United States)

    Lin, Xiaosi; Zhang, Jiaming; Chen, Lingqiu; Chen, Yongjun; Xu, Xiaohui; Hong, Wanjin; Wang, Tuanlao

    2017-07-01

    The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling. Copyright © 2017. Published by Elsevier Inc.

  17. Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

    Czech Academy of Sciences Publication Activity Database

    Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

    2013-01-01

    Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

  18. Tyrosine biosynthesis, metabolism, and catabolism in plants.

    Science.gov (United States)

    Schenck, Craig A; Maeda, Hiroshi A

    2018-05-01

    L-Tyrosine (Tyr) is an aromatic amino acid (AAA) required for protein synthesis in all organisms, but synthesized de novo only in plants and microorganisms. In plants, Tyr also serves as a precursor of numerous specialized metabolites that have diverse physiological roles as electron carriers, antioxidants, attractants, and defense compounds. Some of these Tyr-derived plant natural products are also used in human medicine and nutrition (e.g. morphine and vitamin E). While the Tyr biosynthesis and catabolic pathways have been extensively studied in microbes and animals, respectively, those of plants have received much less attention until recently. Accumulating evidence suggest that the Tyr biosynthetic pathways differ between microbes and plants and even within the plant kingdom, likely to support the production of lineage-specific plant specialized metabolites derived from Tyr. The interspecies variations of plant Tyr pathway enzymes can now be used to enhance the production of Tyr and Tyr-derived compounds in plants and other synthetic biology platforms. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Manzamenones Inhibit T-Cell Protein Tyrosine Phosphatase

    Directory of Open Access Journals (Sweden)

    Jun'ichi Kobayashi

    2006-02-01

    Full Text Available Abstract: Manzamenones A~C (1~3 and E~F (5~6, unique oxylipin metabolites isolated from a marine sponge Plakortis sp., have been found to exhibit inhibitory activity against Tcell protein tyrosine phosphatase (TCPTP. The inhibitory activity of 2 and 5 against TCPTP was 4 times more potent than that against protein tyrosine phosphatase-1B (PTP1B.

  20. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

    DEFF Research Database (Denmark)

    Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

    2004-01-01

    or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

  1. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    Science.gov (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  2. Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): A quantitative immunohistochemical study

    DEFF Research Database (Denmark)

    Møller, Morten; Masson-Pévet, M.; Pévet, P.

    1998-01-01

    Anatomy, neurobiology, sympathetic innervation, tyrosine hydroxylase, colocalization, testosterone, annual variation, Cricetus cricetus......Anatomy, neurobiology, sympathetic innervation, tyrosine hydroxylase, colocalization, testosterone, annual variation, Cricetus cricetus...

  3. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    International Nuclear Information System (INIS)

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-01-01

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G 1 phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  4. Implication of a protein-tyrosine-phosphatase in human lung cancer.

    Science.gov (United States)

    Gaits, F; Li, R Y; Ragab, A; Selves, J; Ragab-Thomas, J M; Chap, H

    1994-07-01

    Protein tyrosyl phosphorylation plays an essential role in regulating cellular events such as proliferation, differentiation and oncogenesis. The recent characterization of the family of protein tyrosine phosphatases (PTPases) suggests that dephosphorylation might be a crucial event in these phenomena. One of the functions of PTPases is to reverse the effect of protein tyrosine kinases (PTKases), many of which are oncogenes, suggesting that they may act as tumor suppressors as described for HPTP gamma. In order to investigate the implication in lung cancer of HPTP beta, a receptor PTPase, we have developed a semi-quantitative method derived from primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labelled nucleotide. We have demonstrated that the expression of HPTP beta mRNA was dramatically decreased in lung adenocarcinomas and lung malpighian carcinomas as compared to normal lung tissue. In addition, HPTP beta was not expressed in the pulmonar adenocarcinoma cell line A427, which proliferates in a deregulated way. These results suggest that the loss of expression of HPTP beta might play a role in neoplasic transformation and thus this molecule could act as a tumor suppressor factor.

  5. Solution structure of the receptor tyrosine kinase EphB2 SAM domain and identification of two distinct homotypic interaction sites.

    OpenAIRE

    Smalla, M.; Schmieder, P.; Kelly, M.; Ter Laak, A.; Krause, G.; Ball, L.; Wahl, M.; Bork, P.; Oschkinat, H.

    1999-01-01

    The sterile alpha motif (SAM) is a protein interaction domain of around 70 amino acids present predominantly in the N- and C-termini of more than 60 diverse proteins that participate in signal transduction and transcriptional repression. SAM domains have been shown to homo- and hetero-oligomerize and to mediate specific protein-protein interactions. A highly conserved subclass of SAM domains is present at the intracellular C-terminus of more than 40 Eph receptor tyrosine kinases that are invo...

  6. Salivary peptide tyrosine-tyrosine 3-36 modulates ingestive behavior without inducing taste aversion.

    Science.gov (United States)

    Hurtado, Maria D; Sergeyev, Valeriy G; Acosta, Andres; Spegele, Michael; La Sala, Michael; Waler, Nickolas J; Chiriboga-Hurtado, Juan; Currlin, Seth W; Herzog, Herbert; Dotson, Cedrick D; Gorbatyuk, Oleg S; Zolotukhin, Sergei

    2013-11-20

    Hormone peptide tyrosine-tyrosine (PYY) is secreted into circulation from the gut L-endocrine cells in response to food intake, thus inducing satiation during interaction with its preferred receptor, Y2R. Clinical applications of systemically administered PYY for the purpose of reducing body weight were compromised as a result of the common side effect of visceral sickness. We describe here a novel approach of elevating PYY in saliva in mice, which, although reliably inducing strong anorexic responses, does not cause aversive reactions. The augmentation of salivary PYY activated forebrain areas known to mediate feeding, hunger, and satiation while minimally affecting brainstem chemoreceptor zones triggering nausea. By comparing neuronal pathways activated by systemic versus salivary PYY, we identified a metabolic circuit associated with Y2R-positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers. The discovery of this alternative circuit that regulates ingestive behavior without inducing taste aversion may open the possibility of a therapeutic application of PYY for the treatment of obesity via direct oral application.

  7. Implications of tyrosine phosphoproteomics in cervical carcinogenesis

    Directory of Open Access Journals (Sweden)

    DeFord James

    2008-01-01

    Full Text Available Abstract Background Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma.

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

    Science.gov (United States)

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

    2015-08-01

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

  9. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

    Science.gov (United States)

    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.

  10. Structure of the ribosomal oxygenase OGFOD1 provides insights into the regio- and stereoselectivity of prolyl hydroxylases.

    Science.gov (United States)

    Horita, Shoichiro; Scotti, John S; Thinnes, Cyrille; Mottaghi-Taromsari, Yousef S; Thalhammer, Armin; Ge, Wei; Aik, WeiShen; Loenarz, Christoph; Schofield, Christopher J; McDonough, Michael A

    2015-04-07

    Post-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Li, Hsiu-Chuan; Huang, Chuan-Chuan; Chen, Sung-Fang; Chou, Min-Yuan

    2005-10-21

    We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.

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

    OpenAIRE

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

    2012-01-01

    The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using...

  13. Involvement of Arabidopsis Prolyl 4 Hydroxylases in Hypoxia, Anoxia and Mechanical Wounding

    OpenAIRE

    Vlad, Florina; Spano, Thodhoraq; Vlad, Daniela; Daher, Firas Bou; Ouelhadj, Akli; Fragkostefanakis, Sotirios; Kalaitzis, Panagiotis

    2007-01-01

    Arabidopsis prolyl 4 hydroxylases (P4Hs) catalyze an important post-translational modification in plants, though the only information on their patterns of expression is solely based on Arabidopsis microarray analysis data. In addition, the expression patterns of plants P4Hs in response to hypoxia, anoxia and other abiotic stresses such as mechanical wounding have never been studied extensively, despite their central role in hypoxic response of several other organisms through the regulation of...

  14. Human prolyl hydroxylase expression in uterine leiomyoma during the menstrual cycle

    OpenAIRE

    Iwahashi, Masaaki; Muragaki, Yasuteru; Ino, Kazuhiko

    2012-01-01

    Abstract Background To investigate the role of prolyl hydroxylase (PH), a key enzyme of collagen synthesis, in human uterine leiomyoma, PH expression was determined in the normal uterine myometrium and the leiomyoma tissues during the menstrual cycle. Methods The tissues were obtained from 40 regularly cycling women (aged 29 to 53 yr) who were undergoing abdominal hysterectomy for symptomatic uterine leiomyoma. Immunohistochemistry for human PH with specific monoclonal antibody was used for a...

  15. Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration

    Directory of Open Access Journals (Sweden)

    Silvina Bartesaghi

    2018-04-01

    Full Text Available In this review we provide an analysis of the biochemistry of peroxynitrite and tyrosine nitration. Peroxynitrite is the product of the diffusion-controlled reaction between superoxide (O2•- and nitric oxide (•NO. This process is in competition with the enzymatic dismutation of O2•- and the diffusion of •NO across cells and tissues and its reaction with molecular targets (e.g. guanylate cyclase. Understanding the kinetics and compartmentalization of the O2•- / •NO interplay is critical to rationalize the shift of •NO from a physiological mediator to a cytotoxic intermediate. Once formed, peroxynitrite (ONOO- and ONOOH; pKa = 6,8 behaves as a strong one and two-electron oxidant towards a series of biomolecules including transition metal centers and thiols. In addition, peroxynitrite anion can secondarily evolve to secondary radicals either via its fast reaction with CO2 or through proton-catalyzed homolysis. Thus, peroxynitrite can participate in direct (bimolecular and indirect (through secondary radical intermediates oxidation reactions; through these processes peroxynitrite can participate as cytotoxic effector molecule against invading pathogens and/or as an endogenous pathogenic mediator. Peroxynitrite can cause protein tyrosine nitration in vitro and in vivo. Indeed, tyrosine nitration is a hallmark of the reactions of •NO-derived oxidants in cells and tissues and serves as a biomarker of oxidative damage. Protein tyrosine nitration can mediate changes in protein structure and function that affect cell homeostasis. Tyrosine nitration in biological systems is a free radical process that can be promoted either by peroxynitrite-derived radicals or by other related •NO-dependent oxidative processes. Recently, mechanisms responsible of tyrosine nitration in hydrophobic biostructures such as membranes and lipoproteins have been assessed and involve the parallel occurrence and connection with lipid

  16. Studies on the mechanism of rapid activation of protein tyrosine phosphorylation activities, particularly c-Src kinase, by TCDD in MCF10A.

    Science.gov (United States)

    Mazina, Olga; Park, Sujin; Sano, Hiromi; Wong, Patrick; Matsumura, Fumio

    2004-01-01

    While the process of the Ah receptor activation leading to cytochrome P450 induction has been well studied, the mechanism and the process through which the Ah receptor activates tyrosine kinases, within a few minutes of its ligand binding, is not known. Previously, it was reported by Tannheimer et al. (Carcinogenesis 1998; 19:1291-1297) that TCDD causes rapid induction of tyrosine phosphorylation activities in the MCF10A human mammary epithelial cell line. To study the mechanistic aspect of this phenomenon, particularly that occurs within a few minutes after administration, we first studied the effect of insulin on MCF10A under serum free conditions with added EGF. The addition of insulin induced a rapid (5 min) tyrosine phosphorylation on several 160-190 kDa proteins which was followed by significant dephosphorylation activities on these proteins by 15 min. TCDD increased the rate of tyrosine phosphorylation on those proteins but at 15 min, the level of phosphorylation was still high. When insulin and TCDD were added together, the ability of insulin to induce de-phosphorylation by 15 min disappeared. Such an action of TCDD was accompanied by an increase in the titer of the activated form of Src kinase (i.e. c-Src protein with 418 tyrosine phosphorylation), and a concomitant decrease in the level of 529 tyrosine phosphorylated form (an inactivated form). The TCDD-induced activation of c-Src could be blocked by pretreated MCF10A cells with antisense oligonucleotides against c-src or with a specific inhibitor of Src kinase, PP-2. These results support the conclusion that c-Src kinase is at least one of the earliest and the most upstream components of toxic signaling of the Ah-receptor activated by TCDD through the post-transcriptional process.

  17. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

    Science.gov (United States)

    Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

    2017-09-01

    Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

  18. Identifying proteins that can form tyrosine-cysteine crosslinks.

    Science.gov (United States)

    Martinie, Ryan J; Godakumbura, Pahan I; Porter, Elizabeth G; Divakaran, Anand; Burkhart, Brandon J; Wertz, John T; Benson, David E

    2012-10-01

    Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

  19. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics

    Science.gov (United States)

    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

  20. Kinetic mechanism and isotope effects of Pseudomonas cepacia 3-hydroxybenzoate-t-hydroxylase

    International Nuclear Information System (INIS)

    Wang, L.H.; Yu, Y.; Hamzah, R.Y.; Tu, S.C.

    1986-01-01

    The kinetic mechanism of Pseudomonas cepacia 3-hydroxybenzoate-6-hydroxylase has been delineated. Double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a constant level of oxygen and several fixed concentrations of NADH yielded a set of converging lines. Similar reciprocal plots of velocity versus NADH concentration at a constant oxygen level and several fixed m-hydroxybenzoate concentrations also showed converging lines. In contrast, double reciprocal plots of initial rate versus NADH concentration at a fixed m-hydroxybenzoate level and several oxygen concentrations showed a series of parallel lines. Parallel lines were also obtained from double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a fixed NADH level and varying oxygen concentrations. These results suggest a sequential binding of m-hydroxybenzoate and NADH by the hydroxylase. The enzyme-bound FAD is reduced and NAD is released. The reduced enzyme subsequently reacts with oxygen leading to the formation of other products. This hydroxylase exhibited a primary isotope effect of /sup D/V = 3.5 for (4R)-[4- 2 H] NADH but no isotope effect was observed with (4S)-[4- 2 H]NADH. An isotope effect of /sup T/V/K = 5.0 was also observed using (4R)-[4- 3 H]NADH. This tritium isotope effect was apparently independent of m-hydroxybenzoate concentration

  1. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

    Science.gov (United States)

    Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

    2005-08-01

    OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

  2. Transcriptional regulation of c-fos

    International Nuclear Information System (INIS)

    Prywes, R.; Fisch, T.M.; Roeder, R.G.

    1988-01-01

    Expression of the c-fos proto-oncogene is induced rapidly and transiently by serum and other mitogenic agents. This rapid induction is therefore likely to involve posttranslational modifications and provides an excellent model for an early nuclear target of the signal transduction process, growth factors that bind to tyrosine kinase receptors. The authors have sought to understand the mechanism of transcriptional induction by each of these agents. The first step in this process was to identify the sequence elements in the c-fos gene responsible for induction by each of these agents. A specific element, termed serum response element (SRE), has been identified by transfection experiments of c-fos promoter constructs. To study regulation via SRE, a nuclear factor that binds to the SRE, termed serum response factor (SRF), has been identified with the gel mobility shift assay

  3. ER Stress Signaling Promotes the Survival of Cancer "Persister Cells" Tolerant to EGFR Tyrosine Kinase Inhibitors.

    Science.gov (United States)

    Terai, Hideki; Kitajima, Shunsuke; Potter, Danielle S; Matsui, Yusuke; Quiceno, Laura Gutierrez; Chen, Ting; Kim, Tae-Jung; Rusan, Maria; Thai, Tran C; Piccioni, Federica; Donovan, Katherine A; Kwiatkowski, Nicholas; Hinohara, Kunihiko; Wei, Guo; Gray, Nathanael S; Fischer, Eric S; Wong, Kwok-Kin; Shimamura, Teppei; Letai, Anthony; Hammerman, Peter S; Barbie, David A

    2018-02-15

    An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells that survive despite effective eradication of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy, a combination previously shown to suppress drug-tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP, and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response associated with STING upregulation, promoting protumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications. Significance: These findings reveal a novel function of the recently described ufmylation pathway, an ER stress survival signaling in drug-tolerant persister cells, which has important biological and therapeutic implications. Cancer Res; 78(4); 1044

  4. Expression of flavonoid 3’-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize

    Directory of Open Access Journals (Sweden)

    Sharma Mandeep

    2012-11-01

    Full Text Available Abstract Background The maize (Zea mays red aleurone1 (pr1 encodes a CYP450-dependent flavonoid 3’-hydroxylase (ZmF3’H1 required for the biosynthesis of purple and red anthocyanin pigments. We previously showed that Zmf3’h1 is regulated by C1 (Colorless1 and R1 (Red1 transcription factors. The current study demonstrates that, in addition to its role in anthocyanin biosynthesis, the Zmf3’h1 gene also participates in the biosynthesis of 3-deoxyflavonoids and phlobaphenes that accumulate in maize pericarps, cob glumes, and silks. Biosynthesis of 3-deoxyflavonoids is regulated by P1 (Pericarp color1 and is independent from the action of C1 and R1 transcription factors. Results In maize, apiforol and luteoforol are the precursors of condensed phlobaphenes. Maize lines with functional alleles of pr1 and p1 (Pr1;P1 accumulate luteoforol, while null pr1 lines with a functional or non-functional p1 allele (pr1;P1 or pr1;p1 accumulate apiforol. Apiforol lacks a hydroxyl group at the 3’-position of the flavylium B-ring, while luteoforol has this hydroxyl group. Our biochemical analysis of accumulated compounds in different pr1 genotypes showed that the pr1 encoded ZmF3’H1 has a role in the conversion of mono-hydroxylated to bi-hydroxylated compounds in the B-ring. Steady state RNA analyses demonstrated that Zmf3’h1 mRNA accumulation requires a functional p1 allele. Using a combination of EMSA and ChIP experiments, we established that the Zmf3’h1 gene is a direct target of P1. Highlighting the significance of the Zmf3’h1 gene for resistance against biotic stress, we also show here that the p1 controlled 3-deoxyanthocyanidin and C-glycosyl flavone (maysin defence compounds accumulate at significantly higher levels in Pr1 silks as compared to pr1 silks. By virtue of increased maysin synthesis in Pr1 plants, corn ear worm larvae fed on Pr1; P1 silks showed slower growth as compared to pr1; P1 silks. Conclusions Our results show that the Zmf3

  5. Protein-bound glycogen is linked to tyrosine residues.

    OpenAIRE

    Aon, M A; Curtino, J A

    1985-01-01

    Tyrosine-glycogen obtained from retina proteoglycogen by exhaustive proteolytic digestion was radiolabelled with 125I. The 125I-labelled tyrosine-glycogen was degraded by amylolytic digestion to a very small radioactive product, which was identified as iodotyrosine by h.p.l.c. The amylolytic mixture used released glucose and maltose that were alpha-linked to the phenolic hydroxy group of p-nitrophenol. No free iodotyrosine was found before or after the intact [125I]iodotyrosine-glycogen was s...

  6. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...

  7. HIV-1 reverse transcription.

    Science.gov (United States)

    Hu, Wei-Shau; Hughes, Stephen H

    2012-10-01

    Reverse transcription and integration are the defining features of the Retroviridae; the common name "retrovirus" derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT.

  8. Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun

    2012-01-01

    Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (...

  9. The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation

    DEFF Research Database (Denmark)

    Richter, H E; Albrektsen, T; Billestrup, Nils

    2003-01-01

    of transcription (STAT)-5 signalling pathway. Within minutes of treatment, GH induced the tyrosine phosphorylation, nuclear localization and DNA binding of STAT5. Importantly, there was no evidence that STAT5 acted via an interaction with peroxisome proliferator-activated receptor gamma. To further understand...

  10. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor

  11. Recent advances in understanding the role of protein-tyrosine phosphatases in development and disease

    NARCIS (Netherlands)

    Hale, Alexander James; Ter Steege, Eline; den Hertog, Jeroen

    2017-01-01

    Protein-tyrosine phosphatases (PTPs) remove phosphate groups from tyrosine residues, and thereby propagate or inhibit signal transduction, and hence influence cellular processes such as cell proliferation and differentiation. The importance of tightly controlled PTP activity is reflected by the

  12. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Xiong Zhang

    2014-11-01

    Full Text Available Embryonic stem (ES cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent

  13. A single amino acid residue, Ala 105, confers 16alpha-hydroxylase activity to human cytochrome P450 17alpha-hydroxylase/17,20 lyase.

    Science.gov (United States)

    Swart, Amanda C; Storbeck, Karl-Heinz; Swart, Pieter

    2010-04-01

    In adrenal steroidogenesis, CYP17 catalyses the 17alpha-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reaction, yielding adrenal androgens. The enzyme exhibits distinctly different selectivities towards these substrates in various species. CYP17 has also been shown to exhibit 16alpha-hydroxylase activity towards progesterone in some species, with only human and chimp CYP17 catalysing the biosynthesis of substantial amounts of 16-OHprogesterone. The 16alpha-hydroxylase activity was investigated by introducing an Ala105Leu substitution into human CYP17. The converse mutation, Leu105Ala was introduced into the baboon, goat and pig enzymes. Wt human CYP17 converted approximately 30% progesterone to 16-OHprogesterone while the Ala105Leu mutant converted negligible amounts to 16-OHprogesterone ( approximately 9%), comparable to wt CYP17 of the other three species when expressed in COS-1 cells. The ratio of 17-hydroxylated products to 16-OHprogesterone of human CYP17 was 2.7 and that of the mutant human construct 10.5. Similar ratios were observed for human and goat CYP17 with the corresponding Ala or Leu residues. Although the Leu105Ala mutation of both baboon and pig CYP17 exhibited the same trend regarding the ratios, the rate of progesterone conversion was reduced. Coexpression with cytochrome b(5) significantly decreased the ratio of 17-hydroxylated products to 16-OHprogesterone in the Leu105 constructs, while effects were negligible with Ala at this position. Homology models show that Ala105 faces towards the active pocket in the predicted B'-C domain of CYP17. The smaller residue allows more flexibility of movement in the active pocket than Leu, presenting both the C16 and C17 of progesterone to the iron-oxy complex. 2010 Elsevier Ltd. All rights reserved.

  14. Polyphenol composition in the ripe fruits of Fragaria species and transcriptional analyses of key genes in the pathway.

    Science.gov (United States)

    Muñoz, Cristina; Sánchez-Sevilla, José F; Botella, Miguel A; Hoffmann, Thomas; Schwab, Wilfried; Valpuesta, Victoriano

    2011-12-14

    Polyphenolics are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions and are beneficial to human health. Seventeen structurally well-defined phenolic compounds including phenylpropanoids, flavonols, flavan-3-ols, and anthocyanins were individually analyzed by LC-MS in the ripe fruits of two cultivars of the commercial strawberry (Fragaria × ananassa Duch., Rosaceae) as well as in accessions of F. vesca, F. moschata, and F. chiloensis. Metabolic analysis revealed that the majority of the compounds analyzed accumulated in a genotype-dependent manner. Transcriptional studies of genes encoding for enzymes of the biosynthetic pathway such as phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, chalcone synthase, and flavonoid 3'-hydroxylase could partially explain the different levels of polyphenolics observed in the Fragaria species. The results can provide a sound basis for selecting markers for the development of cultivars with high phenolic content, which can be of value for the food industry.

  15. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y...

  16. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  17. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances

    NARCIS (Netherlands)

    B.C. Tilly (Bernard); N. van den Berghe (Nina); L.G. Tertoolen; M.J. Edixhoven (Marcel); H.R. de Jonge (Hugo)

    1993-01-01

    textabstractUsing the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded

  18. Auto-thiophosphorylation activity of Src tyrosine kinase.

    Science.gov (United States)

    Cabail, M Zulema; Chen, Emily I; Koller, Antonius; Miller, W Todd

    2016-07-07

    Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low. Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase. Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.

  19. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    5, in Luria Bertani broth ..... bacteria has been used by Larose et al (1993) to identify residues in the platelet derived growth factor ... GCC by EphB1 within bacterial cells suggests that GCC may be a substrate for the Eph family of tyrosine ...

  20. Cloning and Characterization of Secretory Tyrosine Phosphatases of Mycobacterium tuberculosis

    Science.gov (United States)

    Koul, Anil; Choidas, Axel; Treder, Martin; Tyagi, Anil K.; Drlica, Karl; Singh, Yogendra; Ullrich, Axel

    2000-01-01

    Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H37Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis. PMID:10986245

  1. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  2. Role of Bruton's tyrosine kinase in B cells and malignancies

    NARCIS (Netherlands)

    Pal Singh, S. (Simar); F. Dammeijer (Floris); R.W. Hendriks (Rudi)

    2018-01-01

    textabstractBruton's tyrosine kinase (BTK) is a non-receptor kinase that plays a crucial role in oncogenic signaling that is critical for proliferation and survival of leukemic cells in many B cell malignancies. BTK was initially shown to be defective in the primary immunodeficiency X-linked

  3. Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.

    Science.gov (United States)

    Koul, A; Choidas, A; Treder, M; Tyagi, A K; Drlica, K; Singh, Y; Ullrich, A

    2000-10-01

    Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.

  4. Understanding proton affinity of tyrosine sidechain in hydrophobic ...

    Indian Academy of Sciences (India)

    Tyrosine (Tyr), like histidine (His), is known to play a crucial role in a wide range of chemical and biochemi- cal processes3 primarily through (a) deprotonation of the phenolic –OH group present in its sidechain and. #Dedicated to Prof. N Sathyamurthy on his 60th birthday. ∗. For correspondence. (b) formation of hydrogen ...

  5. Isolation of a tyrosine-activating enzyme from baker's yeast

    NARCIS (Netherlands)

    Ven, A.M. van de; Koningsberger, V.V.; Overbeek, J.Th.G.

    1958-01-01

    The extracts of ether-CO2-frozen baker's yeast contain enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. From the extract a tyrosine-activating enzyme could be isolated, which, judging from ultracentrifugation and electrophoretic data, was about 70% pure

  6. D-tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity.

    Science.gov (United States)

    Park, Jisu; Jung, Hyejung; Kim, Kyuri; Lim, Kyung-Min; Kim, Ji-Young; Jho, Eek-Hoon; Oh, Eok-Soo

    2017-11-09

    Although L-tyrosine is well known for its melanogenic effect, the contribution of D-tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L-tyrosine, D-tyrosine dose-dependently reduced the melanin contents of human MNT-1 melanoma cells and primary human melanocytes. In addition, 500 μM of D-tyrosine completely inhibited 10 μM L-tyrosine-induced melanogenesis, and both in vitro assays and L-DOPA staining MNT-1 cells showed that tyrosinase activity is reduced by D-tyrosine treatment. Thus, D-tyrosine appears to inhibit L-tyrosine-mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D-tyrosine inhibited melanogenesis induced by α-MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D-tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D-tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte-derived cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. HIV-1 Reverse Transcription

    OpenAIRE

    Hu, Wei-Shau; Hughes, Stephen H.

    2012-01-01

    Reverse transcription and integration are the defining features of the Retroviridae; the common name “retrovirus” derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral fact...

  8. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Hoff, Tine; Frandsen, Gitte Inselmann; Rocher, Anne

    1998-01-01

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde...... oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis....

  9. The crystal structure of tryptophan hydroxylase with bound amino acid substrate

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Petersen, Charlotte Rode; Christensen, Hans Erik Mølager

    2008-01-01

    of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 Å resolution crystal structure of the catalytic domain (Δ1−100/Δ415−445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino...... acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 Å between...

  10. 17-α-Hydroxylase deficiency: An unusual case with primary amenorrhea and hypertension

    Directory of Open Access Journals (Sweden)

    Sunil Kumar Kota

    2011-01-01

    Full Text Available A 14-year-old girl presented with acute onset quadriparesis and newly detected hypertension. Parental consanguinity, delayed puberty with normal stature form the additional information. Hypokalemia with metabolic alkalosis, low cortisol, high ACTH and FSH pointed to the possibility of CAH with 17α hydroxylase deficiency. 46XX karyotype and high progesterone supported this. Normalization of hypokalemia and hypertension with glucocorticoid treatment confirmed the diagnosis. In summary, the possibility of 17 OHD should be suspected in patients with hypokalemic myopathy, Hypertension and hypogonadism so that appropriate therapy can be implemented.

  11. Variation in tryptophan hydroxylase-2 gene is not associated to male completed suicide in Estonian population.

    Science.gov (United States)

    Must, Anne; Tasa, Gunnar; Lang, Aavo; Vasar, Eero; Kõks, Sulev; Maron, Eduard; Väli, Marika

    2009-04-03

    Dysfunction of the central serotonergic system has been related to a spectrum of psychiatric disorders, including suicidal behavior. Tryptophan hydroxylase isoform 2 (TPH2) is the rate-limiting enzyme in the biosynthetic pathway of serotonin, being expressed in serotonergic neurons of raphe nuclei. We investigated genetic variation in TPH2 gene in two samples of male subjects: 288 suicide completers and 327 volunteers, in order to reveal any associations between 14 single nucleotide polymorphisms and completed suicide. No associations were revealed neither on allelic nor haplotype level. Our finding does not support the hypothesis of TPH2 being a susceptibility factor for completed suicide in males of Estonian origin.

  12. Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6

    DEFF Research Database (Denmark)

    Mantri, M.; Webby, C.J.; Loik, N.D.

    2012-01-01

    The lysyl 5S-hydroxylase, JMJD6 acts on proteins involved in RNA splicing. We find that in the absence of substrate JMJD6 catalyses turnover of 2OG to succinate. H-NMR analyses demonstrate that consumption of 2OG is coupled to succinate formation. MS analyses reveal that JMJD6 undergoes self......-hydroxylation in the presence of Fe(ii) and 2OG resulting in production of 5S-hydroxylysine residues. JMJD6 in human cells is also found to be hydroxylated. Self-hydroxylation of JMJD6 may play a regulatory role in modulating the hydroxylation status of proteins involved in RNA splicing. This journal is...

  13. Expression and localization of sterol 27-hydroxylase (CYP27A1) in monkey retina

    OpenAIRE

    Lee, Jung Wha; Fuda, Hirotoshi; Javitt, Norman B.; Strott, Charles A.; Rodriguez, Ignacio R.

    2006-01-01

    Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols including 7-ketocholesterol (7kCh). CYP27A1 is widely expressed in human tissues but has not been previously demonstrated in the retina. In this study, we examined the expression and localization of CYP27A1 in the monkey retina where it localized mainly to the photoreceptor inner segments. CYP27A1 was also observed in Müller cells with faint immuno staining detected in the RPE and ...

  14. Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy1[W

    Science.gov (United States)

    Lee, Eun-Jeong; Facchini, Peter J.

    2011-01-01

    Tyrosine aminotransferase (TyrAT) catalyzes the transamination of l-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and l-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5′-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for l-Tyr and α-ketoglutarate, with apparent Km values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of l-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde. PMID:21949209

  15. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

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

    Science.gov (United States)

    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...

  17. Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors.

    Science.gov (United States)

    Bai, Yun; Li, Jiannong; Fang, Bin; Edwards, Arthur; Zhang, Guolin; Bui, Marilyn; Eschrich, Steven; Altiok, Soner; Koomen, John; Haura, Eric B

    2012-05-15

    Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. ©2012 AACR.

  18. The Transcription Factor Encyclopedia

    NARCIS (Netherlands)

    Yusuf, Dimas; Butland, Stefanie L.; Swanson, Magdalena I.; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A.; Zhang, Xiao Yu Cindy; Dickman, Christopher T. D.; Fulton, Debra L.; Lim, Jonathan S.; Schnabl, Jake M.; Ramos, Oscar H. P.; Vasseur-Cognet, Mireille; de Leeuw, Charles N.; Simpson, Elizabeth M.; Ryffel, Gerhart U.; Lam, Eric W.-F.; Kist, Ralf; Wilson, Miranda S. C.; Marco-Ferreres, Raquel; Brosens, Jan J.; Beccari, Leonardo L.; Bovolenta, Paola; Benayoun, Bérénice A.; Monteiro, Lara J.; Schwenen, Helma D. C.; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A.; Chakravarthy, Harini; Hoodless, Pamela A.; Mancarelli, M. Michela; Torbett, Bruce E.; Banham, Alison H.; Reddy, Sekhar P.; Cullum, Rebecca L.; Liedtke, Michaela; Tschan, Mario P.; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J.; Eijkelenboom, Astrid; Brown, Philip J.; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L.; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H.; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J.; van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W. Z.; Breslin, Mary B.; Lan, Michael S.; Nanan, Kyster K.; Wegner, Michael; Hou, Juan; Mullen, Rachel D.; Colvin, Stephanie C.; Noy, Peter John; Webb, Carol F.; Witek, Matthew E.; Ferrell, Scott; Daniel, Juliet M.; Park, Jason; Waldman, Scott A.; Peet, Daniel J.; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J.; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M.; Woodcroft, Mark W.; Hough, Margaret R.; Chen, Edwin; Europe-Finner, G. Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; Lebrun, David P.; Biswal, Shyam; Harvey, Christopher J.; Debruyne, Jason P.; Hogenesch, John B.; Hevner, Robert F.; Héligon, Christophe; Luo, Xin M.; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S.; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M.; Bradley, Philip H.; Wasserman, Wyeth W.

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review

  19. The transcriptional landscape

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein codi...

  20. Enhanced hypocholesterolemic effects of interesterified oils are mediated by upregulating LDL receptor and cholesterol 7-α- hydroxylase gene expression in rats.

    Science.gov (United States)

    Reena, Malongil B; Gowda, Lalitha R; Lokesh, Belur R

    2011-01-01

    The concentration of LDL cholesterol in plasma is strongly influenced by the amount and type of lipid in the diet. Our studies have shown that positional changes in the fatty acids in blended oil introduced using lipase-catalyzed interesterification differentially modulate circulating LDL levels in rats compared with those observed in rats given a physical blend of oils. To investigate the molecular basis of these differences, transcriptional profiling of genes involved in cholesterol homeostasis was studied after feeding rats with a semipurified diet containing 10% fat from native oils; coconut oil (CNO), rice bran oil (RBO), or sesame oil (SESO); blended (B); CNO+RBO(B) or CNO+SESO(B) and interesterified oil (I); CNO+RBO(I) or CNO+SESO(I) for 60 d. Hepatic LDL receptor (LDL-R) expression significantly increased in rats fed interesterified oils by 100-200% compared with rats fed blended oils and by 400-500% compared with rats fed CNO. Positional alteration in fatty acids of oils used in the diet induced changes in LDL-R expression, which was accompanied by parallel changes in cholesterol-7α-hydroxylase (CYP7A1) and SREBP-2 genes. This suggested that not only the fatty acid type but also its position in the TG of dietary lipids play an important role in maintaining plasma cholesterol levels by suitably modulating gene expression for LDL-R in rat liver.

  1. Changes in Hypoxia-Inducible Factor-1 (HIF-1) and Regulatory Prolyl Hydroxylase (PHD) Enzymes Following Hypoxic-Ischemic Injury in the Neonatal Rat.

    Science.gov (United States)

    Chu, Hannah X; Jones, Nicole M

    2016-03-01

    Hypoxia leads to activation of many cellular adaptive processes which are regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 consists of HIF-1α and HIF-1ß subunits and levels of HIF-1α protein are regulated by HIF prolyl-hydroxylase enzymes (PHD1, 2, 3). The aim of the current study was to investigate the expression of HIF-1α and PHDs at various time points after hypoxia-ischemia (HI), using a neonatal rat model of HI brain injury. Sprague-Dawley rat pups (postnatal day 7) were anaesthetized and underwent right carotid artery occlusion and were then exposed to 6 % oxygen for 2.5 h at 37 °C. HI injured animals demonstrated a significant reduction in the size of the ipsilateral hemisphere, compared to sham controls. Protein analysis using western blotting and enzyme-linked immunosorbent assay showed that 24 h after HI, there was a significant increase in PHD3 protein and an increase of HIF-1α compared to controls. At the 72 h time point, there was a reduction in PHD3 protein, which appeared to relate to cellular loss. There were no changes in PHD1 or PHD2 protein levels after HI when compared to age-matched controls. Further studies are necessary to establish roles for the HIF-1 regulatory enzyme PHD3 in brain injury processes.

  2. Marmoset cytochrome P450 4A11, a novel arachidonic acid and lauric acid ω-hydroxylase expressed in liver and kidney tissues.

    Science.gov (United States)

    Uehara, Shotaro; Uno, Yasuhiro; Ishii, Sakura; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-07-01

    1. A cDNA encoding novel cytochrome P450 (P450) 4A enzyme was cloned from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR) based on the marmoset genome sequences. The amino acid sequence deduced from P450 4A11 cDNA contained consensus sequences of six substrate recognition sites and one heme-binding domain. 2. Marmoset P450 4A11, highly identical (85-88%) to cynomolgus monkey and human P450 4A enzymes, was grouped into the same cluster as cynomolgus monkey and human P450 4A enzymes by phylogenetic analysis. 3. The tissue distribution analyses by real-time RT PCR and immunoblotting demonstrated that marmoset P450 4A11 mRNA and proteins were expressed in kidneys and livers. Marmoset P450 4A11 enzyme heterologously expressed in Escherichia coli preferentially catalyzed the ω-hydroxylation of arachidonic acid and lauric acid, similar to cynomolgus monkey and human P450 4A11 enzymes. However, lauric acid ω-hydroxylation activity of marmoset P450 4A11 was low compared with those of marmoset liver microsomes. 4. These results indicated that novel marmoset P450 4A11 was also a fatty acid ω-hydroxylase expressed in kidneys and livers, with the same regioselectivity (at ω-position of fatty acid) as cynomolgus monkey and human P450 4A enzymes.

  3. The Arabidopsis nox Mutant Lacking Carotene Hydroxylase Activity Reveals a Critical Role for Xanthophylls in Photosystem I Biogenesis[C][W

    Science.gov (United States)

    Dall’Osto, Luca; Piques, Maria; Ronzani, Michela; Molesini, Barbara; Alboresi, Alessandro; Cazzaniga, Stefano; Bassi, Roberto

    2013-01-01

    Carotenes, and their oxygenated derivatives xanthophylls, are essential components of the photosynthetic apparatus. They contribute to the assembly of photosynthetic complexes and participate in light absorption and chloroplast photoprotection. Here, we studied the role of xanthophylls, as distinct from that of carotenes, by characterizing a no xanthophylls (nox) mutant of Arabidopsis thaliana, which was obtained by combining mutations targeting the four carotenoid hydroxylase genes. nox plants retained α- and β-carotenes but were devoid in xanthophylls. The phenotype included depletion of light-harvesting complex (LHC) subunits and impairment of nonphotochemical quenching, two effects consistent with the location of xanthophylls in photosystem II antenna, but also a decreased efficiency of photosynthetic electron transfer, photosensitivity, and lethality in soil. Biochemical analysis revealed that the nox mutant was specifically depleted in photosystem I function due to a severe deficiency in PsaA/B subunits. While the stationary level of psaA/B transcripts showed no major differences between genotypes, the stability of newly synthesized PsaA/B proteins was decreased and translation of psaA/B mRNA was impaired in nox with respect to wild-type plants. We conclude that xanthophylls, besides their role in photoprotection and LHC assembly, are also needed for photosystem I core translation and stability, thus making these compounds indispensable for autotrophic growth. PMID:23396829

  4. Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARgamma ligands.

    Science.gov (United States)

    Quinn, Carmel M; Jessup, Wendy; Wong, Jenny; Kritharides, Leonard; Brown, Andrew J

    2005-02-01

    CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However, the RXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested, PPAR (peroxisome-proliferator-activated receptor) gamma ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARgamma antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27A1 by PPARgamma may represent a key previously unrecognized mechanism by which PPARgamma protects against atherosclerosis.

  5. Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARγ ligands

    Science.gov (United States)

    2004-01-01

    CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However, the RXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested, PPAR (peroxisome-proliferator-activated receptor) γ ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARγ antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27A1 by PPARγ may represent a key previously unrecognized mechanism by which PPARγ protects against atherosclerosis. PMID:15533057

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Shu Chang

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Timmers, R.J.; Granneman, J.C.; Lambert, J.G.; van Oordt, P.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; 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.

  10. Mechanical Properties of Transcription

    Science.gov (United States)

    Sevier, Stuart A.; Levine, Herbert

    2017-06-01

    The mechanical properties of transcription have recently been shown to play a central role in gene expression. However, a full physical characterization of this central biological process is lacking. In this Letter, we introduce a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase rotation, and DNA supercoiling are coupled. The resulting framework describes the relative amount of RNA polymerase rotation and DNA supercoiling that occurs during RNA elongation. Asymptotic behavior is derived and can be used to experimentally extract unknown mechanical parameters of transcription. Mechanical limits to transcription are incorporated through the addition of a DNA supercoiling-dependent RNA polymerase velocity. This addition can lead to transcriptional stalling and resulting implications for gene expression, chromatin structure and genome organization are discussed.

  11. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK)

    Science.gov (United States)

    Morris, S W; Naeve, C; Mathew, P; James, P L; Kirstein, M N; Cui, X; Witte, D P

    1997-05-08

    Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. Here, we characterize a full-length normal human ALK cDNA and its product, and determine the pattern of expression of its murine homologue in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrated that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicated that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies revealed Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts were confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain; and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system.

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

    Science.gov (United States)

    Guo, Yong; Qiu, Li-Juan

    2013-06-01

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

  13. D-tyrosine affects aggregation behavior of Pantoea agglomerans.

    Science.gov (United States)

    Yang, Jing; Yu, Jiajia; Jiang, Jing; Liang, Chen; Feng, Yongjun

    2017-02-01

    D-amino acids have been proved to disassemble biofilms by disassociating the matrix. Pantoea agglomerans is characterized by the formation of another kind of multicellular structure called symplasmata, which also remains the ability to form biofilms. In this study, a rice diazotrophic endophyte P. agglomerans YS19 was selected as a model strain to explore the effects of D-amino acids on these two kinds of cell aggregate structures. It was discovered that D-tyrosine disassociates biofilm, yet promotes symplasmata formation. D-tyrosine showed no influence on bacterial growth yet promoted the bacterial motility and inhibited the expression of cellular MalE and OmpF proteins, which enriched our knowledge of the biological effect of D-amino acids and expanded the research ideas of symplasmata formation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Receptor tyrosine kinases: the emerging tip of systems control.

    Science.gov (United States)

    Seger, R; Rodeck, U; Yarden, Y

    2008-01-01

    Receptor tyrosine kinases (RTKs) are transmembrane allosteric enzymes: binding of ligand growth factors to their ectodomains stimulates a cytoplasm-facing tyrosine kinase activity, which initiates a plethora of cellular processes. The enormous complexity of RTK signalling, along with rich involvement in pathologies (e.g. cancer and diabetes), motivated the establishment of the international, multi-disciplinary RTK consortium (http://www.rtkconsort.org/) in 2005. In collaboration with the British Society for Proteome Research and the European Bioinformatics Institute, the Consortium held on July 23rd and 24th a Workshop on Proteomics and Phosphoproteomics of RTK Signalling Networks (Hinxton Hall Conference Centre, Cambridge, UK). As highlighted below, systems control (a layered web of regulatory loops summarised in Fig.1) emerged throughout the workshop as a common theme of many presentations.

  15. Novel Bruton's tyrosine kinase inhibitors currently in development

    Directory of Open Access Journals (Sweden)

    D'Cruz OJ

    2013-03-01

    Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

  16. Stress signaling by Tec tyrosine kinase in the ischemic myocardium.

    Science.gov (United States)

    Zhang, Michael J; Franklin, Sarah; Li, Yifeng; Wang, Sujing; Ru, Xiaochen; Mitchell-Jordan, Scherise A; Mano, Hiroyuki; Stefani, Enrico; Ping, Peipei; Vondriska, Thomas M

    2010-09-01

    Nonreceptor tyrosine kinases have an increasingly appreciated role in cardiac injury and protection. To investigate novel tasks for members of the Tec family of nonreceptor tyrosine kinases in cardiac phenotype, we examined the behavior of the Tec isoform in myocardial ischemic injury. Ischemia-reperfusion, but not cardiac protective agents, induced altered intracellular localization of Tec, highlighting distinct actions of this protein compared with other isoforms, such as Bmx, in the same model. Tec is abundantly expressed in cardiac myocytes and assumes a diffuse intracellular localization under basal conditions but is recruited to striated structures upon various stimuli, including ATP. To characterize Tec signaling targets in vivo, we performed an exhaustive proteomic analysis of Tec-binding partners. These experiments expand the role of the Tec family in the heart, identifying the Tec isoform as an ischemic injury-induced isoform, and map the subproteome of its interactors in isolated cells.

  17. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.......We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  18. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    International Nuclear Information System (INIS)

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

    1989-01-01

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

  19. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  20. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. (Univ. of Miami School of Medicine, FL (USA))

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  1. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

    Science.gov (United States)

    Antosiewicz, Jan M; Shugar, David

    2016-06-01

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  2. UV?Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore

    OpenAIRE

    Antosiewicz, Jan M.; Shugar, David

    2016-01-01

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV?Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  3. Role of the pathotype-specific ACRTS1 gene encoding a hydroxylase involved in the biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata.

    Science.gov (United States)

    Izumi, Yuriko; Kamei, Eri; Miyamoto, Yoko; Ohtani, Kouhei; Masunaka, Akira; Fukumoto, Takeshi; Gomi, Kenji; Tada, Yasuomi; Ichimura, Kazuya; Peever, Tobin L; Akimitsu, Kazuya

    2012-08-01

    The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of the rootstock species rough lemon (Citrus jambhiri) and Rangpur lime (C. limonia). Genes controlling toxin production were localized to a 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. We studied the functional role of one of these open reading frames, ACRTS1 encoding a putative hydroxylase, in ACR-toxin production by homologous recombination-mediated gene disruption. There are at least three copies of ACRTS1 gene in the genome and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity; however, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies of the gene. RNA-silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. These results indicate that ACRTS1 is an essential gene in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and is required for full virulence of this fungus.

  4. Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

    Science.gov (United States)

    Conlay, L. A.; Maher, T. J.; Roberts, C. H.; Wurtman, R. J.

    1988-01-01

    Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

  5. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

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

    Science.gov (United States)

    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...

  7. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...

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

    Science.gov (United States)

    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...

  9. Deciphering Transcriptional Regulation

    DEFF Research Database (Denmark)

    Valen, Eivind

    RNA); and ii) translation, in which the mRNA is translated into a protein. This thesis focus on the ¿rst of these steps, transcription, and speci¿cally the initiation of this. Simpli¿ed, initiation is preceded by the binding of several proteins, known as transcription factors (TFs), to DNA. This takes place...... published providing an unbiased overview of the transcription start site (TSS) usage in a tissue. We have paired this method with high-throughput sequencing technology to produce a library of unprecedented depth (DeepCAGE) for the mouse hippocampus. We investigated this in detail and focused particularly...... control spanning the range from completely muted to cranked up to maximum. The volume, in this case, is the production rate of proteins. This production is the result of a two step procedure: i) transcription, in which a small part of DNA from the genome (a gene) is transcribed into an RNA molecule (an m...

  10. The Impact of Tyrosine Kinase Signaling on Breast Cancer Development

    National Research Council Canada - National Science Library

    Marozkina, Nadzeya V

    2006-01-01

    ... of the MMTV promoter are being developed. The MMTV promoter responds transcriptionally to glucocorticoids and steroids and causes expression of the transgene in steroid hormone responsive organs...

  11. Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

    Directory of Open Access Journals (Sweden)

    Alagna Fiammetta

    2012-09-01

    Full Text Available Abstract Background Olive (Olea europaea L. fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism. Results The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF, suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development. Conclusions Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the

  12. Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

    Science.gov (United States)

    2012-01-01

    Background Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism. Results The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development. Conclusions Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data

  13. Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer

    DEFF Research Database (Denmark)

    Hartmann, Stefan; Brands, Roman C; Küchler, Nora

    2015-01-01

    Melanoma-associated antigen (MAGE) has been identified in a variety of types of cancer. The expression of several MAGE subgroups is correlated with poor prognosis and chemotherapeutic resistance. One target of chemotherapeutic treatment in head and neck cancer is the epidermal growth factor...... receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using...... a crystal violet assay. Furthermore, the expression levels of MAGE-A1, -A5, -A8, -A9, -A11 and -A12 were determined by reverse transcription-quantitative polymerase chain reaction. The association between TKI efficacy and MAGE-A expression was analyzed by linear regression. The cell lines revealed...

  14. Cadmium affects the activity of rat liver tyrosine aminotransferase and its induction by dexamethasone

    Energy Technology Data Exchange (ETDEWEB)

    Dundjerski, J. [Department of Ecology, Institute for Biological Research, 29. Novembra 142, Yu-11060 Belgrade (Yugoslavia); Butorovic, B. [Department of Molecular Biology and Biochemistry, Institute for Biological Research, 29. Novembra 142, YU-11060 Belgrade (Yugoslavia); Kipic, J. [Department of Molecular Biology and Biochemistry, Institute for Biological Research, 29. Novembra 142, YU-11060 Belgrade (Yugoslavia); Trajkovic, D. [Department of Molecular Biology and Biochemistry, Institute for Biological Research, 29. Novembra 142, YU-11060 Belgrade (Yugoslavia); Matic, G. [Department of Molecular Biology and Biochemistry, Institute for Biological Research, 29. Novembra 142, YU-11060 Belgrade (Yugoslavia)

    1996-03-01

    The effects of cadmium (Cd) administration to intact rats on hepatic glucocorticoid receptor (GR) steroid binding capacity and DNA-binding ability were examined and correlated with the influence of the metal on rat liver tyrosine aminotransferase (TAT) activity and its induction by dexamethasone. It was found that 24 h after i.p. administration of Cd doses ranging from 0.5 to 4 mg/kg, the GR steroid- and DNA-binding activities were significantly reduced in a dose-dependent manner. The same doses of Cd also affected the basal and dexamethasone-induced level of TAT activity, as well as the concentration of metallothionein in rat liver. The decrease in TAT activity and in its induction by dexamethasone observed in response to low Cd doses was proportional to the alterations of the GR functional properties. Higher doses of Cd, which were more effective in reducing both the GR binding of the hormone and to DNA, however, stimulated TAT activity and potentiated dexamethasone induction of the enzyme. The results led to the conclusion that Cd may alter physiological response of rat liver cells to glucocorticoids interfering with the GR-dependent transcriptional regulation of the TAT gene. (orig.). With 6 figs.

  15. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition

    Science.gov (United States)

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D.; Nencioni, Alessio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. PMID:25909220

  16. Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism1[W][OA

    Science.gov (United States)

    Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A.; Zaharia, L. Irina; Schernthaner, Johann P.; Gesell, Andreas; Abrams, Suzanne R.; Kennedy, James A.; Constabel, C. Peter

    2012-01-01

    Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3′-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3′5′-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation

  17. To Cheat or Not To Cheat: Tryptophan Hydroxylase 2 SNP Variants Contribute to Dishonest Behavior.

    Science.gov (United States)

    Shen, Qiang; Teo, Meijun; Winter, Eyal; Hart, Einav; Chew, Soo H; Ebstein, Richard P

    2016-01-01

    Although, lying (bear false witness) is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology, and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Toward addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2) gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tyfield, L.A. [Southmead Hospital, Bristol (United Kingdom)]|[Univ. of Bristol (United Kingdom); Stephenson, A. [Southmead Hospital, Bristol (United Kingdom); Cockburn, F. [Royal Hospital for Sick Children, Glasgow (United Kingdom)] [and others

    1997-02-01

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

  19. To cheat or not to cheat: Tryptophan hydroxylase 2 SNP variants contribute to dishonest behavior

    Directory of Open Access Journals (Sweden)

    Qiang eShen

    2016-05-01

    Full Text Available Although lying (bear false witness is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Towards addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2 gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

  20. Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    So Yeon Kim

    2015-11-01

    Full Text Available Hypoxia-inducible factor (HIF prolyl hydroxylases (PHDs are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

  1. 2 Novel deletions of the sterol 27-hydroxylase gene in a Chinese Family with Cerebrotendinous Xanthomatosis

    Directory of Open Access Journals (Sweden)

    Tian Di

    2011-10-01

    Full Text Available Abstract Background Cerebrotendinous xanthomatosis (CTX is a rare lipid-storage disease. We investigated the clinic manifestation, histopathology and sterol 27-hydroxylase gene (CYP27A1 in a Chinese family with Cerebrotendinous Xanthomatosis (CTX. Case Presentation A 36-year-old female with typical CTX clinical manifestation had Spindle-shaped lipid crystal clefts in xanthomas and "onion-like demyelination" in sural nerve. The patient was compound heterozygote carrying two deletions in exon 1 (c.73delG and exon 2 (c.369_375delGTACCCA. The family memebers were carriers. Conclusions A Chinese family with Cerebrotendinous Xanthomatosis had typical clinical manifestation. CYP27A1 mutations were found in the proband and all other family members.

  2. Maternal and fetal plasma renin and dopamine-beta-hydroxylase activities in toxemic pregnancy.

    Science.gov (United States)

    Annat, G; Raudrant, D; Chappe, J; Vincent, M; Thoulon, J; Dumont, M; Sassard, J

    1978-08-01

    Toxemic and normotensive pregnant women were compared for plasma renin activity (PRA), aldosterone (PA), and dopamine-beta-hydroxylase (DBH). At term, hypertensive patients exhibited higher levels of PRA and PA, but similar levels of DBH, progesterone, and estradiol. Their elevated blood pressure was significantly correlated to their levels of PRA. During the delivery levels of PRA increased significantly in toxemic patients in spontaneous labor. Venous and arterial cord PRA levels were higher in babies born to hypertensive mothers than in babies born to normotensive mothers. Three days postpartum, maternal PRA level was lower than at term. Seven days postpartum, PRA levels remained higher in toxemic than in normal women. Maternal DBH levels did not change during and after delivery. Levels of DBH were undetectable in cord blood. We conclude that the renin-angiotensin system is involved in the pathogenesis of toxemia.

  3. Alkane Hydroxylase Gene (alkB Phylotype Composition and Diversity in Northern Gulf of Mexico Bacterioplankton

    Directory of Open Access Journals (Sweden)

    Conor Blake Smith

    2013-12-01

    Full Text Available Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM, a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with alkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter. Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables.

  4. Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

    2004-01-01

    The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

  5. Antisense transcription-dependent chromatin signature modulates sense transcript dynamics.

    Science.gov (United States)

    Brown, Thomas; Howe, Françoise S; Murray, Struan C; Wouters, Meredith; Lorenz, Philipp; Seward, Emily; Rata, Scott; Angel, Andrew; Mellor, Jane

    2018-02-12

    Antisense transcription is widespread in genomes. Despite large differences in gene size and architecture, we find that yeast and human genes share a unique, antisense transcription-associated chromatin signature. We asked whether this signature is related to a biological function for antisense transcription. Using quantitative RNA-FISH, we observed changes in sense transcript distributions in nuclei and cytoplasm as antisense transcript levels were altered. To determine the mechanistic differences underlying these distributions, we developed a mathematical framework describing transcription from initiation to transcript degradation. At GAL1 , high levels of antisense transcription alter sense transcription dynamics, reducing rates of transcript production and processing, while increasing transcript stability. This relationship with transcript stability is also observed as a genome-wide association. Establishing the antisense transcription-associated chromatin signature through disruption of the Set3C histone deacetylase activity is sufficient to similarly change these rates even in the absence of antisense transcription. Thus, antisense transcription alters sense transcription dynamics in a chromatin-dependent manner. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  6. Effects of cholecalciferol (vitamin D3)-binding proteins and anti-cytochrome b5 immunoglobulin on cholecalciferol 25-hydroxylase activities of rabbit liver microsomes and mitochondria.

    Science.gov (United States)

    Hiwatashi, A; Nishii, Y; Ichikawa, Y

    1983-11-01

    The effects of cholecalciferol (vitamin D3)-binding proteins and anti-cytochrome b5 immunoglobulin were studied on vitamin D3 25-hydroxylase activities of microsomes and mitochondria under various experimental conditions. The vitamin D3-binding protein in serum as well as in the liver postmicrosomal supernatant (cellular vitamin D3-binding protein), but not serum albumin and ovalbumin, stimulated vitamin D3 25-hydroxylase activities of microsomes and mitochondria. The optimum pH range was from 7.3 to 8.0. Anti-cytochrome b5 immunoglobulin did not affect microsomal vitamin D3 25-hydroxylase activity.

  7. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

    Directory of Open Access Journals (Sweden)

    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  8. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma.

    Science.gov (United States)

    Singh, Dinesh K; Kollipara, Rahul K; Vemireddy, Vamsidara; Yang, Xiao-Li; Sun, Yuxiao; Regmi, Nanda; Klingler, Stefan; Hatanpaa, Kimmo J; Raisanen, Jack; Cho, Steve K; Sirasanagandla, Shyam; Nannepaga, Suraj; Piccirillo, Sara; Mashimo, Tomoyuki; Wang, Shan; Humphries, Caroline G; Mickey, Bruce; Maher, Elizabeth A; Zheng, Hongwu; Kim, Ryung S; Kittler, Ralf; Bachoo, Robert M

    2017-01-24

    Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Tyrosine-rich crystals associated with oncocytic salivary gland neoplasms.

    Science.gov (United States)

    Gilcrease, M Z; Nelson, F S; Guzman-Paz, M

    1998-07-01

    Crystalloids have been identified ultrastructurally within the epithelial cells of Warthin's tumors, but there have been no studies characterizing crystals or crystalloids in Warthin's tumors by light microscopy. The finding of abundant needle-shaped crystals in a fine-needle aspirate of a cystadenoma of the parotid prompted us to examine the prevalence of crystals and crystalloids in oncocytic salivary gland neoplasms. Ninety-seven oncocytic neoplasms (93 Warthin's tumors, 3 cystadenomas, and 1 oncocytoma) excised at our institution between 1950 and 1996 were examined, to identify crystals. Neoplasms with crystals were further characterized by means of a variety of histochemical stains and electron microscopy. Ninety-nine pleomorphic adenomas were similarly reviewed. Seven cases with crystals were identified. Five of these were Warthin's tumors, 1 was a cystadenoma, and 1 was an oncocytoma. The crystals were noted within tumor cysts but were not limited to the neoplasms. The crystals were predominantly either needle-shaped or tabular, but some cases contained mixtures of both as well as intermediate forms. They stained pink with hematoxylin-eosin, although the tabular forms also exhibited a focal yellow hue. The crystals were not discernible under polarized light. They stained a red-brown color with Millon's reagent, which indicated the presence of tyrosine. Trichrome, periodic acid-Schiff stain with diastase, alcian blue (pH 2.5), and Congo red stains were negative. Electron microscopy revealed sharply defined, elongate, electron-dense structures with periodicity, both extracellular and within epithelial cells. No crystals or crystalloids were identified in any of 99 pleomorphic adenomas reviewed. The findings indicate that tyrosine-rich crystals associated with several oncocytic salivary gland neoplasms are morphologically, histochemically, and ultrastructurally distinct from previously described tyrosine-rich crystalloids and collagenous crystalloids of

  10. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    Energy Technology Data Exchange (ETDEWEB)

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  11. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    Energy Technology Data Exchange (ETDEWEB)

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  12. Food for thought: association between dietary tyrosine and cognitive performance in younger and older adults.

    Science.gov (United States)

    Kühn, Simone; Düzel, Sandra; Colzato, Lorenza; Norman, Kristina; Gallinat, Jürgen; Brandmaier, Andreas M; Lindenberger, Ulman; Widaman, Keith F

    2017-12-18

    The fact that tyrosine increases dopamine availability that, in turn, may enhance cognitive performance has led to numerous studies on healthy young participants taking tyrosine as a food supplement. As a result of this dietary intervention, participants show performance increases in working memory and executive functions. However, the potential association between habitual dietary tyrosine intake and cognitive performance has not been investigated to date. The present study aims at clarifying the association of episodic memory (EM), working memory (WM) and fluid intelligence (Gf), and tyrosine intake in younger and older adults. To this end, we acquired habitual tyrosine intake (food frequency questionnaire) from 1724 participants of the Berlin Aging Study II (1383 older adults, 341 younger adults) and modelled its relations to cognitive performance assessed in a broad battery of cognitive tasks using structural equation modeling. We observed a significant association between tyrosine intake and the latent factor capturing WM, Gf, and EM in the younger and the older sample. Due to partial strong factorial invariance between age groups for a confirmatory factor analysis on cognitive performance, we were able to compare the relationship between tyrosine and cognition between age groups and found no difference. Above and beyond previous studies on tyrosine food supplementation the present result extend this to a cross-sectional association between habitual tyrosine intake levels in daily nutrition and cognitive performance (WM, Gf, and EM). This corroborates nutritional recommendations that are thus far derived from single-dose administration studies.

  13. Second-generation inhibitors of Bruton tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Jingjing Wu

    2016-09-01

    Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

  14. Second-generation inhibitors of Bruton tyrosine kinase.

    Science.gov (United States)

    Wu, Jingjing; Liu, Christina; Tsui, Stella T; Liu, Delong

    2016-09-02

    Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.

  15. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  16. The Transcription Factor Encyclopedia

    DEFF Research Database (Denmark)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I

    2012-01-01

    mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written......ABSTRACT: Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130...... and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe....

  17. The transcription factor encyclopedia.

    Science.gov (United States)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

  18. Dielectric Relaxation Behavior of Tyrosine-Derived Polycarbonate.

    Science.gov (United States)

    Suarez, N.; Laredo, E.; Bello, A.; Kohn, J.

    1996-03-01

    Tyrosine-derived polycarbonates represent a new family of polymers that were specifically designed for medical applications. In this work we have used Thermally Stimulated Depolarization Currents (TSDC) to study for the first time the dielectric relaxation behavior of a series of degradable tyrosine-derived polycarbonates. The test polymers differed only in the length of the pendent chain which was increased from two to eight carbons by the use of ethyl, butyl, hexyl and octyl esters as C-terminus protecting groups. The high temperature zone of the spectra shows the glass transition relaxation located at decreasing temperatures as the length of the pendent chain is increased. The low temperature spectrum exhibits a complex dielectric relaxation composed of 4 peaks. The relative intensities of these four peaks are sensitive to packing and entanglement effects caused by the variation in the length of the pendent chain. For data analysis, the Direct Signal Analysis (DSA) procedure was used. This procedure allows the precise determination of the relaxation parameters without having to use peak cleaning techniques. To analyze the whole spectra the Vogel-Fulcher temperature dependence of the relaxation time was used for the glass transition relaxation, and the Arrhenius dependence for the remaining relaxations.

  19. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Feng Liu

    2016-06-01

    Full Text Available Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis.

  20. Tyrosine Sulfation as a Protein Post-Translational Modification

    Directory of Open Access Journals (Sweden)

    Yuh-Shyong Yang

    2015-01-01

    Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

  1. The catalytic mechanism of decarboxylative hydroxylation of salicylate hydroxylase revealed by crystal structure analysis at 2.5 Å resolution.

    Science.gov (United States)

    Uemura, Takuya; Kita, Akiko; Watanabe, Yoshihiko; Adachi, Motoyasu; Kuroki, Ryota; Morimoto, Yukio

    2016-01-08

    The X-ray crystal structure of a salicylate hydroxylase from Pseudomonas putida S-1 complexed with coenzyme FAD has been determined to a resolution of 2.5 Å. Structural conservation with p- or m-hydroxybenzoate hydroxylase is very good throughout the topology, despite a low amino sequence identity of 20-40% between these three hydroxylases. Salicylate hydroxylase is composed of three distinct domains and includes FAD between domains I and II, which is accessible to solvent. In this study, which analyzes the tertiary structure of the enzyme, the unique reaction of salicylate, i.e. decarboxylative hydroxylation, and the structural roles of amino acids surrounding the substrate, are considered. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Dietary Tyrosine Protects Striatal Dopamine Receptors from the Adverse Effects of REM Sleep Deprivation.

    Science.gov (United States)

    Hamdi, A; Brock, J W; Payne, S; Ross, K D; Bond, S P; Prasad, C

    1998-01-01

    L-Tyrosine is a non-essential amino acid that is produced as an intermediary metabolite in the conversion of phenylalanine to 3,4-dihyroxyphenylalanine (DOPA), and is a precursor of the neurotransmitter dopamine. In previous studies, tyrosine pretreatment was shown to protect against the neurochemical and behavioral deficits of acute stress caused by tail shock or cold exposure in rodents. The present study addressed the hypothesis that tyrosine administration may be an effective counter-measure to dopamine-mediated behaviors induced by rapid eye-movement sleep deprivation (RSD). In order to test the hypothesis, Sprague-Dawley rats were divided into 9 treatment groups: RSD-treated rats on normal-protein diet (20% casein: 1% tyrosine, 1% valine); tank control (TC) rats on a normal diet; cage control (CC) rats on normal diet; RSD-treated rats on 4% tyrosine diet; TC rats on 4% tyrosine diet; CC rats on 4% tyrosine diet; RSD-treated rats on 4% valine diet; TC rats on 4% valine diet; CC rats on 4% valine diet. In the RSD group receiving tyrosine, there was no apparent change in Bmax for binding of the dopamine D2 receptor ligand [(3)H]YM-09151-2 in the striata as compared to the respective TC and CC groups; whereas RSD-treated rats maintained on the normal diet and valine supplementation demonstrated expected increases in Bmax for ligand binding. The TC group on the tyrosine diet showed attenuated catalepsy compared to the corresponding CC group, while the RSD group consuming tyrosine showed a catalepsy that was significantly increased, and similar to that of cage control animais on a control diet. These data suggest that the tyrosine-supplemented diet significantly attenuated RSD-induced changes in striatal dopamine D2 receptors, and the effect appeared sufficient to influence RSD-induced behaviors.

  3. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

    Science.gov (United States)

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

  4. Transcriptional profiles of hybrid Eucalyptus genotypes with contrasting lignin content reveal that monolignol biosynthesis-related genes regulate wood composition

    Directory of Open Access Journals (Sweden)

    Tomotaka eShinya

    2016-04-01

    Full Text Available Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected three-year old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis genotypes (AM063 and AM380 that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0% and 48.2%, -cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA and sucrose synthase (SUSY were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase (UGP and xyloglucan endotransglucoxylase (XTH than those in AM380. Most monolignol biosynthesis- related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase (PAL, cinnamate-4-hydroxylase (C4H and 4-coumarate-CoA ligase (4CL. Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents

  5. Tyrosine agonists reverse the molecular defects associated with dominant-negative mutations in human peroxisome proliferator-activated receptor gamma.

    Science.gov (United States)

    Agostini, Maura; Gurnell, Mark; Savage, David B; Wood, Emily M; Smith, Aaron G; Rajanayagam, Odelia; Garnes, Keith T; Levinson, Sidney H; Xu, H Eric; Schwabe, John W R; Willson, Timothy M; O'Rahilly, Stephen; Chatterjee, V Krishna

    2004-04-01

    Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPARgamma mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPARgamma mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPARgamma mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPARgamma target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.

  6. Andrographolide inhibits hypoxia-induced HIF-1α-driven endothelin 1 secretion by activating Nrf2/HO-1 and promoting the expression of prolyl hydroxylases 2/3 in human endothelial cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lu, Chia-Yang; Lin, Ai-Hsuan; Lin, Wan-Chun; Liu, Chin-San; Yang, Ya-Chen; Wang, Hsiu-Miao; Lii, Chong-Kuei; Chen, Haw-Wen

    2017-03-01

    Andrographolide, the main bioactive component of the medicinal plant Andrographis paniculata, has been shown to possess potent anti-inflammatory activity. Endothelin 1 (ET-1), a potent vasoconstrictor peptide produced by vascular endothelial cells, displays proinflammatory property. Hypoxia-inducible factor 1α (HIF-1α), the regulatory member of the transcription factor heterodimer HIF-1α/β, is one of the most important molecules that responds to hypoxia. Changes in cellular HIF-1α protein level are the result of altered gene transcription and protein stability, with the latter being dependent on prolyl hydroxylases (PHDs). In this study, inhibition of pro-inflammatory ET-1 expression and changes of HIF-1α gene transcription and protein stability under hypoxia by andrographolide in EA.hy926 endothelial-like cells were investigated. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2. We found that hypoxia stimulated the production of reactive oxygen species (ROS), the expression of HIF-1α mRNA and protein, and the expression and secretion of ET-1. These effects, however, were attenuated by co-exposure to andrographolide, bilirubin, and RuCO. Silencing Nrf2 and heme oxygenase 1 (HO-1) reversed the inhibitory effects of andrographolide on hypxoia-induced HIF-1α mRNA and protein expression. Moreover, andrographolide increased the expression of prolyl hydroxylases (PHD) 2/3, which hydroxylate HIF-1α and promotes HIF-1α proteasome degradation, with an increase in HIF-1α hydroxylation was noted under hypoxia. Inhibition of p38 MAPK abrogated the hypoxia-induced increases in HIF-1α mRNA and protein expression as well as ET-1 mRNA expression and secretion. Taken together, these results suggest that andrographolide suppresses hypoxia-induced pro-inflammatory ET-1 expression by activating Nrf2/HO-1, inhibiting p38 MAPK signaling, and promoting PHD2/3 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 918-930, 2017. © 2016 Wiley

  7. Machine Dictation and Transcription.

    Science.gov (United States)

    Harvey, Evelyn; And Others

    This instructional package contains both an instructor's manual and a student's manual for a course in machine dictation and transcription. The instructor's manual contains an overview with tips on teaching the course, letters for dictation, and a key to the letters. The student's manual contains an overview of the course and of the skills needed…

  8. Automatic Music Transcription

    Science.gov (United States)

    Klapuri, Anssi; Virtanen, Tuomas

    Written musical notation describes music in a symbolic form that is suitable for performing a piece using the available musical instruments. Traditionally, musical notation indicates the pitch, target instrument, timing, and duration of each sound to be played. The aim of music transcription either by humans or by a machine is to infer these musical parameters, given only the acoustic recording of a performance.

  9. Bayesian Music Transcription

    NARCIS (Netherlands)

    Cemgil, A.T.

    2004-01-01

    Music transcription refers to extraction of a human readable and interpretable description from a recording of a music performance. The final goal is to implement a program that can automatically infer a musical notation that lists the pitch levels of notes and corresponding score positions in any

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

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, S.P. (Univ. of South Carolina School of Medicine, Columbia (USA))

    1991-01-01

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

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

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

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

  12. Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors.

    Science.gov (United States)

    Tan, Suat Cheng; Carr, Carolyn A; Yeoh, Kar Kheng; Schofield, Christopher J; Davies, Kay E; Clarke, Kieran

    2012-04-01

    Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.

  13. Unexpected roles for ancient proteins: flavone 8-hydroxylase in sweet basil trichomes is a Rieske-type, PAO-family oxygenase.

    Science.gov (United States)

    Berim, Anna; Park, Jeong-Jin; Gang, David R

    2014-11-01

    Most elucidated hydroxylations in plant secondary metabolism are catalyzed by oxoglutarate- or cytochrome P450-dependent oxygenases. Numerous hydroxylations still evade clarification, suggesting that they might be performed by alternative enzyme types. Here, we report the identification of the flavone 8-hydroxylase (F8H) in sweet basil (Ocimum basilicum L.) trichomes as a Rieske-type oxygenase. Several features of the F8H activity in trichome protein extracts helped to differentiate it from a cytochrome P450-catalyzed reaction and identify candidate genes in the basil trichome EST database. The encoded ObF8H proteins share approximately 50% identity with Rieske-type protochlorophyllide a oxygenases (PTC52) from higher plants. Homology cloning and DNA blotting revealed the presence of several PTC52-like genes in the basil genome. The transcripts of the candidate gene designated ObF8H-1 are strongly enriched in trichomes compared to whole young leaves, indicating trichome-specific expression. The full-length ObF8H-1 protein possesses a predicted N-terminal transit peptide, which directs green fluorescent protein at least in part to chloroplasts. The F8H activity in crude trichome protein extracts correlates well with the abundance of ObF8H peptides. The purified recombinant ObF8H-1 displays high affinity for salvigenin and is inactive with other tested flavones except cirsimaritin, which is 8-hydroxylated with less than 0.2% relative activity. The efficiency of in vivo 8-hydroxylation by engineered yeast was improved by manipulation of protein subcellular targeting. blast searches showed that occurrence of several PTC52-like genes is rather common in sequenced plant genomes. The discovery of ObF8H suggests that Rieske-type oxygenases may represent overlooked candidate catalysts for oxygenations in specialized plant metabolism. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  14. Suppression of the β-carotene hydroxylase gene increases β-carotene content and tolerance to abiotic stress in transgenic sweetpotato plants.

    Science.gov (United States)

    Kang, Le; Ji, Chang Yoon; Kim, Sun Ha; Ke, Qingbo; Park, Sung-Chul; Kim, Ho Soo; Lee, Hyeong-Un; Lee, Joon Seol; Park, Woo Sung; Ahn, Mi-Jeong; Lee, Haeng-Soon; Deng, Xiping; Kwak, Sang-Soo

    2017-08-01

    β-carotene, a carotenoid that plays a key photo-protective role in plants is converted into zeaxanthin by β-carotene hydroxylase (CHY-β). Previous work showed that down-regulation of IbCHY-β by RNA interference (RNAi) results in higher levels of β-carotene and total carotenoids, as well as salt stress tolerance, in cultured transgenic sweetpotato cells. In this study, we introduced the RNAi-IbCHY-β construct into a white-fleshed sweetpotato cultivar (cv. Yulmi) by Agrobacterium-mediated transformation. Among the 13 resultant transgenic sweetpotato plants (referred to as RC plants), three lines were selected for further characterization on the basis of IbCHY-β transcript levels. The RC plants had orange flesh, total carotenoid and β-carotene contents in storage roots were 2-fold and 16-fold higher, respectively, than those of non-transgenic (NT) plants. Unlike storage roots, total carotenoid and β-carotene levels in the leaves of RC plants were slightly increased compared to NT plants. The leaves of RC plants also exhibited tolerance to methyl viologen (MV)-mediated oxidative stress, which was associated with higher 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical-scavenging activity. In addition, RC plants maintained higher levels of chlorophyll and higher photosystem II efficiency than NT plants after 250 mM NaCl stress. Yield of storage roots did not differ significantly between RC and NT plants. These observations suggest that RC plants might be useful as a nutritious and environmental stress-tolerant crop on marginal lands around the world. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. Expression of serotonin, chromogranin-A, serotonin receptor-2B, tryptophan hydroxylase-1, and serotonin reuptake transporter in the intestine of dogs with chronic enteropathy.

    Science.gov (United States)

    Bailey, Candice; Ruaux, Craig; Stang, Bernadette V; Valentine, Beth A

    2016-05-01

    Serotonin regulates many intestinal motor and sensory functions. Altered serotonergic metabolism has been described in human gastrointestinal diseases. The objective of our study was to compare expression of several components of the serotonergic system [serotonin (5-HT), serotonin reuptake transporter protein (SERT), tryptophan hydroxylase-1 (TPH-1), 5-HT receptor2B (5-HT2B)] and the enterochromaffin cell marker chromogranin-A (CgA) in the intestinal mucosa between dogs with chronic enteropathy and healthy controls. Serotonin and CgA expression were determined by immunohistochemistry using banked and prospectively obtained, paraffin-embedded canine gastrointestinal biopsies (n = 11), and compared to a control group of canine small intestinal sections (n = 10). Expression of SERT, TPH-1, and 5-HT2B were determined via real-time reverse transcription (qRT)-PCR using prospectively collected endoscopic duodenal biopsies (n = 10) and compared to an additional control group of control duodenal biopsies (n = 8, control group 2) showing no evidence of intestinal inflammation. Dogs with chronic enteropathies showed strong staining for both 5-HT and CgA. Mean positive cells per high power field (HPF) were significantly increased for both compounds in dogs with chronic enteropathies (p < 0.001 for 5-HT; p < 0.05 for CgA). The number of 5-HT-positive and CgA-positive cells/HPF showed significant correlation in the entire group of dogs, including both diseased and healthy individuals (Pearson r(2) = 0.2433, p = 0.016). No significant differences were observed for SERT, TPH-1, or 5-HT2B expression; however, dogs with chronic enteropathy showed greater variabi